WO1981001883A1

WO1981001883A1 - Chemical luminescence amplification substrate system for immunochemistry

Info

Publication number: WO1981001883A1
Application number: PCT/US1980/001485
Authority: WO
Inventors: Y Wong; R Mandle
Original assignee: Electro Nucleonics
Priority date: 1979-12-19
Filing date: 1980-11-06
Publication date: 1981-07-09
Also published as: ES8203152A1; ES497867A0; IT1151457B; BE886751A; IT8026043A0; CA1166133A; ZA807274B

Abstract

A system for the detection of a biological analyte of interest which comprises contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an energy source which is capable of activating the fluorescer. A method for the qualitative and/or quantitative detection of a biological of interest is disclosed, which comprises: (1) labeling an immunological specie specific to the analyte of interest with a fluorescer material which is biologically compatible with such specie; (b) contacting the fluorescer labeled specie and the biological of interest; (c) separating the fluorescer labeled specie/biological complex; (d) contacting the separated fluorescer labeled specie/biological complex of (c) with an energy source which is capable of activating the fluorescer label; and (e) determining the presence of and/or measuring the quantum of chemiluminescent light emitted.

Description

CHEMICAL LUMINESCENCE AMPLIFICATION SUBSTRATE SYSTEM FOR IMMUNOCHEMISTRY

This invention relates to a system for the detection of a biological analyte of interest which comprises contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an energy source which is capable of activating the fluorescer.

Background Of The Invention

The clinician is concerned with detecting the presence of, and quantitatively measuring, a variety of substances via the use of many different analytical techniques. The most commonly used techniques employ absorbtiometry, both at visible and ultraviolet wavelengths, however, emission, flame photometry and radioactivity are also commonly used. A novel technique, thus far relatively unexplored in chemistry, is that employing the phenomenon of luminescence.

Analyses based on the measurement of emitted light offer several distinct advantages over conventionally employed techniques, including high sensitivity, wide linear range, low cost per test, and relatively simply and inexpensive equipment.

It has been predicted that the phenomenon of luminescence, and more particularly chemiluminescence, could have a major impact in two main aras of clinial analysis. First, it may have an important role as a replacement for conventional colorimetric or spectrophotometric indicator reactions in assays for substrates of oxidases and dehydrogenases. In this type of assay the sensitivity of the luminescence indicator reaction may be used to quantitate substrates not easily measured by conventional techniques (e.g., prostaglandins and vitamins).

The second major clinical application of luminescense might be in the utilization of luminescent molecules as replacements for radioactive or enzyme labels in immunoassay. In each of these major clinical application areas, chemiluminescent reactions can provide a means to achieve a high level of analytical sensitivity.

Chemiluminescence may be simply defined as the chemical production of light. In the literature it is often confused with fluorescence. The difference between these two phenomena lies in the source of the energy which promotes molecules to an excited state. In chemiluminescence this source is the energy yielded as the result of a chemical reaction. The subsequent decay of molecules from the excited state back to the ground state is accompanied by emission of light, which is called luminescence. In contrast, in fluorescence, incident radiation is the source of the energy which promotes molecules to an excited state.

From an analytical point of view, the types of luminescence that have engendered the most interest are chemiluminescence and bioluminescence. The latter being the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the luminescent reaction. Bioluminescent reactions such as the enzymatic firefly process, have been very useful analytically and convert chemical energy to light with a quantum efficiency of 88%.

In contrast to bioluminescence with, the longevity and efficiency of the firefly, the history of chemiluminescence (hereinafter referred to as CL) , especially that occurring in the non-aqueous phase, is remarkably short. The important aqueous CL substances luminol and lucigenin were discovered in 1928 and 1935, respectively. A series of organic soluble CL materials were developed in the early 1960's based upon a study of the luminescent reactions of a number of oxalate compounds. A typical organic system useful for CL was disclosed by Bollyka et al., United States Patent No. 3,597,362, and claimed to exhibit a quantum efficiency of about 23% compared with about 3% for the best known available aqueous systems.

