CA1143654A - Application of water-dispersible hydrophobic dyes as labels in immunoassays - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Processes, reagents and test kits for the demonstration or determination of an immunochemically reactive component, in which one or more labelled com-ponents are used, that are obtained by direct or indirect coupling of such a component or components to particles of an aqueous dispersion of a hydrophobic dye or pigment, or of polymer nuclei coated with such a dye or pigment.
During the reaction or after an adequate reaction time the nature and/or the quantity of the dye is determined, in the test medium, or optionally in one of the fractions obtained after separation of the bound and free labelled components. This immunoassay is more sensitive than other known techniques, can be performed more rapidly, it is more easily automated, also it enables the simultaneous determination of several immuno components.

Description

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The present invention relates to a method for the demonstration and determination of an immunochemically reactive component such as a hapten, antigen or antibody in an aqueous test medium, employing the principle of the specific inter-action between such immunochemically reactive components-A large number of methods are known whereby substances can be demonstrated or determined, based on the formation of specific immuno complexes. A variety of analytical techniques are available for the direct or indirect detection of the finally-formed immune complexes. Apart from eye reading methods, physical methods are often widely used, such as spectrophotometry, fluorimetry, nephelometry and electron-/
dark field microscopy. These methods can be combined with those using a label or tracer. Instead of detecting the actual immune complex, it is the label which is coupled with one of the components of the complex which is detected, so that it is possible to reach a considerably lower detection limit.
Examples of qualitative immunochemical techniques include the classical precipitin reaction (Heidelberger and Kendall, 1930) and immuno-diffusion - similarly based on immuno-precipitation - (Ouchterlony, 1948) followed in 1953 by immuno-electrophoresis developed by Grabar. In the latter-mentioned two methods antigen and antibody encounter each other via diffusion in an agar gel. The resultant precipita-tion line can, either after a previous colouring or not, be perceived by the naked eye. Gne disadvantage of these simple methods is that diffusion is rather time consuming and that the detection limit is relatively high.
Methods of demonstration based sn the principle of immuno-precipitation were developed by Mancini (1965, radial immuno-diffusion) and Laurell (1966, rocket-electrophoresis).

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These methods suffer from disadvantages because, as in the previously mentioned methods, they need a rather long deter-rnination period and/or a relatively high detection limit.
Apart from these non-labelled immunochemical tech-niques, throughout the years a number of labelled techniques have been developed, among which we can mention the haemagglu-tination test where one of the components is attached to the surface of erythrocytes, the technique of immunofluorescence where one of the components is labelled with a fluorescent compound (fluorophore), the radio-immunoassay developed by Yalow and Berson around 1959 wherein, instead of fluorophore, a radio-active atom or radio-active group is used as the tracer' and the most recent technique known as the enzyme-immunoassay, on which the first publications appeared in 1971 from two groups working independently on each other: the Swedish investigators Engvall and Perlmann and the Dutchmen Schuurs and van Weemen. In principle, the latter assay is analogous to the known radio-immunoassays except that instead of radioactive tracing, an enzyme is used as label.
Radio-immunoassays, which are widely used, have undoubtedly shown great value. ~Iowever, they are afflicted by a number of significant shortcomings such as the risk factor associated with working with radioactive materials, the high costs of reagents and equipment, the short stability of radioactively-labelled reagents, and the requirement that only qualified personnel may perform such assays.
The enzyme-immunoassay does not suffer from these disadvantages. Nevertheless, it is desirable that new assay techniques be developed which are even more sensitive, can be performed more rapidly, can be more easily automated, and/or enable the simultaneous determination of several immuno com-ponents.

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3~54 The present invention relates to an immunoassay which, with respect to the final detection of the immunocomplex, is characterised by the application of, for one or more labelled immunochemically reactive components, obtained by direct or in-direct coupling of such a component or components to particles of an aqueous dispersion of a hydrophobic dye or pigment, or of polymeric nuclei coated with such a dye of pigment, whereby during or after a certain reaction time for the immunochemical reaction, possibly after separation of the free and bound labelled component(s), the nature and/or the quantity of the dye is determined in the test medium, or in one of the frac-tions obtained after separation using known methods, the de termination giving a qualitative or quantitative indication of the immunochemical reactive component(s) to be determined.
The"dispersed dye immunoassay" (DIA) according to the present invention is considerably simpler than the "Radio-Immunoassay" (RIA), because the final detection can be made by a simple eye reading and/or by means of a simple colori-meter. As compared to the "Enzyme-Immunoassay" (EIA, ELISA
EMIT R ), the determination is simpler and more rapid because the enzyme/substrate incubation step can be omitted. Further-more, it is possible to demonstrate and determine simultaneous-ly two or more components if the labels that are used are chromophores, that are clearly distinguishable by spectrophoto-metry. Finally, the advantage of using a dye as a label ia that it can be synthetically reproduced, it can be character-ized exactly by analytical and/or chemical methods and it is stable (in the form of a colloidal particle). This is ob-vious when comparing this type of label with radioactive and enzyme labels which are of limited stability, and/or variable batch quality. On the other hand, the detection limits are ~3~

at least equivalent. Sols of dispersed dyes have some advan-tages over metal sols (e.g. gold), in colorimetric assays, due to the considerably higher molar absorbances of the dye sols as compared to the metal sols, for example:
. gold sol (par-ticle size 50 nm): 3300 l.mole l.cm 1 . dispersed dyes (5.000-80.000 l.mole l.cm 1, cf.
K. Venhataraman: "The Analytical Chemistry of Synthetic Dyes", Wiley & Sons, 1977).
In addition, the colour can be intensified (increase in absorbance) during the final determination of the dye label by dissolving the dye sol particles into an organic solvent (e~g. ethanol, methanol, iso-propanol). For example:

label ~ max(sol) ~ maX(EtOH) ~

Palanil ~ (nm) (nm) max Luminous Yellow G 496 70.77 28300 , " " 464110.36 44100 Palanil Luminous Red G 520 43.33 19300 .. , ., 544 88.27 35300 * 1 g~l Cm-l ** 1 1 -1 -1 The group of coloured organic compounds which can be used in the form of a hydrophobic sol according to the inven-tion described herein, include all the hydrophobic organic dyes and pigments which are insoluble in water or are soluble there-in only to a very limited extent~
These coloured organic compounds should also include water-soluble organic dyes, insofar as, in suitable concentra-tions they form association colloids which, either after a previous cross-linking or not, can be stabilised. Furthermore, ~;~ -4-3~54 it is also possible to use leuco-vat dyes which are soluble in alkaline aqueous medium, and which can be converted by oxida-tion into their original coloured and water-insoluble form, also included are leuco-vat dyes which are water soluble and are skabilised in the form of a sulphate half-ester. Another useful group is the group of dye components which are soluble as such in water and whether or not they are coloured, after coupling to each other in situ, for example via oxidation or diazo coupling, can be converted into water insoluble dyes. me following groups can be mentioned as examples of the above-mentioned dyes, using for this purpose the Official Colour Index nomenclature: "disperse dyes, solvent dyes, pigments, vat dyes, sulphur dyes, mordant dyes, solubilised (leuco) vat dyes, solubilised (leuco) sulphur dyes, azoic dyes, oxidation bases, ingrain dyes" and "transfer dyes" which have not yet been officially named.
me colloidal dye particles which may be used as labels can be prepared by a large number of methods which are known in the art' see for example: Kruyt (Ed.) (1952) "Colloid Science", Vol. I, Elsevier, Amsterdam, Venkataraman (Ed.):
"The Chemistry of Synthetic Dyes), Academic Press, New York, Vol. I (1952), II (1952), III (1970), IV (1971), V (1971), VI
(1972), VII (1974), VIII (1978), Dollmetsch (1976): "Unter-suchungen ~ber die Ursachen der Agglomeration von Dispersions-farbstoffen durch Farbstoffhilfsmittel beim Farben", Forschungsbericht Neue Serie No. 2, Institut f~r Chemiefasern der Institute f~r Textil- und Faserforschung Stuttgart, Leube (1978) Textil Praxis International, Heft 6, 733-737, Heft 7, 823-831.
The process according to the present invention is particularly suitable for the demonstration and determination of an immunochemically reactive component, such as a hapten, 3~

antigen or antibody which is present in an aqueous test medium.
It can also be used for the histological or cytological deter-minations of such components.
For this reason the invention also relates to the new immunochemical reagents, consisting of an aqueous dis-persion of particles of a hydrophobic dye or pigment, normally organic in nature, or of polymeric nuclei coated with such a dye or pigment, to which an immunochemically reactive component has been attached either directly or indirectly.
The invention also relates to new test kits contain-ing such an immunochemical reagent.
By directly or indirectly coupling the immunochemical-ly reactive component to the particle, there is meant any che-mical, physical or physico-chemical bonding, such as a chemical covalent bond, via hydrogen bridges, polar attraction, or ad-sorption, also including bio-specific adsorption~
The particles of the aqueous dispersion of a hydro-phobic dye or pigment, or of polymeric nuclei coated with such a dye or pigment, have a particle size of at least 5 nm, and preferably from 10 to 500 nm. mese dispersions are normally sols, but other types of dispersions can also be used.
The dye sol particles carry a charge, which gives a stabilising effect by mutual repulsion. By adding mainly strong electrolytes, the charge pattern is modified, so that aggre-gation and flocculation take place. I'his can be prevented by coating the particles with macromolecules which possess polar groups, such as proteins, polysaccharides, polyethyleneglycols, polyvinyl alcohols etc.
me protective proteins which can be used include antigens, antibodies or polypeptide fragments thereof which are still immunochemically active. Furthermore it is possible t;S~

to envisage haptens attached to macromolecules (e.g. proteins, polysaccharides) which do not interfere with the other compo-nents during the immunoassay. At the same time there is ob-tained the dyestuff sol-labelled, immunochemically-active component.
It is possible that, in order to stabilise the dyes dyestuff sol, there is required such a high concentration of, for example, antibody on the surface of the colloidal particles, that the effective immunochemical activity of this immobilised protein is affected, for example by steric hindrance. In such a case the coating can be performed in two stages:
1) coating with an optimum quantity, to be determined, of, for example, an antibody, followed by 2) coating with a macromolecular compound (e.g. a protein, a polysaccharide, polyethyleneglycol, polyvinyl alcohol) which, during the immunoassay per se, does not interfere with the other components. This "subsequent coating", e.g. with bovine serum albumin, can at the same time serve to reduce possible non specific adsorption effects.
Another possible protective protein is protein A or the group consisting of lectins (e.g. Con A). After initially coating the sol particles with these proteins, because of the specific affinity of these proteins, it is possible to select-ively provide a second layer by adsorption of immunoglobulins via the-Fc part, and glycoproteins (also including immuno-globulins), via the sugar residue(s) present.
Another possibility is to first coat the dyestuff sol particles with a polymer or co-polymer which is inert in the final immunoassay, after which, subsequently by adsorption and/or covalent bonding, an immunochemically-active component can be attached to the layer of coating material. During the ~ . . ,~

5~ -coating of the sol particles with the inert polymer of co-polymer each particle can be enveloped separately, but it is also possible for several colloid particles to be included inside one and the same polymeric layer.
Covering of the dyestuff sol particles with the inert polymer can take place in two ways: by bringing the dyestuff sol in contact with the polymer, followed by adsorp-tion and/or covalent bonding to the sol particles, or by bring-ing the sol in contact with one or more monomers, and poly-- merising or co-polymerising the latter in situ. Polymerisation can take place for example by radiation or by the addition of a suitable initiator, such as for example a persulphate.
The wrapping of a dyestuff sol particle by polymerisation in situ of the monomeric solution, in which the particle is pre-sent, under the influence of an inorganic initiator such as a persulphate, involves practical difficulties, be.cause the sol -when such an initiator is added - flocculates out. However, it was established that such coating is nevertheless possible by first of all protecting the sol particles, and then placing the protected particles in the monomeric solution, and only then polymerisation being initiated. The compounds mentioned above can be employed as protective agents for this purpose.
Coating of the colloidal dyestuff particles for the purpose of stabilising the sol, applying an immunochemically-reactive component, eliminating non-specific adsorption effects, and/or applying an intermediate polymer or co-polymer layer respectively, can be performed by direct-indirect adso adsorption at the colloidal dyestuff particles, but also by covalent chemical bonding. The latter is governed by the presence of suitable functional groups in the coating material and in the dyestuff. For example one can envisage the ~ '~