Chemiluminescence has become increasingly attractive for its potential in the clinical laboratory, especially for use in the analysis of a number of biologically associated materials, and its known applications have been the subject of thorough, reviews, see for example: Whitehead et al. (1979) Analytical Luminescence: Its Potential In The Clinical Laboratory, Clin. Chem., 25, 9 1531-1546; Gorus et al. (1979) Applications Of Bio- And Chemiluminescence In The Clinical Laboratory, Clin. Chem., 25, 4 512-519; Isacsson et al. (1974) Chemiluminescence In Analytical Chemistry, Analytical Chemica Acta, 68, 339-362.

With few exceptions, most published CL clinical analytical applications have made use of the less efficient but well known diacylhydrazides, acridinium salts, pyrogallol, or lophine structures. It is important to appreciate that due to the nature of the chemical decomposition of the above chemiluminescent structures in the presence of hydrogen-peroxide, or generators of H₂O₂, as compared to that of the oxidation reaction of diaryloxalate structures, the latter has over 20 times the quantum yield of chemiluminescence, although its requirement for hydrogen peroxide is greater than the former. Hydrogen peroxide, an essential component in the chemiluminescent reaction, has usually been the species selected for use in detecting the analyte of interest. For example, in the determination of glucose - Auses et al. (1975), Chemiluminescent Enzyme Method For Glucose. Analytical Chemistry, 47, No. 2, 244-248 employed the oxidation of glucose in the presence of glucose oxidase as the source of H₂O₂ which, in turn, was reacted with luminol to produce chemiluminescence in proportion to the initial glucose concentration. A limit of detection of 8 x 10-⁹M peroxide was obtained with this system. Williams et al. (1976), Evaluation Of Peroxyoxalate Chemiluminescence For Determination Of Enzyme Generated Peroxide. Anal. Chem., 48, 7 1003-1006 in a similar reaction concluded the limit of sensitivity of the peroxyoxalate system is an order of magnitude poorer than that of the luminol system.

Therefore, until now the oxalic ester system (pxalate system) was generally thought to have little utility for analytical purposes due to its inefficient conversion of hydrogen peroxide.

The present invention overcomes this deficiency of H₂O₂ dependence by. making use of the large chemiluminescent reservoir of energy in the oxalate system's chemistry. By using a suitable quantity of hydrogen peroxide and oxalate, a vast amount of energy may be concentrated in a form which is then released as chemiluminescence upon the introduction of a conjugated fluorescer.

Thus, the oxalate, acting in a fashion which can be visualized as analogous to a charged chemical battery, releases the stored energy to the fluorescer-conjugate in the same manner as an electrical switch in a circuit releases the energy of a battery to a lamp. This "switch" action causes chemiluminescence and, by conjugating the fluorescer to a detector of the analyte of interest, one can employ the reaction to trigger a detection system both qualitatively and quantitatively related to the analyte to be measured.

It is, therefore, an object of the present invention to provide for a system for the detection of a biological analyte of interest comprising contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an energy source which is capable of activating the fluorescer.

A further object of the present invention is to provide for a qualitative method for the detection of a biological analyte of interest comprising: (a) labeling an immunological specie specific to the analyte of interest with a fluorescer material which is biologically compatible with such specie;

(b) contacting the fluorescer labeled specie and the biological of interest; (c) separating the fluorescer labeled specie/ biological complex;

(d) contacting the separated fluorescer labeled specie/biological complex of (c) with an energy source which is capable of activating the fluorescer label; and (d) determining the presence or absence of chemiluminescent light emitted from the activated fluorescer. A further object of the present invention is to provide for a quantitative method for measuring the amount of a biological analyte of interest comprising

(a) labeling an immunological specie specific to the analyte of interest with a fluorescer material which is biologically compatible with such specie;

(b) contacting the fluorescer labeled specie and the biological of interest;

(c) separating the fluorescer labeled specie/ hiological complex;

(d) contacting the separated fluorescer labeled specie/biological complex of (c) with an energy source which, is capable of activating the fluorescer label; and

(e) determining the amount of chemiluminescent light emitted from the activated fluorescer using appropriate instrumentation.