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diazotization or aromatic amino groups followed by diazo attachment to an activated aromatic ring system; carboxyl groups can be activated by a carbodiimide and then, possibly via an active ester, be attached to a primary amino component.
Aliphatic primary amino groups and hydroxyl groups can be ac-tivated for example by cyanogen bromide or halogen-substituted di- or tri-azines, after which attachment with a primary amino component or with for example a component containing a -SH, -OH or imidazolyl group can take place. Bifunctional reactive compounds can also be used. For example glutaral-dehyde can be used for the mutual coupling of primary amino components, whilst for example a hetero-bifunctional reagent such as N-succinimidyl 3-(2-pyridyldithio) propionate can be employed for the coupling of a primary amino component to a component containing a thiol group.
In this context, there should only be mentioned reactive dyestuffs which are dispersible and other reactive dyestuffs which are insoluble in water, where the dye consists of a chromophore covalently coupled to a group which as such is already reactive, such as for example halogen-substituted di- and triazines, epoxy groups, vinyl-sulphonic groups and dihaloquinoxalines (see Siegel, (1972) in~ Venkataraman (Ed.):
"The Chemistry of Synthetic Dyes", Academic Press, New York, Vol. VI, Harms, 1979 in: ~anks (Ed.): "Organofluorine Chemicals and their Industrial Applications", Ellis Horwood Ltd., Chichester; pp. 188-204.
Usually the immunochemically-reactive components labelled with colloidal dye particles are employed as re-agent in combination with other reagents, for the demonstra-tion and/or determination of for example haptens, antigensand antibodies, for which all types of immunochemical tech-, g_ 3~S~

niques can be considered such as those used for RIA and EIA.
Hence the invention also relates to "test kits" for use in such immunochemical techniques, which, as their most important component, contain an immunochemically-reactive com-ponent labelled with a dyestuff sol, consisting of a dyestuff sol, the particles of which are coated directly or indirect-ly, adsorptively and/or covalently, with the immunochemically-reactive component.
One of the conventional immunochemical techniques is competitive immunoassay, which can be used for the demonstra-tion and/or determination of any immunochemically-reactive component. For the demonstration, for example, of a certain antigen, this method consists of bringing a test sample, containing an unknown quantity of antigen, into contact either with a certain quantity of the corresponding antigen labelled with a dye sol and an antibody attached to an insoluble carrier, which is directed against this antigen, or a certain quantity of antigen attached to an insoluble carrier and an antibody labelled with a dyestuff sol directed against this antigen.
2 At the end of the reaction the nature and/or the quantity of the dyestuff is determined in the bound and/or the free fraction, which provides a qualitative and/or quanti-tative indication of the antigen to be determined. Mutatis mutandis, an analogous procedure applies to the determination of other immunochemically-reactive components.
Widespread use is also made of the "sandwich tech-nique" which can also appropriately be used with immuno-chemically-reactive components labelled with colloidal dyes-tuff particles. Using this technique, such a component, e.g.
an antibody in cases where an antigen has to be determined, is fixed on an insoluble carrier material. This carrier material h~ -10-~ . .

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can for example be the interior surface of the reaction vessel in which the imrnunochemical reaction is performed, it is also possible to employ carrier materials in the f~ of beads or small rods. After initial incubation with the specimen con-taining the antigen, possibly followed by a washing step, a second incubation takes place with an antibody labelled with a dyestuff sol, after which the dyestuff is determined in the bound and/or the free phase.
Apart from the techniques mentioned for this purpose there are also innumerable other immunochemical techniques where the immunochemically-reactive component labelled with the dyestuff sol can be employed as reagent. We are thinking here especially of an immunochemical test based on the agglu-tination principle. Here for example an antibody labelled with a dyestuff sol is added to a liquid specimen containing the antigen to be determined. In this case, the separation of the bound and frae fractions of labelled components can be avoided, because the detection is based on a visual assess~
ment of the dyestuff sol or on a spectrophotometric/colori-metric determination.
The present invention also makes it possible to si-multaneously demonstrate the presence in a test specimen of different immunochemically reactive components, such as for example haptens, antigens and antibodies or combinations thereof, by employing, for each of the components to be de-monstrated, a corresponding imrnunochemically reactive compo-nent which has been labelled with a colloidal dyestuff parti-cle which is characterising of that component.
Determination of the nature and/or the concentration of the dyestuff at the end of the test can be performed using various techniques known in the art. Examples of these tech-niques include visual assessment which is highly suitable for ~4~365~L

a demonstration precisely when employing dyestuffs, for deter-mination use can for example be made of colorimetry/spectro-photometry. These methods are also suitable for the ultimate detection in the agglutination test, where not so much the concentration of the dyestuff is important, but instead the external appearance of the dyestuff sol (a greater or lesser degree of aggregation, possible flocculation, spectral modifi-cations caused thereby).
Furthermore for the determination of the dyestuff ~or dyestuffs, during simultaneous determination) one can resort to fluorimetry and - in the case of metal complex dyes-tuffs and/or pigments, to "normal" and/or flameless atomic absorption spectrophotometry.
The invention will now be described in greater detail with the aid of the following examples and Figures 1 to 6 in which:
Figure 1 represents standard curves for HCG in phos-phate buffer and urine according to a "DIA-sandwich" technique, Figure 2 demonstrates the possibility of a simul-taneous DIA/sandwich for HCG and HPL, Figure 3 shows spectra of a Palanil ~ Red BF sol/
rabbit anti-HCG immunoglobulin conjugate, in the presence of 0, I, and 5 IU HCG/ml, respectively, after incubation during 20 hours at room temperature;
Figure 4 shows the effect of particle si~e on con-jugate reactivity' Figure 5 is a scanning electron microscope photo of a sol of the transfer dye Lurafix Red BF prepared by the method described in Example 9.2.2.9., and Figure 6 is a scanning electron microscope photo of a sol of the transfer dye Lurafix Red BF prepared by the method described in Example 9.2.2.6.
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Colorimetric and/or visual determination of human chorionic gonadotropin (HCG) in accordance with the DIA principle des-cribed ("Sandwich test").
1.1. Preparation of the dye sol Palanil ~ red BF (BASF, 7 g) was dispersed in dis-tilled water (140 ml). The dispersion was stirred for 45 minutes at room temperature and then centrifuged (30 min., 125 g = 1,225 N/kg). The supernatant portion was transferred into other centrifuge tubes and centrifuged (30 min., 7,500 g =
73,500 N~kg). The supernatant portion was removed and the pellet was washed three times with distilled water (3 x 140 ml, centrifuge: 30 min., 7,500 g - 73,500 N/kg). The pellet was resuspended in distilled water (70 ml), and subsequently enough glass beads (dia. = 3 mm and dia. = 4 mm, mixture 1:2) are added that the liquid level is the same as that of the beads. Then rolling was carried out for 5 days at room temper-ature on a roller bench. The liquid was decanted and centri-fuges (30 min., 300 g = 2,940 N/kg)~ The supernatant portion was then transferred into other centrifuge tubes and again centrifuged (30 min., 1,000 g = 9,800 N/kg). From this last supernatant portion, 3/4th (52.5 ml) wa~ carefully aspirated and this concentrated dyestuff sol was stored at room temper-ature. The extinction at 533 ( = ~ max) nm of a 20 x diluted sample of this sol was 1.57.
1.2. Preparation of the rabbit anti-HCG immunoqlobulin/dye sol conjuqate A sample (0.8 ml of the dye sol described in 1.1 above, was diluted with distilled water (4.2 ml) and the pH
was adjusted to 7.0 using 0.1 mol/l NaOH or HCl. The extinc-tion of this sol is 5.0 at 533 nm (= ~max). Subsequently a ~365~

rabbit anti-HCG immunoglobulin solution (0.1 ml)* was added;
the reaction mi~ture was shaken every 15 minutes during one hour at room temperature, after which a solution of bovine serum albumin (BSA) was added (1 ml, 307.2 g BSA + 0.1 g sodium merthiolate -~ 5 mmol NaCl/l, pH 7.0). The dispersion was shaken every 15 minutes during one hour at room temperature and was then centrifuged (30 min., 4,000 g = 39,200 N/Xg). l'he supernatant portion was removed and the pellet was resuspended up to a volume of 5 ml in a solution having the following com-position: 51.2 g BSA + 0.1 g sodium merthiolate + 5 mmol NaCl/l (pH 7.0 with 0.1 mol/l NaOH).
1.3. "Coatinq'' of Microelisa ~ _plates with rabbit anti-HCG
immunoqlobulin Pho_phate buffer (FFB)-0.04 mol/l Na2HPO4 and 0.04 mol/l NaH2PO4 were mixed to give a solution having pH 7.4, then NaCl was added up to a concentration of 0.15 mol/l.
Solution A
__________ Rabbit anti-HCG immunoglobulin (see 1~2) was dis-solved in FFB Up to a concentration of 30 mg/l.

*) The solution of rabbit anti-HCG immunoglobulin was prepared as follows: The immunoglobulin fraction of rabbit anti-HCG
serum was isolated via the known Na2SO4 precipitation method.
The precipitate was dissolved in and dialysed against an aqueous solution of 5 mmol/l NaCl, the pH of which has been adjusted to 7.0 with solid Na2CO3. The dialysed solution was finally diluted to a protein concentration of 1 mg/ml (in accordance with the Warburg-Kalckar formula:
protein concentration (mg/ml) = 1.45 x A128Co - 0.75 x A26Co ), or 0.65 mg/ml (according to A1280 ~ 1% = 14.5 for IgG).

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Solution B
_ _ _ _ 20 g BSA + 0.1 g sodium merthiolate/l FFB.
Solution A (0.11 ml) was pipetted into the wells of Microelisa ~ plates, after which the plates were incubated for 16 hours at 0-4C. After aspiration, solution B (0.11 ml) I was added to all the wells, after which the plates were incubated i for 30 minutes at room temperature. Finally the wells were aspirated and washed three times with distilled water, after which the plates were dried (16 hours at room temperature over ` 10 pre-dried silica gel), packaged in aluminium laminate-bags (with silica gel sachet) and stored at 4C.
1.4. Determination of_a standard curve for HCG in phosphate buffer and in blank urine Phos~hate buffer (FFB) ____ _________________ As described in 1.3, but with 1 g BSA/l, and 1 g sodium merthiolate/l.
Washing buffer I
Phosphate buffer.
Washing buffer II
0.1 mol TRIS ( (HOCH2)3CNH2 ) + 0.1 mol NaCl + 0.5 g Tween ~ -20 + O.l g sodium merthiolate/l, adjusted to pH 7.4 with 4 mol/l HCl.
Ethanol P.A., 96% (v/v).
HCG
_ _ _ A solution of human chorionic gonadotropin with a content of 1,000 IU (immunoassay)/ml FFB.
Blank urine ___________ Urine from non-pregnant women, filtered over Hyflo and subsequently frozen, prior to use filtered through folding filter.

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Con2ugate The dye sol/anti-HCG conjugate, prepared as described in 1.2, (9 ml) was mixed with concentrated phosphate buffer (1 ml, 10 x concentrated FFs).
Procedure:
1. The following dilution series of the HCG solution were made in FFB and urine respectively:

4,000, 1,000, 250, 62.5, 15.6, 3.9 m IU (immunoassay)/ml.
2. The 0.1 ml of these solutions, and of blank FFB and urine, was pipetted into the wells of a Microelisa ~ plate which has previously been "coated" as described in 1.3 with rabbit anti-HCG immunoglobulin (see 1.3). All this is~
done in duplicate.
3. Close the plate with a suitable cover, and incubate for 3.5 hours at 37C in an atmosphere saturated with water vapour.
4. Aspirate the wells, pipette wash buffer I (0.3 ml) into each well and aspirate again.