A further object of the present invention is to provide for a novel class of fluorescer materials which, may he conjugated to an immunological specie specific to a biological of interest in order to provide for the detection of such biological.

A furtber object of the present invention is to provide for a novel class of conjugated fluorescer/ biological compositions useful in the detection of various hiologicals of interest. Summary Of Invention

According to the present invention, there is provided a system for the detection of a biological analyte of interest comprising contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an energy source which is capable of activating the fluorescer.

There is also provided a method for the qualitative and/or quantitative method for the detection of a biological of interest comprising:

(b) contacting the fluorescer labeled specie and the biological of interest;

(c) separating the fluorescer labeled specie/ biological complex;

(d) contacting the separated fluorescer labeled specie/biological complex of (c) with an energy source which is capable of activating the fluorescer label; and

(e) detecting the presence of and/or measuring the quantum of chemiiuminescent ligh.t emitted.

Additionally, there is provided for novel fluorescer and conjugated fluorescer/immunological specie compositions useful in the detection of various biological analytes of interest.

Witb respect to Charts I, II, and III, Rauhut et al. (1969), Chemiluminescence From Concerted Peroxide Decomposition Reactions, Accounts of Chemical Research, Vol. 2 , 80-87, it can be seen that one mole of H₂O₂ is necessary to convert one mole of luminol into one mole of the energized or excited molecule. This exicted molecule then reverts to its ground state and emits light. Of interest is the fact that the CL compound, in Chart I, luminol or its derivatives, is also capable of converting the chemical energy of the system to light. Thus, the luminol acts as a source of CL energy and also as a fluorescer to absorb the energy and produce visible light. The luminol system is, therefore, not particularly useful in the context of the present invention since no differentiation between the light emitted upon fluorescer addition and that generated by the luminol itself can be made.

Charts II and III illustrate the fact that for the oxalate system, hydrogen peroxide does not always produce a species which gives rise to an excited state producing light. Some peroxide may be lost in side reactions which are "dark", thus, there is no predictable stoichiometric relationship between the H

₂O₂ consumption and the quanta of emitted light.

A major difference between the luminol system, which has been used to detect the presence or the quantity of H₂O₂, and the oxalate system is the requirement that the oxalates have an additional fluorescer to absorb the chemical energy generated in the reaction and then convert that energy to visible light. If the specified fluorescer is absent, the energy generated by the reaction will be dissipated without emitting visible light. The oxalate system is generally employed in an organic solvent and this requirement also has made its use in CL analytical methods less desirable than other CL materials, which are soluble in an aqueous medium, due to the incompatibility of biological anti-analytes to such organic solvents.

The present invention dramatically differs from the prior art utilizing CL for analytical purposes in the way the generated CL energy is employed. The present invention makes use of the CL system as a substrate or reservoir of chemical energy which emits light upon the addition of another compound, i.e. the fluorescer. We have found that by conjugating this fluorescer compound to the anti-analyte of interest it is possible to quantify the analyte's concentration in terms of the amount of emitted light. CL as thus applied becomes competitive as a highly sensitive, replacement for radioimmunoassay techniques (RIA).

The comparison of Table 1 shows various analytical systems employing CL and illustrates the manner in which components of different reactions may be used to achieve detection. An analyte may be determined using CL by coupling the detector for the analyte to either: I. A catalyst for generation of the H₂O₂ CL reaction, such as glucose-oxidase, or II. A CL compound which generates CL energy and itself emits light, such as luminol, or III. A fluorescer which absorbs chemical energy and emits light, such as a perylene derivative.