5. Pipette the conjugate (0.1 ml) into each well.

6. Incubate as described in item 3, but now for 18 hours.

7. Aspirate the wells, assess the colour in the wells visual-ly and/or pipette wash buffer II (0.3 ml) into the wells.
Aspirate and repeat this procedure twice further.

8. Add ethanol (0.12 ml) to all the dry wells, shake gently and assess the colour by eye and/or measure the extinction at 516 nm (= ~ max).
If, using the procedure described, samples having an unknown HCG content are also included, the HCG concentration can easily be estimated by eye' a more accurate determination can if required be performed by extinction measurement and com-parison with the standard curve. Standard durves for HCG in A

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urine and FFB determined in this manner are shown in Figure 1.
The detectlon limit, defined as (blank + 2 x standard deviation) is for FFB 3.9 m IU (immunoassay)/ml, and for urine 15.6 IU (immunoassay)/ml.
1.5. Determination of HCG in the urine of women in order to detect pre~nancy~
Rea~ents see section 1.4.

Procedure:
As described in 1.4., but now with the inclusion of the corre~ponding urine specimens, which may or may not be diluted. The results are shown in the following table:

_ _ Pregnos. _ __ SpeClmen Dllutioll ~ IA Visual 516 HCG conc.

1374 Undiluted + +00588 4 000 1374 1:1~ + + 0.59540 000 1130 Undiluted + ~ 0.659 4 000 305 Undiluted ~ ~ 0.707 4 000*
726 Undiluted _ _ 0.057 80 546 Undiluted _ _ 0.043 65 1123 Undiluted _ 0.047 65 *in the undiluted specimen.
Ihe conclusion as to whether or not pregnancy exists corresponds with the results of the pregnancy test Pregnosticon ~ "All-in". In this latter test "~
1000 IU/ml and "-" = ~ 500 IU/l.

As example 1, except that disperse dye/anti-HCG
con~ugates were prepared using Resolin ~ Brilliant Blue RRL
and the fluorescent disperse dyes Samaron ~ Brilliant Red H6GF, Samaron ~ Brilliant Yellow HlOGF, Palanil ~ Luminous Red G and Palanil ~ Luminous Yellow G as colloidal labels.
(for corresponding ~ max values see Example 9).

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Dilution series of HCG were made in buffer. Reduc-tion of the incubation periods as compared to the standard procedure was investigated: HCG incubation 2.5 h and conjugate incubation 2.5 h instead of 6.5 and 18 h, respectively. A
detection limit of 0.02-0.25 mIU HCG/ml was obtained. This compares very well with other test systems:
- RIA : 2 mIU HCG/ml (DL:HCG concentration at (90%
binding + 2 x SD)) - EIA : 20 mIU HCG/ml (DL: as for RIA) - SPIA : 0.25-l mIU/ml (DL: blanc + 2 x ~D) colorimetric detection) - rev-HAI : 10 - 20 mIU HCG/ml (DL: HCG concentration giving a significant change in pattern).
Additionally, the total test period was decreased from 24.5 h to 5 h, with only a limited effect on the detection limit (in the case of Samaron Brilliant Red H6GF).
The fluorescent disperse dyes were investigated in order to improve the detection limit by measuring fluorescence instead of absorbance. A significant improvement was obtained in the case of Samaron ~ Brilliant Yellow HlOGF and Palanil Luminous Yellow G, whereas no effect was found in the case of Samaron ~ Brilliant Red H6GF, and Palanil ~ Luminous Red G.

DIA for Hepatitis B surface Antigen ( B sAg); sand-wich system.
3.1. Preparation of dye sol See section l.l., the disperse dyes Palanil ~ Red BF
and Samaron ~ Brilliant Red H6GF were used as colloidal labels. (for corresponding ~ max values see Example 9).
3.2. Preparation of the sheep-(anti-HBsAg) IgG/dye conjugate.
See section 1.2., but use sheep-(anti- BsAg) immuno-3~S4 globulin instead of rabbit anti-HCG IgG.
3.3. Coating of Microelisa ~ plates with sheep-(anti-HBsAg) IgG.
See section 1.3., use the sheep instead of the rabbit immunoglobulins.
3.4. Determination of standard curves for HgsAg (subtype ad and ay).
Dilution series of HBsAg (ad and ay) were made using human negative control serum as diluent, in the range 4 - 1000 ng/ml. Samples (0.1 ml) of these dilutions, and of the negative control serum, were assayed accord-ing to the procedure described in section 1.4. (steps 3-8), for ~ max (ethanol) see Example 9.
Detection limits of 16-23 ng/ml (ad) and 24-38 ng/ml (ay) were obtained with the Samaron ~ dye/conjugate. For comparison:
- EIA (Hepanostika ~ ): 3 ng/ml - EIA (Hepanostika-T ~ ): 0.7 ng/ml (DL: mean negative value +
5 x SD) - SPIA: 20-40 ng/ml (DL: blank + 2 x SD) (colorimetric detec-tion).
me DIA/sandwich system was also used to compare several samples of monoclonal autibodies, with a standard pre-paration of heterogeneous sheep anti-HBsAg IgG. Three samples gave dose response curves similar to the standard, whereas the other preparations were of a distinctly poorer quality.

DIA for Human Placental Lactogen (HPL), sandwich system.
4.1. Preparation of dye sol.
See section 1.1., the disperse dyes Palanil ~ Red BF

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and Palanil ~ Yellow 3G were used as colloidal labels (for corresponding ~max values see Example 9).
4.2. Preparation of the rabbit (anti-~IPL) IgG/dye conjugate.
See section 1.2., but use anti-HPL instead of anti-HCG.
4.3. Coating of Microelisa ~ plates with rabbit anti-HPL
IgG.
See section 1.2., but use anti-HPL instead of anti-HCG.
4.4. Determination of Standard curves for HPL.
Dilution series of HPL were made in FFB (see section 1.2) ~ 10 - in the range 0.4-100 ng/ml. Samples (0.1 ml) of these ¦ dilutions, and of FFB, were assayed according to the ¦ procedure described in section 1.4. (steps 3-8); for ¦ ~max (ethanol) see Example 9.
A detection limit of 0.2-1.7 ng HPL/ml was obtained.
For comparison:
- RIA : 0.03-0.14 ng/ml - EIA : 2 ng/ml - SPIA : 0.12 ng/ml (colorimetry).

DIA for anti-Rubella, sandwich system.
' 5.1. Preparation of dye sol.
See section 1.1., the disperse dyes Palanil ~ Red BF
and Resolin ~ Brilliant Blue RRL were used as colloidal labels (for corresponding ~max values see Example 9).
5.2. Preparation of the sheep anti-(human IgG) IgG/dye conjugate.
See section 1.2., but use the sheep immunoglobulin instead of the rabbit material.
5.3. Coating of Microelisa ~ plates with inactivated Rubella viral antigen (obtained from tissue culture).
See section 1.3., but use the Rubella antigen instead of ' -20-;5~

the immunoglobulin.
5.4. Determination of standard curves for human anti-Rubella.
Dilution series of human anti-Rubella were made using sheep negative control serum as diluent, in the range 0.4-320 IU/ml. Samples (0.1 ml) of these dilutions, and of the negative control serum, were assayed accord-ing to the procedure described in section 1.4. (steps 3-8), for ~max (ethanol) see Example 9.
The detection limit, defined as (BL + 2 SD), was 2.5 IU/ml, which compares favourably with an estimation of the detection limit of Rubenostika of ~ 10 IU/ml.

DIA for human Prolactin (PRL), sandwich system.
6.1. Preparation of dye sol.
See section 1.1., the disperse dyes Palanil ~ Luminous Red G and Palanil ~ Luminous Yellow G were used as colloidal labels (for corresponding ~ max values, see Example 9).
6.2. Preparation of the monoclonal (anti-PRL) IgG/dye con-jugate.
See section 1.2, but use the monoclonal IgG instead of the rabbit material.
6.3. Coating of the Microelisa ~ plates/strips with mono-clonal (anti-PRL)IgG.
See section 1.3., but use the monoclonal IgG instead of the rabbit material NOTE: Immunoglobulins from different clones were used for the preparation of the conjugate (6.2.) and for the coating of the plates/strips.
6.4. Determination of standard curves for PRL.
Dilution series of PRL were made in FFB (~ee section 1.4.) in the range 0.4-100 ng/ml. Samples (0.2 ml) of ~3~

these dilutions, and of FFB, were assayed by the proce-dure described in section 1.4. (steps 3-8), with the following modifications:
- incubation of antigen (PRL): 20 h, room temperature - incubation of conjugate: 20 h, room temperature.
For ~max tethanol) see Example 9.
A detection limit of 1 4 ng/ml was obtained.
For comparison: EIA 1-4 ng/ml SPIA 6-10 ng/ml.

Simultaneous determination of HCG and HPL, accord-ing to the DIA principle, sandwich system.
7.1. Preparation of the dye sols.
See section 1.1., the disperse dyes Resolin ~ Brilliant Blue RRL and Palanil ~ Yellow 3G, were used as colloidal labels (for corresponding ~max values see Example 9).
7.2. Preparation of the rabbit (anti-HCG) IgG/- and rabbit (anti-HPL) IgG/dye conjugates.
See Section 1.2., prepare the following combinations:
- Resolin ~ Brilliant Blue RRL/anti-HCG
- Palanil ~ Yellow 3G/anti-HPL.
- ~he combined conjugate is prepared by mixing equal volu-mes of the two conjugates, yielding a final absorbance of 5 (at ~ max) for each dye-conjugate.
7.3. Coating of Microelisa ~ plates with rabbit anti-HCG, rabbit anti-HPL, and with a mixture of both.
See section 1.3., plates for the simultaneous assay were prepared using the following coating mixture:
rabbit anti-HCG 15 ng/l rabbit anti-HPL 15 ng/l.

~3~4 7.4. Simultaneous determination o-f HCG and HPL.
Generally, the assay procedure described in section l.~.
was used (for ~ max values in ethanol, see Example 9):
a) The single and combined conjugates were tested in microtitre plates which were coated only with rabbit anti-HCG or anti-HPL. The combined conjugate and the anti-HCG conjugate gave an equal response in a dilu-tion series of HCG in FFB (see section 1.4), whereas the anti-HPL conjugate did not react. The anti-HPL
conjugate gave a higher response than the combined conjugate in a dilution series of HPL in FFB, whereas the anti-HCG conjugate did not react.
b) Finally, samples of HCG, HPL and (HCG + HPL) in FFB
were incubated in the wells of a microtitre plate coated with rabbit anti HCG and anti-HPL simultaneously. The second incubation was performed with the combined con-jugate. T~e results are presented in Figure 2, which demonstrates the possibility of a simultaneous DIA/
sandwich.

DIA for testosterone.
8.1. Method l.
This method is based on the detection of free anti-testosterone on the solid phase, after incubation with the testosterone containing sample.
8.1.1. Preparation of the dye sol See section 1.1.
8.1.2. Preparation of testosterone~ -hemisuccinyl-BSA/dye sol conjuqate Testosterone-ll ~-hemisuccinate is dissolved in 2 ml dimethylformamide (DMF) and then the solution is cooled to -15 C.
~' ~ 23-5~

Bovine serum albumin (BSA, 140 mg) is dissolved in distilled water (3 ml), after which 40 ~1 of 4 mol/l NaOH and 2 ml DMF is added. The solution is then cooled to -15C.
- Now 12.5 ~1 of N-methylmorpholine and 12.5 ~1 of isobutyl chloxoformiate is added to the solution of testo-sterone derivative. After three minutes this reaction mix-ture is added to the BSA solution. Whilst stirring the reac-tion mixture is held for 1 ho~r at -15C and then for 3 hours at 0C. men the reaction mixture is dialysed against distilled water for 16 hours at 4C, with the distilled water being re-gularly renewed. The dialysed solution is then centrifuged and the clear supernatant is freeze-dried.
The testosterone~ -hemisuccinyl-BSA/dye sol conjugate is now prepared using the method described for the immobilisa-tion of rabbit anti-HCG immunoglobulin on a Palanil ~ Red BF
sol whilst using the same concenkration for the testosterone-sSA derivate as for the rabbit anti-HCG immunoglobulin.
8.1.3. "Coatinq" of Microelisa ~ plates with rabbit anti-testosterone immunoqlobulins The coating was carried out as described in section 1.3.
using the immunoglobulin fraction isolated from a rab rabbit antiserum, obtained by immunisation with testo-sterone-ll-~-hemisuccinyl-BSA.
8.1.4. Determination of testosterone -Using the reagents and the test procedure as described for HCG in section 1.4., a standard curve was deter-mined for testosterone by means of which the testo-sterone concentration of unknown samples was subse-quently calculated via the A516 of the samples, obtain-ed in accordance with the said test procedure. The detection limit of this determination is 1 ng/ml.