In each case, for the purpose of simplicity in this comparison, the analyte is assumed to be surface antigen to Hepatitis B (HB_SA_g) in human serum and is determined by a solid phase "sandwich" technique. This system is presently widely used with I ¹²⁵, a radioactive isotope, as the label or indicator.

I I-* U1

Schroeder et al. (1979) Immunoassay For Serum Throxine

Monitored By Chemiluminescence.

Journal Of Immunological Methods, 25, 275-282.

Olsson et al. (1979) Luminescence Immunoassay (LIA) A Solid Phase Immunoassay Monitored By Chemiluminescence. Journal of Immunological Methods, 25, 127-135.

In order to detect the antigen-antibody reaction the indicator in all cases illustrated in the comparison of Table 1 is taken to be the emission of light from CL. In the "sandwich technique" the following steps are taken: anti-HB_S (Goat) is coated to controlled pore glass (CPG) particles in tablet form (solid phase). Patient serum is added to a tube containing a CPG tablet. During incubation the tablet disintegrates. If Hepatitis B Surface Antigen is present in the serum tested, it will combine with the antibody on the glass particles. After incubation, the serum is removed and the glass beads rinsed. A label, as discussed below, conjugated to an anti-body specific to HB_SAg is then added. The labeled antibody combines with the antigen bound to the antibody on the glass particles forming the "sandwich". The labeled antibody then reacts in a specified manner in the CL system to give light as an indication of antigen presence. This CL assay is a qualitative test for the presence of Hepatitis B Surface Antigen in serum. In general, however, the greater the amount of HB_SAg in a sample, the greater the intensity of emitted light.

The reaction sequence and procedures used in carrying out the Methods illustrated in Table 1 were as follows:

Method I - Enzyme Chemiluminescent Immunoassay Label: Antibody to Hepatitis B Surface Antigen conjugated with glucose-oxidase (GLO).

Reaction: (1) Glass. ab. ag + ab. GLO + glucose → H₂O₂ (2) Luminol + NaOH + H₂O₂ (from reaction 1) → light Procedure: After incubation of the oxidase label to form the "sandwich" as described above, the complex is washed to remove excess label. The washed complex is then incubated for a fixed time with a standard glucose solution to allow the glucose substrate to form H₂O₂, the quantity of which is proportional to the original GLO present in the sandwich. An aliquot of the solution is then added to a standard catalyzed alkaline luminol solution with the light emission proportional to the HB_SAg in the original sample.

Method II - Chemiluminescent-labeled Immunoassay Label: Antibody to Hepatitis B Surface Antigen labeled with, luminol.

Reaction: (1) Glass .ab .ag. ab .luminol + H₂O₂ + hemin → light

Procedure: After incubation of the luminol label to form the "sandwich" as described above, the complex is washed to remove excess label. To the washed complex is added a standard hydrogen peroxide alkaline hemin reagent. The light emission is proportional to the HBgAg in the original sample. It is noteworthy that Hersh. et al. (1979) Luminol-Assisted, Competitive-Binding Immuno-Assay Of Human Immuno-Globulin, G. Anal. Biochem., 93, 267-271, end their paper describing a similar use of luminol with the following summary:

"The luminol-based chemiluminescent label can be employed as a substitute for radiolabels in immuno assay for serum components at concentrations greater than 10 -⁹ mol/liter. The main factor limiting the sensitivity of the method is the relatively low overal chemiluminescent efficiency (CE) of the luminol tag. The CE of underivatized luminol is reported to be 1.5% (5). Our luminol-IgG label had a final efficiency of about 0.3%. It is possible that a more efficient means of coupling luminol, if found, would increase sensitivity by a maximum of 600%. The most efficient chemiluminescent system reported to date (not involving enzymes) is the hydrogen peroxide-oxalate ester reaction (6). This reaction has an overall chemiluminescence efficiency of 23%. The use of the oxalate ester as a chemiluminescent label would provide the more substantial gain of 1500% compared to the luminol system."

Thus, while earlier investigators recognized the quantum efficiency of the oxalate system for CL, they, like others, never appreciated the most efficient way to use this oxalate as a source of energy, would be by controlling the "switch" and not the "source" of the energy.

Method III - Chemiluminescent Labeled Light Amplification

System

(The method of the present invention. - "CLASSIC")

Label: Antibody to Hepatitis B Surface Antigen conjugated to a perylene derivative fluorescer.

Reaction: (1) Glass. ab. ag. ab. perylene + TCPO + H₂O₂ → light.

Procedure: After incubation of the perylene label to form the "sandwich." as described above, the complex is washed to remove excess label. The "sandwich." is then washed with tertiary butanol to remove excess buffer salts. Then an excess of bistrichlorophenyl oxalate and hydrogen peroxide in dimethylphthalate are added to cause the fluorescer conjugate to emit light. The light emission is proportional to the HB_SAg in the original sample. The light intensity may be measured qualitatively by eye, or quantitatively by using a photodiode in the same manner that a photomultiplier in proximity to a sodium iodide crystal responds to the photons released by the gamma rays from the I ¹²⁵ label.

Discussion Of Methods I, II and III

The use of an oxidizer conjugated to an antibody (Method I) is in reality an adaptation of the wellknown enzyme-immunoassay systems of Syva Corporation (United States Patent No. 3,817,837) and Organon Company (United States Patent No. 3,654,090) but here using CL as a light indicator instead of a dye color change. We are not aware of an analogous system incorporating all the solid phase sequences suggested herein. Nonetheless, the detection limit of this method is governed by the ability of the oxidase enzyme conjugate to liberate sufficient H₂O₂ as in the above enzyme immunoassays. Some increase in detection level may be achieved by using CL because of the better sensitivity of CL vs. dye color change, this sensitivity does not however approach the detection level of the fluorescer conjugate of Method III.

In Method II a number of analysts have suggested labeling the analyte detector with a CL compound or derivative. This method is inferior to Methods I or III in that the amount of light emitted can never be more than the total energy content of the amount of CL compound conjugated - i.e., luminol or oxalate. A further disadvantage in coupling the CL compound directly to the antibody, for example, is the loss in CL capacity of the conjugate and the continued loss of light as the compound is consumed in the reaction. Finally, the entire loss of the consumed CL compounds before test completion prevents the analyst from repeating or rechecking the sample's CL.

Method III, alternatively referred to as "CLASSIC", the method of the present invention, overcomes the inherent disadvantages of Methods I and II. With

"CLASSIC" it is possible to achieve the highest order of activity and specificity of the analyte detector because one can carefully select the preferred attachment site on the biological to be labeled. It is also possible to design the linkage of an efficient and durable fluorescer to conjugate with the biological effectively at this site without damaging the biological. Damage in specificity and activity of biologicals from I ¹²⁵ labeling, and damage to enzymes by conjugation is well known and an accepted fact in the preparation of immunodiagnostic reagents. A fluorescent label of preferred utility in CL, by its very structure, must be stable to the oxidizing conditions of the oxalate reaction. This inertness augers well in making fluorescers a particularly efficient form of label for immunochemical analyses.

The various levels of sensitivity and variations in different types of amplification is evaluated in a 1976 review by G. Wisdom, Enzyme-immunoassay, Clin. Chem., 22, 1234-1255. These systems provide the amplification for enzyme labels since enzyme catalytic properties allow them to act as amplifiers, and many enzyme molecules can catalyze the formation of more than 10⁵ product molecules per minute.

To be suitable as a label, an enzyme must meet the several criteria set forth by Wisdom (1976) (supra) which are as follows:

(1) Available cheaply in high purity.

(2) High specific activity.