, ~
, . ..

~3q~5~

It is easy to estimate the testosterone contents by reading with the naked eye (comparison of the colour intensity of standard and unknown samples a-fter stop-ping the reaction).
8.2. Method 2.
This method is based on a competitive binding of testo-sterone (T) and T -BSA to anti-(T -BSA), which was fixed onto the solid phase (e.g. polystyrene tube, or micro titre plate).
Detection followed by a second incubation with an anti-(T -BSA)/dye conjugate.
NOTE: .T -BSA: testosterone-3-O-carboxymethyloxime, covalently coupled to a NH2-function of BSA
(amide bond) .T -BSA: testosterone-ll-hemisuccin~te, covalent-ly coupled to a NH2 function of BSA (amide bond).
8.2.1. Preparation of dye sol.
See section 1.1, the disperse dyes Samaron ~ Brilliant Red H6GF and Samaron ~ Brilliant Yellow HlOGF were used as colloidal labels (for corresponding max values, see Example 9).
8.2.2. Preparation of the rabbit (anti-T -BSA) IgG/dye conju-gate.
See section 1.2., but use the rabbit anti-Tll-BSA
instead of anti-HCG.
8.2,3. Coating of Microelisa ~ plates and polystyrene tubes with rabbit (anti-Tll-BSA) IgG.
See section 1.3., but use the rabbit (anti-Tll-BSA) instead of the anti-HCG.
NOTE: 1 ml of solutions A and B are used when coating the tubes.

.

~3~5~

8.2.4. Determination of standard durves for testosterone.
Solutions of T and T -BSA were made in FFB (see sec-tion 1.4.).
a) The concentration of T3-BSA to be used in the com-petitive assay, was investigated by incubation of a dilution series of T3-BSA only, followed by the conjugate; 1 ml volumes per tube, or 0.1 ml volumes per microtitre plate well. Procedure as described in section 1.4. (steps 3-8): for ~ max ~ethanol) see Example 9. For the total competitive assay con-centrations of T3-BSA corresponding with 2 and 16 pmol T/ml total test volume, were chosen b) The competitive assay was performed using constant concentrations of T -BSA corresponding with 2 and 16 pmol T/ml total test volume, and a dilution se-ries of 0-64 pmol T/ml sample, for the tubes: 0.9 ml of testosterone containing sample and 0.1 ml of a T -BSA solution (corresponding with ~0, respect-ively, 160 pmol T/ml); for the microtitre plate the respective volume are changed to 0~09 and 0.01 ml.
Further procedure as described in section 1.4.
(steps 3-8), for ~max (ethanol) see Example 9.
The detection limit, defined as (BL - 2 x S~), was 0.2 -0.4 pmol T/ml sample, using a T3-BSA concentration correspond-ing to 2 pmol T/ml total test volume. The total detection range was about 0-8 pmol T/ml sample. In RIA and EIA, 50%
binding is reached at, respectively, 1 pmol T/ml and 0.7 pmol T/ml.

Alternative methods for the preparation of dye sols.

~' ~ -26-~3~5~

9.1. Disperse dyes.
Instead of the Palanil ~ Red BF (BASF) mentioned in section 1.1. other disperse dyes have also been used for preparing dyestuff sols including:

_ _ A (nm) ~ (nm)c) max measurinq watera) ethanol~) ethanol _ Palanil~ Violet 6R BASF 623 571 __ Yellow 3G 415 443 443 Luminous ) Yellow Gd - 496 464 443 " Lumina~s Red G 520 544 540 Ter asil~ Brd~liant Flavin 8G~F Ciba-Geig~ 488 461 443 Terasil~ Brilliant Pink 4BN Ciba-Geig~ 571 571 540 Cibacet~
Violet 2R Ciba-Geig~ 538 592 549 Foron~ Brilliant Flavin S8GFd) Sandoz 433 427 _~
Resolin~ Brilliant Blue RRL Bayer 670 578 600 Procinyl~ Blue R ICI 672 __ __ Samaron~ ~rilliant Red H6G~d Hoechst 512 510 510 Samaro ~ Brilliant Yellow HlOGFd) Hoechst 451 458 443 Samaro ~ Br~lliant .
Orange HFRd Hoechst 508 499 492 Samaron~ Violet HFRL Hoechst 566 543 540 a) as colloidal solution b) as molecular solution c) these values were used due to the presently available filters.
d) representatives of disperse dyes which can also be detected by fluorometry e~ a representative of reactive disperse dyes.
Sols were prepared from the commercial dyestuffs ~36~

starting with a 5% (w/v) dispersion of the dyestuff in dis-tilled water, in case of dry, powdered products, in case of liquid preparations experiments were started with a 5% (v/v) dispersion in distilled water.
I Fractionation of the dye dispersion in water was ¦ carried out by centrifuging as described in 1.1. Fractiona-I tion to particle size has also been carried out with the aid ¦ of filters having a defined pore size' in this way usable sols ! are obtained, but the yield of dyestuff was considerably less than with centrifuging, whilst furthermore, the method is ex-tremely time-consuming.
Hydrodynamic chromatography and the use of gradients during centrifuging form a useful supplement to the methods mentioned above.
9.2. Transfer dyes Transfer dyes are dyes which are used during transfer printing, whereby a coloured pattern is transferred from one surface to another, generally from paper to textile. The "sublistatic-, sublimation-, dry heat-, or thermopr1nting process" makes use of sublimable organic dyes which are generally insoluble in water and soluble in organic environments. Sols of this type of dye have also been made up in water, via the "condensation method" (see J.Th.G. Overbeek in: H.R. Kruyt (Ed.) "Colloid Science", Vol. I, pp. 59-60, 1952, Elsevier, Amsterdam).
9.2.1. Lurafix 3 Blue FFR (BASF) __________________________ Solutions (1 ml) of Lurafix ~ Blue FFR in acetone with the following concentrations: 2, 1.5, 1.0, 0.8, 0.6, 0.~, and 0.2 g/l were added, during intensive stirring, al-ways to 49 ml distilled water. The suspensions obtain-ed are centrifuged (30 minutes, 1000 g = 9,800 N/kg) ~3~

and the pellets are washed with distilled water (50 ml) and again centrifuged under the above-mentioned condi-tions. Then the pellets are suspended in such a volume of distilled water that the final concentration is 0.1 mg/ml. ~he final dye sols were obtained by subjecting the various suspensions to an ultrasonic treatment (Branson Sonifier B-12, 2 minutes, 70 Watts). The ab-sorption spectrum of these dye sols was recorded in the region 750-360 nm. The value of ~max dropped from 716 to 617 nm, starting from the sol corresponding to an original dye/acetone concentration of 2 g/l down to 0.2 g/l. These spectral changes are indicative of a decreasing particle size (see ~.R. Kruyt: "Colloids", P. 132, 1930, Wiley, New York, G.H. ~onker in H.R.
Kruyt (Ed.): "Colloid Science", Vol. I, p. 102, 1952, Elsevier, ~nsterdam, F.B. Gribnau, Dissertation, utrecht 1935).
9.2.2. Lurafix ~ Red BF (BASF) ________________________ a. A solution of Lurafix ~ Red BF in acetone (1 ml, 0.5 g/l) is added, under vigorous stirring, to distilled wa-ter (24 ml). Then, under vacuum and at room temperature, the acetone is gently evaporated. Via an initially stable sol there is finally obtained a precipitate. The suspension is centrifuged (30 minutes, 1000 g - 9800 N/kg) and the pellet is resuspended in distilled water (25 ml) followed by an ultrasonic treatment (Branson Sonifier B-12, 2 minutes, 70 Watts).
b. A solution of Lurafix ~ Red BF in acetone (1 ml, 5 g/l) is added under vigorous stirring to distilled water (249 ml, 50C). After 1 minute at 50C, the mixture is cooled down to room temperature. After standing for one day at room temperature the initially stable sol starts 365~

to partially flocculate.
Figures were taken of both freshly prepared sols by means of a scanning electron microscope, see Figures 5 and 6.
9.3. Fat dyes (solvent dyes) A group of hydrophobic organic dyes which are insoluble in water but soluble in organic solvents or mixtures thereof. Using condensation methods as described in 9.2, sols can be made from these dyes in aqueous media.
9,4. Vat dyes mese water-insoluble anthraquinoid or indigoid dyes can be converted by reduction in alkaline medium into the corresponding, water-soluble leuco compounds. From these it is then possible to prepare dye sols by control-led oxidation. The leuco compounds (solubilised vat dyes) stabilised as sulphate ester can also be used for this purpose.
9.5. Orqanic piqments These compounds which by definition are insoluble in water and organic media can be converted via a dispersion me-thod (see P. Nylen and E. Sunderland: "Modern Surface Coatings", Interscience Publishers, London 1965) into colloidal "solutions.

Variations on the preparation of dye sol/immunoglobulin conjugates, as described in Example 1 (1.2.).

10.1. Immobilisation of immunoqlobulin on colloidal dye par-ticles 10.1.1. Lurafix ~ Blue FFR (BASF) __________________________ A solution of Lurafix ~ Blue FFR in acetone ~5 ml, 1 g/l) is added under vigorous stirring to distilled wa-~3 ter (245 ml). After centrifuging (30 minutes, 1000 g =

365~

9800 N/kg) the supernatant is removed and the pellet is washed with distilled water (50 ml). The pellet is resuspended in distilled water (up to 50 ml) and the suspension is treated ultrasonically (Branson Sonifier B-12, 2 minutes, 70 Watt). The sol obtained is diluted to give a AllC7m of 1Ø
Rabbit anti-HCG immunoglobulin solution (0.2 ml, 1 mg/ml in a solution of 5 mmol NaCl/l, pH 7.0) is added to 10 ml of this sol and the mixture is stored for 16 hours at room temperature. Then a solution of Carbowax ~ -20M (0.2 ml, 10 g/l in a solution of 5 mmol NaCl/l, pH 7.0) is added and after being kept for 30 minutes at room temperature the sol is cen-trifuged (30 min. 4000 g = 39,200 N/kg). The pellet is washed twice with a Carbowax ~ -20M solution (0.2 g/l in a solution of 5 mmol NaCl/l, pH 7.0) and subsequently resuspended therein to give an end volume of 5 mlO
me procedure described above is repeated, but now using normal rabbit immunoglobulin. The immuno-activity of - the conjugates is established in the following manner Z0 Specimens (2 ml) of each conjugate are diluted using the latter-mentioned Carbowax ~ -20M solution. To this 0.1 ml of a solution of HCG labelled with horse-radish peroxidase HRP
is added, and the reaction mixture is incubated for two hours at room temperature. Then this is centrifuged (30 mins., 4000 g = 39,200 N/kg), the pellets are washed twice with the latter-mentioned Carbowax ~ -20M solution and resuspended in a solution of chromogen/substrate (o-phenylene-diamine/urea peroxide). After one hour at room temperature the enzyme reaction is stopped with 4 mol/l sulphuric acid, and the A492 of the supernatants is measured after centrifuging (30 mins., 4000 g = 39,200 N/kg).