(3) Stable under assay and storage conditions. (4) Soluble. (5) Assay method that is simple, sensitive, rapid, and cheap.

(6) Absent from biological fluids.

(7) Substrate, inhibitors, and disturbing factors, absent from biological fluids.

(8) Capable of retaining activity while undergoing appropriate linkage reactions.

(9) Capable of inhibition or reactivation when antibody binds to the enzyme-hapten conjugate. (10) Assay conditions compatible with hapten antibody binding.

These specifications are easily met by fluorescent organic compounds which may be readily coupled as labels capable of absorbing the chemical energy from the oxalate "substrate". In addition, as has been shown by Rauhut, certain selected fluorescer structures are capable of catalyzing the peroxyoxalate reaction products, thus providing the type of amplification available with enzymes. Whether such amplification does in fact take place has been question by Hastings et al. (1976) Photochem., Photobiol., 23, 461. The CL system of the present invention, "CLASSIC", also has certain advantages over fluorescent antibody techniques which make use of the ability of a fluorescent tag to emit light of a particular wave length when excited by radiant energy of a lower wave length. A number of clinical analyses which utilize fluorescent "probes" or tags have been described in a recent review by Soini (1979) Fluoroimmunoassay: Present Status And Key Problems. Clin. Chemistry, 25, 353-361. In general, the detection level, or sensitivity, of fluoroimmunoassay techniques is greater than enzyme immunoassay techniques and approaches the capability of radioimmunoassay systems.

The use of fluorescent probes to replace radioactive isotopes is hindered by the decreased sensitivity obtained with fluorescence. This is due, to a great extent, to the sample's or serum's own fluorescence. The intensity of this background is affected by many fluorescing compounds, such as protein which may be present in the sample, and which also increase scattering caused by the specimen.

Fluorescence methods are now extensively applied in immunology, mainly in fluorescence microscopy, for studying various types of tissues, cells, bacteria, viruses and so on. A number of fluorescent materials and procedures for coupling them to the above biologicals and haptens is well developed.

To take advantage of the full scope of this invention, special high intensity fluorescent molecules are required. These must be capable of biological coupling with protein, polysaccharide and hapten substances, especially immunoglobulins - i.e., I_gG and antigens without disturbing the specificity or activity of these biological materials.

Bellin (1968) Photophysical and Photochemical Effects of Dye Binding. Photochem. and Photobiol., 8, 383-342 and Porro et al. (1963 and 1965) Fluorescence And Absorption Spectra Of Biological Stains. Stain Technology, Vol. 38, and Fluorescence And Absorption Spectra Of Biological Dyes (II). Stain Technology, Vol. 40, No. 3, 173-175, respectively, have shown that there is a reduction in efficiency in the light output of fluorescers as a result of bonding or conjugation to proteins as compared to the output of these fluorescers in free solution. Our work has shown a similar loss in output, however, the energy efficiency of the oxalate system compensates for this loss. While this loss in light output effects all other known applications of conjugated fluorescers, the analytical method of the present invention requires a conjugate only during the biological antibody/antigen formation phase of the analysis. Procedures are well known for preparing a conjugate of a fluorescer in a manner which permits the conjugate to be subsequently separated at will by changing the pH, or other parameter, of the conjugate solution. It should also be noted that the immunochemical reaction of CLASSIC, Method III, may be carried out in the environment best suited for the optimum detection of the analyte of interest. After the label has been identified with the analyte one may then separate the label, the fluorescer, from the conjugate which allows the fluorescer to enter the solvent phase of the CL system to yield the maximum light efficiency. In general, it is desirable to provide the high quantum efficiency of fluorescing aromatic and substituted hydrocarbons, heterocyclic compounds, dyestuffs, and metal chelates with the ease of conjugation to the biological now available for microscopy reagents. We have found that we can couple the fluorescer using known procedures currently accepted for use with the fluorescent conjugates such as set forth in Soini (1979) supra, the teachings of which are incorporated herein by reference.