~4365~

Dye/anti-HCG Ig conjugate: A492 = 0.S56 ~ye/normal Ig conjugate: A492 = 0.27S.
10.1.2. Palanil ~ Red sF tBASF) Immobilisation of for example proteins on solid car-rier materials can be obtained via adsorption and via, direct or indirect, covalent coupling. me latter de-pends on the presence of suitable functional groups in the chemical structure of the dye. For example it is possible to use aromatic amino groups in a diazo coupling, whilst carboxyl groups can be activated by means of a carbodiimide. Aliphatic primary amino groups and hydroxyl groups can be activated by means of cyanogen bromide. Bi-functional compounds can also be used, mus it is possible to use glutaraldehyde for the coupling of amino components.
It is also possible in this context to use reactive dispersion dyes, these being dyes in which the chromophore is attached to a group which is already reactive as such, e.g.
halotriazines and halo-pyrimidines.
I`he following examples have been performed with the dispersion dye Palanil ~ Red sF (sASF) using a fraction which was isolated in the following manner:
A dispersion of this dye in water (62.5 g/l) is cen-trifuged (30 min., 750 g = 7,350 N/kg). Ihe pellet is washed five times with distilled water and twice with a solution of poly (vinyl alcohol) (PVA) in water (1 g PVA/l, PVA: Mowiol ~
28-99, Hoechst). Finally the pellet is resuspended to a final volume of 2 1 in a PVA solution (0.1 g PVA/l in a solution of 5 mmol NaCl/l, pH 7.0). Samples (25 ml) of this dye sol, optionally after processing as described in greater detail be-low, were mixed with a solution of sheep anti-HCG immuno-~ .

~3~4 globulin (0.65 ml, 40 mg/ml in a solution of 5 mmol NaCl/l, pH
7.0) Adsorption and/or attachrnent of this protein to the colloidal dye particles was carried out for 16 hours under the specified conditions:
10.1.2.1. Adsorption a. Adsorption at pH 4.0 and 0 mol NaCl/l b. Adsorption at pH 4.0 and 0.1 mol NaCl/l c. Adsorption at pH 7.0 and 0 mol NaCl/l d. Adsorption at pH 7.0 and 0.1 mol NaCl/l e. Adsorption at pH 6.0 ancl 0 mol NaCl/l (sol and protein were mixed at pH 4.0) f. Adsorption at pH 6.0 and 0.1 mol NaCl/l (sol and protein were mixed at pH 4.0).
10.1.2.2. Diazo cou~linc~
The dye was diazotised at 4C using NaN02/HCl and subsequently the pH of the reaction mixture was ad-justed to 8.6 using solid Na2CO3. Finally protein was added, the coupling was performed at 4C.
a. NaN02 : 0.1 mol/l HCl: 0.1 mol/l b. NaN02 : 0.05 mol/l: HCl: 0.05 mol/l c. NaN02 : 0.01 mol/l HCl: O . 01 mol/l 10.1.2.3. Cross-llnking by_means of glutaraldehyde The glutaraldehyde concentration of the dye sol was set at 1 and 10 mmol/l respectively, after which the pH was adjusted to 7.4 and 10.0 respectively. After 1 hour at room temperature the pH 10 was reduced to 9Ø Finally protein was added. The reaction was performed at room temperature.
a. Glutaraldehyde: 1 mmol/l pH 7.4 b. " : 10 mmol/l pH 7.4 c. " : 1 mmoljl pH 10 . O

~1~3~5~

~ 10.1.2.4. CNBr activation -- _ _ _ The dye sol was centrifuged and the pellet was wash-ed with a solution of 5 mmol NaCl/l, pH 7Ø This was followed by resuspension in the same solution, or in a solution of PVA (10 g/l in a solution of 5 mmol NaCl/l, pH 7.0). The CNBr concentration of the various sols was adjusted to 0.045 and 0.005 mol/l respectively, after which the pH was adjusted to lloO using 4 mol/l NaOH. After 12.5 min. at room temperature the reaction mixtures were centrifuged and the pellets were washed twice with 0.1 mol/l NaHC03, pH 8.5 (4C). This was followed by resus-pension to the initial volume in 0.025 mol/l NaHC03, pH 8.5. The protein coupling was performed at 4C.
a. PVA concentration: O g/1 CNBr conc.: 0.045 mol/l b. ., : 10 g/1 " : 0.045 mol/l c. " : 10 g/l " : 0.005 mol/l After 16 hours, the various reaction mixtures were centrifuged (30 min., 1000 g = 9,800 N~kg). The supernatants were removed and the pellets were washed twice with a solution of 5 mmol NaCl/l, pH 7.0 and resuspended to a final volume of 20 ml in the same solution.
The immuno-activity of the various conjugates was determined by incubating 5 ml of each conjugate (16 hours, 4C, in the dark) with HCG, labelled with horse-radish peroxidase (HRP). Then the reaction mlxtures are centrifuged (1000 g =
9,800 N/kg, 30 min.). The pellets are washed twice with 3 ml of a solution of 5 mmol NaCl/l, pH 7.0 and subsequently re-suspended in a solution of chromogen/substrate (o-phenylene-diamine/urea peroxide~. After one hour reaction time at roomtemperature (in the dark) the enzyme reaction is stopped with A _34_ mol/l sulphuric acid and after centrifuging (30 min., 1000 g =
9,800 N~kg) the A492 of the supernatants is measured.
'~he above-mentioned procedure was also performed u~sing HRP alone, to check specificity.

__ Conjugate A492 492 A492 (HCG-HRP) *) (HRP)*) (HCG-HRP) **) .
10.1.2.1-a. 0.195 0.094 b. , 0.420 0~098 0.750 c. 1.738 0.109 1.970 d. 0.942 0.109 1.280 e. 0.230 0.109 f~ 1.129 0.103 1.496 10.1.2.2-a. 0.738 0.120 1.260 b. 0.568 0.113 1.316 c. 0.777 0.086 1.505 10.1.2.3-a. 1.097 0.062 1.920 b. 00489 0.073 c~ 0.4~3 - 0.065 d. 0.436 0.118 10.1.2.4-a. 1.900 0.088 2.153 b. 1.300 0.137 2.020 c. 1.022 0.118 1.671 *)The conjugates were tested on three different days:
Day 1: 10.1.2.1-a/f Day 2: 10.1.2.2-a/f and 10.1.2.3-a/c Day 3: 10.1.2.4-a/c ~-**) 'rhese conjugates were tested on the same day.
0 10.2. 'rhe effect of "subsequent coatin~/ of dye sol/immuno-qlobulin coniuqates on immuno-activity 'rhe experiments were performed using as example Palanil Red BF (BASF), using the type of sol already prepared in accordance with the method described in 1.1. above. This sol was adjusted to pH 7.0 using 0.1 mol/l NaOH or HCl and the extinction (at 533 nm) was set at 5Ø

~3~

Samples (37 ml) of this sol are mixed with a solu-tion of rabbit anti-HCG immunoglobulin (0.74 ml, 1 g/l in a solution of 5 mmol NaCl/1, pH 7.0). ~he Ig concentration in the reaction mixture is then 20 mg/l. After 1 hour incuba-tion at room temperature the following are added to the sols:
a. Nothing b. Carbowax ~ -20M up to a concentration of 0.2 g/1 c. BSA up to a concentration of 0.2 g/l.
After a further 1 hour incubation at room tempera-ture the sols are centrifuged (30 min., 4000 g = 39,200 N/kg).
me pellets are re-suspended to a final volume of 37 ml in a solution of 5 mmol NaCl/l with either no extra additive, 0.2 g Carbowax ~ -20M/l, or 0.2 g BSA/l.
me various conjugates and the blank dye sol were tested in a DIA ("Sandwich test") using the procedure described in section 1.4. me dilution series for HCG employed was 0, 25 250, 1000 and 4000 IU (immunoassay)/l phosphate buffer.

. ___ Conjugate A5l6 HCG ~IU/l) 250 1000 4000 10.2-a 0.579 0~773 0.722 0.480 b 0.1650.17~3 0.165 0.166 __________________ 0 621 _________ 1.300 1O591 Blank dye sol 0.056 10.3. Purification methods for isolation of the conjuqate after preparation Inter alia the following techniques can be considered as methods of purification:

- centrifugation - gel chromatography - affinity chromatography - membrane filtration - partial precipitation (flocculation, followed by - . ~
3~

washing and reconstitution of the sol), Centrifugation and gel chromatography were investi- -gated in detail and the results were compared with those for a conjugate which had not been purified but otherwise identically prepared~ For ~his purpose we used non-purified conjugate 10.2-c.
10.3-a Gel chromatography 4 ml conjugate (A513C3m = 5.0) is passed through a Sepharose ~ CL 2B column (Pharmacia K 16/20, bed volume 35 ml, equilibrated with a solution of 5 mmol NaCl + 0.2 g BSA + 1.0 g NaN3/1, pH 7.0). The column is eluated with equilibration buffer (room temperature, 30 ml/hour), detection of the eluate by measuring the A280. Fractions of 1.3 ml are collected, the main fractions from the dye peak are mixed.
10.3-b Centrifugation 4 ml conjugate (A533 = 5.0) is centrifuged (30 min., 4000 g = 39,200 N/kg) and re-suspended to give a final volume of 4 ml using a solution of 5 mmol NaCl + 0.2 g BSA + 1.0 g NaN3/1, pH 7Ø
10.3-c No purification The unpurified conjugate 10.2-c is used for this purpose.
~he three conjugates are tested in a DIA (Sandwich test) using the procedure described in section 1.4. Ihe HCG
dilution series in section 10.2. was employed.
. ._ _. . .~
Conjugate A5l6 HCG (IU/l) 0 250 lOOO 4000 Yield(%) 10.3-a 0.228 0.551 0.713 0.505 4.3 b 0.218 0.339 0.456 0.557 93.6 c 0.234 0.418 0.583 0.700 lOO.0 ~, -37-10.4. Effect of the immunoqlobulin concentration used durinq the preparation of the conjuqate on the final immuno-activit~
Samples (5 ml) of a dye sol prepared from Palanil ~ Red BF (BASF) by the method described in section 1.1. are mixed with 0.1 ml of a rabbit anti-HCG immunoglobulin solution (5 mmol NaCl/l, pH 7.0) resulting in final concentrations of:
10.4-a 10 mg/l 10b 20 mg/l c 40 mg/l d 80 mg/l After incuvation for one hour at room temperature, 0.1 mol of a BSA solution (5 mmol NaCl + 20 g BSA/l, pH 7.0) is added, resulting in a final concentration of 0.4 g BSA/l.
After incubation for 1 hour at room temperature the sols are centrifuged (30 min., 4000 g = 39,200 N/kg)l the supernatants are removed and the pellets are re-suspended up to a final vo-lume of 5 ml using a solution of 5 mmol NaCl + 0.4 g BSA +
0.1 g sodium merthiolate/l, pH 7Ø
The immuno-activity of the conjugates is ascertained by a DIA (Sandwich test) in accordance with the procedure des-cribed in section 1.4., the HCG dilution series from section 10.2. was employed.

Conjugate 1- __ _ _ HCG (IU/l) ¦ 0 250 1000 4000 10.4-a 0.2680~4990.761 1.000 b 0.3620.6580.9021.141 20 c 0.5110.7410.9131.107 d 005070.6810.8470.955 3~54 10.5. Effect of the type of anti-HCG immunoqlobulin on the immuno-activity of the_conju~ate, the use of anti-HCG
immunoqlobulin isolated from sheep and rabbit anti-HCG
serum and of monoclonal mouse anti-HCG immunoqlobulin The anti-HCG immunoglobulin/dye sol conjugates were prepared by coating samples (5 ml, A1S3C3 = 5.0) of a Palanil ~ Red BF sol, made by the method described in section 1.1., in the following manner:
10.5-a Sheep anti-HCG immunoglobulin The protein solution (0.1 ml, 1 g/l in a solution of 5 mmol NaCl/l, pH 7.0) is added to the sol and the reaction mixture is incubated for 1 hour at room tem-perature. Then a BSA solution (0.1 ml, 20 g/l in a solution of 5 mmol NaCl/l, pH 7.0) is added and after incubation for 1 hour at room temperature the sol is centrifuged (30 min., 4000 g = 39,200 N/~g). Ihe supernatant is removed and the pellet is re~suspended up to a final volume of 5 ml in a solution of 5 mmol NaCl + 0.4 g BSA + 0.1 g sodium merthiolate/l, pH 7Ø
10.5-b Rabbit anti-HCG immunoglobulin The same as 10.5-a, but now with the rabbit immuno-globulin.
10.5-c Monoclonal mouse anti-HCG immunoglobulin As 10.5-a, but now with the mouse immunoglobulin and using the following quantities:
10.5-c-1: 0.1 ml immunoglobulin solution (1 g/l, S rnmol NaCl/l, pH 7.0) 10.5-c-2: 0.1 ml irnrnunoglobulin solution (0.25 g/l, 5 mmol NaCl/l, pH 7.0).
The immuno-activity of the conjugates was ascertain-ed using a DIA (Sandwich test) by the procedure described in ~3~

section 1.4. me following HCG dilution series was used: O, 15.6, 62.5, 250, 1000 and 4000 IU (immunoassay)/l.

t Conjugate 516 . .
HCG (IU/l) O 15.6 62.5 250 1000 4000 _ 10.5-a 0.4830.5090.552 0.618 0.789 1.032 b 0.5560.6080.649 0.710 0.877 1.114 c-1 0.4060.4320.450 0.503 0.536 0.586 c-2 0.4370.4950.497 0.528 0.559 0.653 10.6. Effect of the BSA concentration offered durinq "sub-sequent coatinq" on the immuno-activity of the_coniuqate Conjugates of the dispersion dye Palanil ~ Red BF (BASF) were made in accordance with the procedure described in sections 1.1. and 1.2., with however the following varia-tions in the BSA concentrations:

BSA conc. in thë added BSA conc. in the solution (g/1) reaction mixture (g/1) . _ 10.6-a 9.6 1.6 b 19.2 3.2 c 38.4 6.4 d 76.8 12.8 e 153.6 2506 f 307.2 51.2 The conjugates were isolated as described in sec-tion 1.2. and finally re-suspended in a solution having the following com~osition: 5 mmol NaCl + 0.1 g sodium merthiolate + g BSA/l 9pH adjusted to 7.0 with 0.1 mol/l NaOH); the BSA
concentration is always equal to that during the "subsequent coating", and is thus respectively: x = 1.5, 3.2, ..... , 51.2 g/l (see table).
The immuno-activity of the conjugates was ascertain-ed by a DIA ("Sandwich test") in accordance with the procedure described in section 1.4. During this the following HCG

i5'~

dilution series was used: 0, 3.9, 15.6, 62.5, 250, 1000, 4000, 16000 IU immunoassay/l.

Con]ugate A516 l _ HCG (IU/l) 0 1 3.9 15.6 62.5 250 1000 4000 16000 ..
10.6-a 0.322 0.397 0.378 0.436 0.565 0.773 0.954 1.034 b 0.254 0.282 0.286 0.344 0.489 0.707 0.892 0.960 c 0.137 0.154 0.175 0.235 0.397 0.661 0.812 0.906 d 0.062 0~078 0.095 0.147 0.297 0.594 0.778 0.852 e 0.039 0.040 0.054 0~110 0.283 0.572 0.759 0.843 f 0.021 0.029 0.044 0.111 0.310 0.629 0.803 0.882 Colorimetric and/or visual determination of human cho-rionic gonadotropin (HCG according to the DIA principle (agglutination test; method 1).

11.1. Preparation of the dye sol See 1.1.
11 2 Preparation of the rabbit anti-HCG immunoglobulin/dye sol coniuqate See 1.2., in this case however the BSA solution contains 2.4 g/l, because the pellet is resuspended in a solution having the described composition, but with 0.4 g BSA/l.
The conjugate is finally further diluted up to a value of A 533 = 2-2-11.3. Determination of HCG in phosphate buffer or urine TRIS
TRIS buffer ___________ 1 mol TRIS + 1 mol NaCl -t 10 g BSA/l, adjusted to pH 7.4 with 4 mol/l HCl.
HCG
_ _ _ See 1.4.
Urine _____ See 1.4.

,1, ~3~S4 Phos~hate_buffer (FFB) See 1.4.
Con]ugate See 11.2.
Procedure 1. HCG solution of 5,000 and 1,000 IIJ (immunoassay)/l are made up by diluting the standard HCG solution with FFs or urine.
2. Pipette into a cell (1 cm light path): conjugate (1 ml), TRIS buffer (0.1 ml) and HCG solution (0.11 ml). Mix well and immediately scan the absorption spectrum in the region 750-360 nm, with FFB and urine respectively as reference.
3. Allow the cells to stand at room temperature for 20 hours. Then assess the contents by eye and shake the cells and then once again establish an absorption spectrum in the region 750-360 nm.
Representative spectra are given in Figure 3, while numerical data are provided in the following table:

(IU/l) Visual ) (A533 0 ( 533 x (A575)0-(A575) O _ _ __ _ 1000 + 0.15 0.14 5000 ++ 0.40 0.21 *) "-" : no agglutination/aggregation. Dye sol stable.

"+" : incipient agglutination/aggregation. The super-natant is lighter in colour than with the correspond-ing blank (0 IU HCG/l) due to sedimentation.
"++" : complete agglutination/aggregation. The dye sol has flocculated completely and the dye is precipitated.

**) 0 : 0 IU HCG/l x : 1 and 5 respectively, IU HCG/l.

i4 -Colorimetric de-termination of human chorionic gonado- -tropin (HCG) according to the DIA principle (agglutination test; method 2).

12.1. Preparation of dye sol.
See section 1.1.
12.2. General test procedure for DIA/aqqlutination The dye-immunoglobulin conjugate (2 ml) was pipetted into a glass or polystyrene cuvette and mixed with 0.2 ml of the antigen sample dissolved in FFB (see section 1.4.), or 0.2 ml FFB only, and 0.2 ml of distilled water or of a solution of MgSO4 in distilled water. The cuvettes were maintained at room temperature (without agitation) and the absorbances were determined (without prior agi-tation) after regular time intervals. The antigen causes agglutination of the dye sol particles, which yields a decrease in absorbance due to the increased effective par-ticle size per se and due to the concomitant sedimentation of the aggregates.
All further experiments are concerned with the determina-tion of HCG.
12.3. Preparation of the rabbit (anti HCG) IqG/dye conjuqate Standard procedure: see section 11.2 Optimalization was performed by investigating the follow-ing parameters:
12.3.1. Secondary coating of conjugates with sSA
___________________________________~____ .
Conjugates for DIA/sandwich are secondarily coated with sSA in order to reduce non-specific effects; this extra coating of the conjugate will certainly also improve its stability and will be disadvantageous therefore for conjugates to be used in DIA/aqglutination. Con-5~

jugates coated with varying amounts of BSA, and also without BSA, were compared in the agglutination assay.
The conjugate without BSA yielded the highest sensiti-vity and lowest detection limit in the shortest test period, and was still sufficiently stable to give a constant blank value.
12.3.2. O~timal IgG concentration during coniugate preparation Palanil ~ Red BF/anti-HCG conjugates were prepared using different IgG concentrations during the coating, and were screened in the agglutination assay using a constant HCG concentration of 5 IU/ml sample (or: 0.42 IU/ml total test volume). The optimal IgG concentra-tion during conjugate preparation appeared to be 0.033 mg/ml reaction mixture at a sol concentration corres-ponding with a A153Com = 5.
12.3.3. Effect of incubation of_I~G at ~H 2.0 ~rior to coniu-~ate ~re~aration ____ __ _______ A solution of rabbit anti-HCG IgG (4.5 mg/ml in 5 mmol NaCl/l pH 7.0) was adjusted to pH 2.0 and incubated for 1 h. at 4C, the pH was readjusted to 7.4 and the IgG
solution was used for conjugate preparation. The con-centration of "pH 2 treated IgG" during the conjugate synthesis was varied, and the various conjugates were screened as described in 12.3.2.
A concentration of 0.033 mg "pH 2 treated IgG"/ml reaction mixture, at a sol concentration corresponding with A153com = 5, appeared to be the optimal value.
Comparison of conjugates based on native IgG and "pH 2 treated" IgG clearly demonstrated the advantage of the latter with respect to reactivity in the agglutination test:

-4~-~3~5~

(HCG)Decrease in A (%) IgG in con~ugate(IU/ml) 530 after 4 h. after 18 h.
native IgG 2.513 63 pH-2 treated IgG 2.5 14 74 5 _ 28 100 12.3.4. Effect of additives on the reactivity of coniugates Addition of a destabilizing agent (e.g. MgS04) to the conjugate prior to addition of the sample may yield a decrease in reaction time and/or detection limit of the agglutination assay, particularly in case of con-jugates high stable per se. Anti-HCG/Palanil ~ RED
sF conjugates, based on the dye batches 5228513 (powder) and 4742893 (wet dispersion), were incubated with a dilution series of MgS04 yielding a final con-centration range of 0-20 mmol MgS04/1 total test mix-ture, and A53Com was determined at regular time inter-vals. Concentrations of 1.2 and 9.5 mmol MgS04/1 total test mixture, respectively, werefound to be compatible with a still suitable stability of the conjugates (de-crease of A53Com less than 0.1-0.2 after 2 h).
A dilution series of HCG in FFs (see section 1.4., 0-5 IU/ml sample) was incubated with anti-HCG/Palanil Red BF (4742893) conjugate with an without 9.5 mmol MgS04/1 total test mixture. The conjugate without MgS04 gave only a slight decrease in A1530Cm, as compared to the blank, whereas in the presence of MgSO4 a de-crease in absorbance, significantly differing from the blank, was observed:

~3~5'~L

[MgS04] (HCG) decrease in Al cO
(~mol/ml total test (IU/ml) (after 2 5 h) 53 mixture) 2.5 9.5 2.5 13 0 5.0 4 9.5 5.0 21 12.3.5. Reactivity of_anti-HCG/Palanil ~ Red BF conjugate, based on "~H 2 treated" I~G, and in the ~resence of __________ ______________ ______________ __________ M~S04 The combined effects, described in 12.3.3./12.3.4.
were investigated with Palanil ~ Red BF conjugates (based on the wet and dry commercial preparation), and a HCG dilution series in FFB (0-4 IU/ml sample). The detection limits obtained (defined as the concentration of HCG yielding a response equal to (BL - 2 x SD) are 10summarized below.

- Palanil ~ Red BF 120 IU/l (20 h) 5228513 (powder) 400 IU/l ( 4 h) - Palanil ~ Red BF 280 IU/l (20 h) 4742893 (dispersion) 1300 IU/l ( 4 h) The detection limit of SPIA/agglutination is 100 IU/l (colorimetric detection after 2 h).
12.3.6. Effect of ~article size on coniu~ate reactivity All experiments up to now were performed with only one fraction of the total dye dispersion, viz. the ma--terial remaining in the supernatant at 1000 - 1100 g (9800 - 10,800 N/kg), which corresponds roughly to a particle size ~ 0.2 ~m diameter. me effect of par-ticle size was further investigated by preparing anti-HCG conjugates of different "g-fractions" of Palanll ~
Red BF, and testing them in an HCG agglutination assay.
Significant effects were observed (Figures 4a and 4b), .

.

~L43~5~

yielding optimal results for the dye fraction isolated between 1500 and 2500 g.

i Lyophilization i.f disperse dye/immunoglobulin conjuga-tes, stability.
.` A Palanil ~ Red BF/rabbit anti-HCG conjugate was lyo-philized from an aqueous dispersion ~A530Cm ~5) containing the following constituents:
5 mmol NaCl ~ lO 5 g BSA
i l g sodium merthiolate per litre 2.5 g dextran (Mw 40 000 Dalton) pH 7.0 5.0 g lactose The reconstituted dry conjugate showed no loss in reactivity as compared to the original wet preparation. The conjugate retains its immunoreactivity for at least 2 l/2 months at -20C, 4C and room temperature. Some loss in ac-tivity was found after 2 l/2 months/37C, a considerable loss was found after 2 l/2 months/45C.