The following Tables 2 and 3 from Soini (1979) supra, set forth data on various fluorescent materials which can be advantageously employed as labels in the environment of the present invention.

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Footnotes to Table 3 a Measurement results have been obtained with some commonly used probes as conjugates of bovine serum albumin, IgG, thyroxine, and digoxin. Conjugation was by common methods described in the literature (47, 37, 46, 44). No attempt was made to optimize measurement in any way, this was done directly at emission maxima for bandwidths of 10 nm. No cut-off filters were used. It would probably have been possible to reduce the detection limits of some probes considerably by altering the slit-values and by adjusting the measurement wavelengths, and by using suitable cut-off filters. (The emission of fluorescein for example is usually measured at 540 nm, although the emission maximum occurs at 515 nm.) The fluorescence and detection limits for different probe conjugates were measured with, a Perkin-Elmer fluorescence spectrometer, Model MPF-2A. The detection limits were measured in the regions of excitation and emission maxima, and the values compared with the background fluorescence values of diluted serum at the same wavelengths and with the same instrument sensitivity.b No reaction in IgG, SH-groups. c Serum background, may bind to different proteins. d Interference by protein fluorescence. e Interference by serum fluorescence, own fluorescence weak. BSA, bovine serum albumin; hlgG, human immunoglobulin G. Typical of fluorescers which provide derivatives to which the biological may be coupled are the following from Pringsheim (1946) Luminescence Of Liquids And Solids And Its Practical Applications. Interscience Publishers, Inc., New York, New York, as Tables 4-6:

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I to

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In addition to the organic fluorescers listed above, a number of metal organic materials have been suggested for laser fluorescent assay systems: Ruthenium (IΙ)-tri(bipyridyl) complex has been identified by Curtis et al. (1977), Chemiluminescence; A New Method For Detecting Fluorescent Compounds Separated By Thin Layer Chromatography, J. Chromatography, 134, 343-350 for CL applications; Metal Complexes by Sherman (1978), Analytical Applications Of Peroxyoxalate Chemiluminescence, Analytical Chim. Acta, 97, 21-27, and Soini (1979) supra. Weider United States Patent No. 4,058,732 disclosed and suggested their immunofluorescent application. It is also well known, Van Uitert (1960), Factors Influencing The Luminescent Emission States Of The Rare Earths. J. Electrochem. Soc., 107, 803, that small additions of the rare earth and/or transition metals function as promotors, activators or coactivators in inorganic and organic phosphors. Thus, it is not unexpected that trade impurities will behave in a similar manner in other organic and metallo-organic systems and have a profound effect on the quantum efficiency of the fluorescer.

The discussion has thus far centered around the novel analytical use of a fluorescer-biological conjugate activated by the chemical energy from a peroxyoxalate CL system. The preferred peroxyoxalate system is advantageous for CL because of its quantum efficiency and because there is no background light in the absence of a fluorescer conjugate. This system is particularly "noise free" when certain intensity control additives are eliminated, such as are disclosed by Bollyky (1972) Chemiluminescent Additives, United States Patent No. 3,704,231. A system for analytical purposes need only provide light of high intensity for a short period, that is, for example, under about 30 minutes.

While peroxyoxalates which are "noise free", or nonfluorescent are preferred, other naturally self fluorescent oxalate esters or CL compounds are also useful with, the proper selection of a barrier filter and use of a conjugate fluorescer of longer wavelength. Such esters include 2-napthol-3,6,8,-trisulfonic acid, 2-carboxyphenyl, 2-carboxy-6-hydroxyphenol, 1,4-dihydroxy-9, 10-diphenylanthracene, 2-napthol, as well as aqueous CL materials such as luminol, lophine, pyrogallol, luciferin, and related compounds.

Other systems besides those mentioned are also capable of activating a CL fluorescer-conjugate.