~ '' ~ -47-;

Claims (68)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for the qualitative and/or quantitative determination of an immunochemically reactive component selected from the group consisting of a hapten, antigen, and antibody in an aqueous test medium using the immunochemical reactivity of said component comprising:
(1) providing a known amount of at least one labelled immunochemically component obtained by the direct or indirect attachment of an immunochemically reactive component to parti-cles of an aqueous dispersion of (a) a hydrophobic dye or pig-ment, or (b) polymer nuclei coated with said dye or pigment, said particles having a particle size of at least 5 nm;
(2) providing at least one non-labelled immuno-chemically reactive component;
(3) mixing said components (1) and (2) with a sample containing said test medium;
(4) allowing the immunochemical reaction to proceed to form free and bound labelled component(s);
(5) optionally separating the free and bounded labelled component(s) in the test medium or in one of the fractions obtained after separation; and (6) determining the presence and/or the quantity of the dye or pigment, said determination providing a qualitative and/or quantitative indication of the immunochemically reac-tive component(s) to be determined.

2. The process of claim 1, wherein the determination (6) takes place by the detection and determination of the physico-chemical changes of a dye sol.

3. The process of claim 1 or claim 2, wherein two or more immunochemically reactive components are determined simultaneously, wherein for each component to be determined there is present a corresponding immunochemically reactive component that is labelled with a dye sol particle respective for that component, and further wherein respective chromo-phores are used that can clearly be distinguished from each other spectrally and/or visually.

4. The process of claim 1, further comprising obtaining the labelled component by adding to the dye or pigment dispersion a predetermined quantity of the immuno-chemically reactive component to be labelled, wherein the latter at least partially envelopes the dispersed particles and optionally supplementary coating said particles with a polar macromolecule that is immunochemically inert in the corresponding determination.

5. The process of claim 1, wherein the labelled component is obtained by adding to said dispersion of dye or pigment at least one macromolecule that is immuno-chemically inert in the corresponding determination and that coats the dispersion particles and thereafter attaching by adsorption, possibly bio-specific, or via covalent attachment the immunochemically reactive component to the coating material.

6. The process of claim 1, wherein the labelled component is obtained by placing a dye and/or organic pigment sol in a monomer environment and causing the latter to be polymerised in situ, resulting in envelopment of the sol particles, and subsequently adsorbing or covalently attaching the immunochemically reactive component to the polymer material.

7. The process of claim 1, wherein the labelled component is obtained by covalently attaching the immuno-chemically reactive component to colloidal dye particles, either by prior chemical activation of suitable functional groups in the dye and/or the immunochemically reactive component, or by the use of conjugates of organic hydro-phobic chromophores and reactive groups, known as reactive (dispersion) dyes.

8. The process of claim 5, wherein the dye and/or organic pigment sol particles are first protected by a hydrophilic macromolecule after which (co-)polymerisation takes place in the presence of an organic initiator.

9. A test kit for the determination of one or more immunochemically reactive components in an aqueous medium comprising:
(a) a known amount of at least one labelled immunochemically reactive component obtained by the direct or indirect attachment of an unlabelled immunochemically reactive component to particles of an aqueous dispersion of (i) a hydrophobic dye or pigment or (ii) polymer nuclei coated with said dye or pigment, said particles having a particle size of at least 5 nm, (b) other immunochemical reagents, and (c) directions for use of said test kit.

10. A process for the preparation of an immuno-chemical reagent comprising directly or indirectly attaching an immunochemically reactive component to particles of an aqueous dispersion of a hydrophobic dye or pigment, or of polymer nuclei coated with such dye or pigment.

11. An immunochemical reagent consisting of an aqueous dispersion of particles of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with said dye or pigment directly or indirectly attached to an immunochemically react-ive component, said particles having a particle size of at least 5 nm.

12. A freeze-dried or spray dried reagent containing at least one labelled immunochemically reactive component obtained by direct or indirect attachment of an unlabelled immunochemically reactive component to particles of an aqueous dispersion of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with such a dye or pigment, said labelled immuno-chemically reactive component being freeze-dried or spray dried and said particles having a particle size of at least 5 nm.

13. A process for the detection and/or determination of at least one immunochemically reactive component in an aqueous test medium comprising:
providing a known amount of at least one labelled immunochemically reactive component having attached thereto particles of an aqueous dispersion of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm;
providing an aqueous test medium containing at least one immunochemically reactive component to be detected or determined;
contacting said labelled immunochemically reactive component(s) with said immunochemically reactive component(s) to be detected or determined; and measuring the presence or quantity of said hydrophobic dye or pigment to provide a measure of the immunochemically reactive component(s) to be detected or determined.

14. The process of claim 13, wherein the immuno-chemically reactive components are selected from the group consisting of an antigen, an antibody, and a hapten.

15. The process of claim 13, wherein said aqueous dispersion of a hydrophobic dye or pigment is a sol.

16. The process of claim 15, wherein said hydrophobic dye or pigment in said sol has a particle size of at least 5 nm.

17. The process of claim 16, wherein said hydro-phobic dye or pigment has a particle size of about 10 to about 500 nm.

18. An immunochemical reagent comprising an immunochemically reactive component having coupled thereto a member selected from the group consisting of particles of (a) a hydrophobic dye or pigment or (b) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm.

19. The immunochemical reagent of claim 18, wherein said particles of a hydrophobic dye or pigment are provided by an aqueous dispersion of said particles.

20. The immunochemical reagent of claim 19, wherein said aqueous dispersion is a sol.

21. The immunochemical reagent of claim 20, wherein said particles have a size of at least 5 nm.

22. The immunochemical reagent of claim 21, wherein said particles have a size of about 10 to about 500 nm.

23. The immunochemical reagent of claim 18 which is freeze-dried.

24. The immunochemical reagent of claim 18 which is spray-dried.

25. A test kit for the determination of at least one immunochemically reactive component in an aqueous medium, comprising:
(a) a known amount of at least one immunochemical reagent comprising an immunochemically reactive component having coupled thereto a member selected from the group consisting of particles of a hydrophobic dye or pigment and a polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm, (b) other immunochemical reagents; and (c) directions for use of said test kit.

26. A process for the detection and/or determination of at least one component of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous test sample, by applying the known binding affinity of such said protein and said substances for one another, comprising:
(a) mixing a known amount of one or more labelled components, obtained by coupling directly or indirectly the desired-component of said reaction with particles of an aqueous dispersion of a hydrophobic dye or pigment or polymer nuclei coated with a hydrophobic dye or pigment, said parti-cles having a particle size of at least 5 nm, and (b) detecting and/or determining during the reaction or after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of said dye or pigment and/or a formed agglomerate containing said dispersed particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.

27. The process of claim 26, whereby the component or components of the reaction between a specific binding protein and the corresponding bindable substances are immunochemical components selected from the group consisting of haptens, antigens, and antibodies, comprising:
(a) employing a known amount of labelled components, obtained by coupling directly or indirectly the desired immunochemical component to particles of an aqueous dispersion of (i) a hydrophobic dye or pigment or (ii) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm, and (b) detecting or determining after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of said dye or pigment and/or a formed agglomerate containing said dye or pigment particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the immunochemical component or components to be detected and/or determined.

28. A test kit, to be used for the determination of one or more components of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous medium according to a predetermined protocol, comprising:

(a) a known amount of a hydrophobic dye or pigment-labelled component that has been obtained by coupling a component of said reaction to particles of an aqueous dispersion of a hydrophobic dye or pigment or polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm;
(b) other immunochemical reagents, and (c) directions for the performance of said protocol.

29. A freeze-dried reagent for use in an immuno-assay, containing a hydrophobic dye or pigment-labelled component, that has been obtained by coupling directly or indirectly the desired component to particles of an aqueous dispersion of a hydrophobic dye or pigment or a polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm.

30. A method for the determination of a first component of an immunochemical reaction in an aqueous medium selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces an immunochemical component which is at least immuno-chemically equivalent to said first component to be determined, and (2) a known amount of an insolubilized second component capable of reacting with either said first component to be determined or said reagent:

(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component, and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

31. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces (i) an immunochemical component which is at least immunochemically equivalent to said first component to be determined and (ii) an immunochemically inert macro-molecule, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent, (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

32. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first com-ponent to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment disper-sion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it an immunochemical component which is at least immuno-chemically equivalent to said first component, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent' (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

33. A method for the determination of a first com-ponent of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first com-ponent to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment disper-sion particles having a particle size of at least 5 nm, which particles have attached to their surfaces a second immuno-chemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized second immunochemical component which is at least immunochemically equivalent to said first component and capable of reacting with said reagent, (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

34. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding prot-ein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first com-ponent to be determined with (1) a known amount of a reagent, said reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces (i) a second immunochemical component capable of reacting with said first component to be determined and (ii) an immunochemically inert macromolecule, and (2) a known amount of an insolubilized second immunochemical component which is at least immunochemically equivalent to said first component to be determined and capable of reacting with said reagent;
(b) allowing sufficient time for the reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (Z) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

35. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized third immunochemical component and capable of reacting with said reagent;

(b) allowing sufficient time for the reaction to take place, whereby a fraction of said first component to be determined bound to said insolubilized component and a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the dye or pigment of the dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.

36. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) binding a known amount of a second immuno-chemical component of said reaction capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier, (b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a parti-cle size of at least 5 nm, which particles have attached to their surface an immunochemical component capable of reacting with said first component to be determined;
(e) allowing sufficient time for reaction to take place, to bind a fraction of said reagent to that part of said bound second immunochemical component which has undergone the reaction step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.

37. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) binding a known amount of a second immuno-chemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of step (c) with an immunochemical excess of a reagent consisting essen-tially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having attached to their surfaces (i) an immunochemical component capable of re-acting with said first component to be determined and (ii) an immunochemically inert macromolecule;
(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the dye or pigment dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said component to be determined in said sample.

38. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) binding a known amount of a second immuno-chemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product to step (c) with an immunochemical excess of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm and having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it an immunochemical com-ponent capable of reacting with said first component to be determined;
(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the dye or pigment dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.

39. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substances, comprising:
(a) contacting a sample containing the first com-ponent to be determined with a known amount of a reagent, said reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having attached to their surfaces a second immunochemical component of said reaction capable of reacting with said first component to be determined;
(b) allowing sufficient time for reaction to take place; and (c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.

40. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first com-ponent to be determined with a known amount of a reagent consisting essentially of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having attached to their surfaces (i) a second immunochemical component of said reaction capable of reacting with said first component to be deter-mined and (ii) an immunochemically inert macromolecule;
(b) allowing sufficient time for a reaction to take place; and (c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.

41. A method for the determination of a first component of an immunochemical reaction in an aqueous test medium selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprlsing:
(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting of hydrophobic dye or pigment dispersion particles having a particle size of at least 5 nm, said particles having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined;
(b) allowing sufficient time for a reaction to take place; and (c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.

42. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of rabbit and anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG
in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Palanil Red BF disper-sion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immuno-logical reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Palanil Red BF in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

43. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of rabbit anti-HCG
immunogiobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG
in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Resolin Brilliant Blue RRL dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Resolin Brilliant Blue RRL in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

44. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, form.ing a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Samaron Brilliant Red H6GF dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immuno-logical reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Samaron Brilliant Red H6GF in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

45. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;
(d) separating the first and second solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Samaron Brilliant Yellow H10GF dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immuno-globulin attached to their surfaces;

(f) allowing sufficient time for a second immuno-logical reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Samaron Brilliant Yellow H10GF in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

46. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;

(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Palanil Luminous Red G
dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immuno-logical reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Palanil Luminous Red G
in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

47. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HCG to be determined;
(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with an immunochemical excess of a reagent, said reagent consisting essentially of Palanil Luminous Yellow G dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immuno-globulin attached to their surfaces;
(f) allowing sufficient time for a second immuno-logical reaction to take place, to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Palanil Luminous Yellow G in the second liquid phase or the second solid phase, which is a measure of the amount of HCG in said sample solution.

48. A method for the immunochemical determination of hepatitis Surface B antigen (HBsAg), comprising:
(a) binding a known amount of sheep anti-HBsAg immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HBsAg to be determined;
(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and the HBsAg in the sample solution in step (b) to take place, to bind the HBsAg to be determined to the insolubilized sheep anti-HBsAg, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HBsAg therein with an immunochemical excess of particles of Palanil Red BF or Samaron Brilliant Red H6GF particles hav-ing anti-HBsAg immunoglobulin attached to their surfaces, said particles having a particle size of at least 5 nm;
(f) allowing sufficient time for a second immuno-chemical reaction to take place, to bind said reagent to that part of said HBsAg solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Palanil Red BF or Samaron Brilliant Red H6GF in the second liquid phase or the second solid phase, which is a measure of the amount of HBsAg in said sample solution.

49. A method for the immunochemical determination of human placental lactogen (HPL) in a liquid sample, comprising:
(a) binding a known amount of rabbit anti-HPL
immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound rabbit anti-HPL with a sample solution containing the HPL to be determined;
(c) allowing sufficient time for an immunological reaction between the bound rabbit anti-HPL immunoglobulin and the HPL in the sample solution in step (b) to take place to bind the HPL to be determined to the insolubilized rabbit anti-HPL, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HPL therein with a known amount of particles of a Palanil Red BF or Palanil Yellow 36 to form a particle-HPL con-jugate, said particles having a particle size of at least 5 nm;
(f) allowing sufficient time for a second immuno-chemical reaction to take place to bind said reagent to that part of said solid phase bound rabbit anti-HPL immuno-globulin which has not undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Palanil Red BF or Palanil Yellow 36 in the second solid phase or the second liquid phase, which is a measure of the amount of testosterone in said liquid sample.

50. A method for the immunochemical determination of human anti-Rubella sera, comprising:
(a) binding a known amount of Rubella viral antigen to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound Rubella viral antigen with a human serum sample containing the human anti-Rubella sera to be determined;
(c) allowing sufficient time for an immunological reaction between the bound Rubella viral antigen and the serum containing the human anti-Rubella sera to be determined to take place to bind the anti-Rubella sera to the insolubilized Rubella viral antigen, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound anti-Rubella titer therein with a known amount of a reagent, said reagent consisting essentially of dispersion particles of Palanil Red BF or Resolin Brilliant Blue RRL, said particles having a particle size of at least 5 nm and having sheep anti-human immunoglobulin attached to their surfaces to form a particle-sheep anti-human immunogiobulin conjugate;
(f) allowing sufficient time for a second immunological reaction to take place to bind said reagent to the bound anti-Rubella titer solid phase, resulting in the binding of said reagent to that part of said bound antigen which has undergone the reaction in step (c), to form a second solid and a second liquid phase;
(g) separating the second liquid and solid phases;
and (h) determining the amount of Palanil Red BF or Resolin Brilliant Blue RRL in the second solid phase or the second liquid phase, which is a measure of the human anti-Rubella serum to be determined in said sample.

51. A method for the immunochemical determination of human prolactin (PRL), comprising:
(a) binding a known amount of monoclonal (anti-PRL) immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound monoclonal (anti-PRL) immunoglobulin with a sample solution containing the PRL

to be determined;

(c) allowing sufficient time for an immunological reaction between the monoclonal (anti-PRL) immunoglobulin and the PRL in the sample solution in step (b) to take place, to bind the PRL to be determined to the insolubilized mono-clonal (anti-PRL) immunoglobulin, forming a first solid phase and leaving a first liquid phase;
(d) separating the first solid phase from the first liquid phase, (e) contacting said first solid phase and bound PRL with a known amount of a reagent consisting essentially of dispersion particles of Palanil Luminous Red G or Palanil Luminous Yellow G, said particles having a particle size of at least 5 nm and having monoclonal (anti-PRL) immunoglobulin attached to their surfaces, (f) allowing sufficient time for a second immuno-logical reaction to occur to bind said reagent to that part of said PRL solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid and second liquid phases, and (h) determining the amount of Palanil Luminous Red G or Palanil Luminous Yellow G in the second solid phase or the second liquid phase, which is a measure of the amount of the PRL to be determined in said sample solution.

52. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) contacting an aqueous sample containing the HCG to be determined with a known amount of a reagent consist-ing essentially of dispersion particles of a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm and having rabbit anti-HCG immunoglobulin attached to their surfaces;

(b) allowing sufficient time for an immunological reaction between said reagent and any HCG in the sample solution to take place; and (c) determining the color of the sample solution, which is a measure of the amount of HCG to be determined in said sample solution.

53. The method of claim 52, wherein said hydrophobic dye or pigment is Palanil Red FF.

54. A method for the simultaneous determination of human chorionic gonadotropin (HCG) and human placental lactogen (HPL), comprising:
(a) binding a known amount of rabbit anti HCG
immunoglobulin and a known amount of rabbit anti-HPL immuno-globulin to the surface of a water insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulins with a sample containing the HCG and the HPL to be determined, (c) allowing sufficient time for an immunological reaction between the bound immunoglobulins and said HCG
and said HPL in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG and to bind the HPL to be determined to the insolubilized rabbit anti-HPL, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound HCG therein with a known amount of a reagent, said reagent consisting essentially of Resolin Brilliant Blue RRL

dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immunoglobulin attached to their surfaces;

(f) contacting said first reacted solid phase and bound HPL therein with a known amount of a reagent, said reagent consisting essentially of Palanil Yellow 36 dispersion particles, said particles having rabbit anti-HPL
immunoglobulin attached to their surfaces;
(g) allowing sufficient time for a second immuno-logical reaction to take place, to bind the reagent containing Resolin Brilliant Blue RRL dispersion particles to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c) and to bind the reagent containing Palanil Yellow 36 dispersion particles to that part of said HPL solid phase bound immunoglobulin which has undergone the reaction in step (c);
(h) separating the second solid phase from the second liquid phase; and (i) determining the presence of Resolin Brilliant Blue RRL and Palanil Yellow 36 in the second liquid phase or the second solid phase, which is an indicator or HCG
and HPL respectively in the sample solution.

55. A method for the immunological determination of testosterone in a liquid sample, comprising:
(a) binding a known amount of rabbit anti-testosterone immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound rabbit anti-testosterone with a sample solution containing the testosterone to be determined;
(c) allowing sufficient time for an immunological reaction between the bound rabbit anti-testosterone immuno-globulin and the testosterone in the sample solution in step (b) to take place to bind the testosterone to be determined to the insolubilized rabbit anti-testosterone, forming a first solid phase and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound testosterone therein with a known amount of particles of Palanil Red BF, said particles having a particle size of at least 5 nm and having testosterone 11.alpha.-hemisuccinyl bovine serum albumin attached to their surfaces to form a particle-testosterone-11.alpha.-hemisuccinyl serum albumin conjugate;
(f) allowing sufficient time for a second immunological reaction to take place to bind said reagent to that part of said solid phase rabbit anti-testosterone immunoglobulin which has not undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of Palanil Red BF in the second solid phase, which is a measure of the amount of testosterone in said liquid sample.

56. A method for the immunological determination of testosterone in a liquid sample, comprising:
(a) contacting an aqueous sample containing the testosterone to be determined with (1) a known amount of a reagent consisting essentially of Samaron Brilliant Red H6GF
or Samaron Brilliant Yellow HlOGF particles, the particles having a particle size of at least 5 nm and having attached thereto rabbit anti-T11-bovine serum albumin immunoglobulin and (2) a known amount of T3-bovine serum albumin;

(b) allowing sufficient time for a reaction to take place, whereby a first fraction of testosterone bound to said T3-bovine serum albumin and a fraction of testosterone free of T3-bovine serum albumin are produced; and (c) determining the amount of Samaron Brilliant Red H6GF or Samaron Brilliant Yellow HlOGF particles in one of said fractions, which is a measure of the amount of testosterone in said sample.

57. A method for the determination of human chorionic gonadotropin (HCG) 9 comprising:
(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound receptor protein with a sample solution containing the HCG to be determined;
(c) allowing sufficient time for an immunological reaction between the insolubilized HCG receptor protein and the HCG to be determined;
(d) adding a known amount of reagent to said sample solution, said reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment and having HCG
attached to their surfaces, said particles having a particle size of at least 5 nm;
(e) allowing sufficient time for a reaction to take place between (1) the dispersion reagent and (2) the in-solubilized HCG receptor protein not bound to HCG, to form a solid phase containing a fraction of dispersion reagent bound to insolubilized HCG receptor protein, and a liquid phase of unbound and free dispersion reagent;
(f) separating the liquid and solid phases; and (g) determining the amount of dye or pigment in either the solid or liquid phase, which is a measure of the amount of HCG in said sample solution.

58. A method for the immunochemical determination of human chorionic gonadotropin (HCG), comprising:
(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound receptor protein with a sample solution containing the unknown HCG to be determined;
(c) allowing sufficient time for an immunochemical reaction between the bound receptor protein and the HCG in the sample solution to take place, to bind the HCG to be determined to the insolubilized HCG receptor protein, forming a first solid phase and leaving a first liquid phase;
(d) separating the first solid phase from the first liquid phase;
(e) contacting said first solid phase and in-solubilized HCG therein with a known amount of a reagent consisting essentially of dispersion particles of a hydro-phobic dye or pigment having a particle size of at least 5 nm and having rabbit anti HCG immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immuno-logical reaction to occur to bind said reagent to that part of said insolubilized HCG which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of dye or pigment in the second solid phase or the second liquid phase, which is a measure of the HCG to be determined in said sample solution.

59. A test kit, to be used for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and the corresponding bindable substance, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surfaces either a component immunochemically similar to said first component to be determined, or a binding partner of the first component; and (b) directions for the performance of said protocol.

60. A test kit, to be used for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and the corresponding bindable substance, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule; and (b) directions for the performance of said protocol.

61. A test kit, to be used for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and the corresponding bindable sub-stance, according to a predetermined protocol, comprising:

(a) a reagent consisting essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surfaces a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component selected from the group consisting of a component immunochemically equivalent to said first component, or a binding partner to said first component; and (b) directions for the performance of said protocol.

62. A freeze-dried reagent for use in an immuno-assay, consisting essentially of dispersion particles of a hydrophobic dye or pigment having attached to their surfaces an immunochemical component, said particles having a particle size of at least 5 nm.

63. The reagent in claim 62, wherein said dye is Palanil Red BF and said immunochemical component is rabbit anti-HCG immunoglobulin.

64. A freeze-dried reagent for use in an immuno-assay to determine a first immunochemical component, consist-ing essentially of dispersion particles of a hydrophobic dye or pigment having a particle size of at least 5 nm and having attached to their surface (i) a component immunochemically equivalent to the first component to be determined and (ii) an immunochemically inert macromolecule.

65. A freeze-dried reagent for use in an immuno-assay to determine a first immunochemical component, consist-ing essentially of dispersion particles of a hydrophobic dye or pigment having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component which is immunochemically similar to said first component, and said particles having a particle size of at least 5 nm.

66. A test kit, to be used for the detection and/or determination of at least one component of the reaction between a specific binding protein and a corresponding bindable substance thereto in an aqueous medium according to a predetermined protocol, comprising:
(a) a known amount of a dye- or pigment-labelled component obtained by coupling a component of said reaction to (1) particles of a hydrophobic dye or pigment or (2) polymer nuclei coated with a hydrophobic dye or pigment, said particles having a particle size of at least 5 nm, (b) a known amount of at least one additional immunochemical reagent, at least one of said reagents being a ligand or an immobilized ligand, which ligand is selected from the group consisting of:
(aa) a ligand capable of binding with the dye- or pigment-labelled component (a);
(bb) a ligand capable of binding with a binding partner of the dye- or pigment-labelled component (a), (cc) a ligand capable of binding with at least one of the component(s) to be determined, and (dd) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and (c) directions for the performance of a protocol for the detection and/or determination of at least one component of an immunochemical reaction in an aqueous medium between a specific binding protein and a corresponding bindable substance thereto.

67. A reagent for use in an immunoassay to determine a first immunochemical component, consisting essentially of dispersion particles of a hydrophobic dye or a pigment having attached to their surface (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule, said particles having a particle size of at least 5 nm.

68. The reagent of claim 67, wherein said dye is Palanil Red BF, the component (1) is rabbit (anti-HCG) immunoglobulin, and said immunochemically inert macromolecule is bovine serum albumin.