These include: (1) Ozone, which has been shown by Randhawa (1967), Ozonesonde For Rocket Flight, Nature, 213, 53, to activate Rhodamine-B. (2) Keszthelyi et al. (1969), Electrogenerated Chemiluminescence: Determination Of Absolute Luminescence Efficiency, etc., A. Chem., 47, 249-256, has demonstrated electrogenerated CL in 9,10diphenylanthracene, thianthrene, and rubrene with some systems. Thus, Ozone or electro-generated CL in the presence of the fluorescer-conjugate can provide other useful energy sources for the CL fluorescer systems of the present invention. In addition, other known energy sources such as have been found useful in applications involving the distortion of various polymers by mechanical energy and other similar systems which yield free radicals are also useful in the present invention. It should be understood that many analytical system variations are possible, but all have in common the use of a labelled immunological specie specific to the analyte. The analyst has the latitude in selecting a procedure which provides the detection level required from a minimum amount of sample and which uses the least expensive and most reliable instrument. The detection level required is a function of the antigen, antibody or hapten concentration in the analyte and its clinical significance.

For clinically significant dosage testing, i.e. Digoxin, standard curves are obtained from known samples analyzed together with the unknown and run under carefully controlled duplicate analyses on highly calibrated instruments. While a presumative test for an immunoglogulin requires a much lower level of sophistication, it is highly advantageous for a single analytical system to be able to cover this analytical spectrum.

The sophisticated analytical requirements may be met by using a Centrifugal Fast Analyzer such as that made by Electro-Nucleonics, Inc. Burtis et al. (1975) Development Of A Multipurpose Optical System For Use With A Centrifugal Fast Analyzer. Clinical Chemistry, 21, 1225-1232. For the N^th nations lacking the ability or need for such sophistication, or for presumptive testing at the physician's office or clinic, no instrument is required. The "CLASSIC" system of the present invention delivers sufficient intensity to the labeled biological to enable the clinician to make a simple go-no-go determination by "eyeballing". The clinician may also modify the role of the labeled specie used in carrying out the analyses. While solid phase techniques have been used as examples to illustrate the advantages of the present invention, it should be recognized that homogeneous and heterogeneous assays also will benefit from the use of the "CLASSIC" system. Acceptable alternative variations in test procedure include:

(1) Competitive binding of labeled antigen.

(2) Competitive binding of labeled antibody.

(3) Quenching analyses.

(4) Immunoprecipitant reactions.

(5) Ion exchange methods.

(6) Ion exclusion methods.

Description Of Invention

The major components for the preferred "lightswitch" or "light indicator" of the present invention are similar to those disclosed in United States Patent No. 3,597,362. They include an oxalic ester, a hydroperoxide, a fluorescer (or fluorescent compound) and a diluent. Furthermore, in order to generate maximum intensity of light, the employment of an additional catalytic accelerator is sometimes necessary. The choice and the concentration and other parameters of a suitable catalytic accelerator is also described in United States Patent No. 3,704,231.

The present invention differs from the teaching of United States Patent No. 3,597,362 in that the fluorescent compound (or fluorescer) employed in this invention is covalently bonded to a biological material, such as inmmunoglobulin, enzymes, proteins, bacteria, and so on; or to an organic material, such as haptens or polymers; or to an inorganic material, such as glass, silica, ceramic, or the like. The organic and inorganic materials to which suitable fluorescer may be bonded can be in the form of particles, crystals, tubes, rods, plates, blocks and the like, or in solution. The fluorescent compound, or fluorescer, bonded to the above mentioned substances can then be utilized as a label in place of radioactive materials or an an indicator in place of color dye, for use in various well-known assays.

Especially suitable fluorescent compounds, or fluorescers, for use in the present invention are those which have a spectral emission falling between 260 millimicrons and 1,000 millimicrons. The structure of the fluorescent compounds or fluorescers useful in the present invention must possess one or more functional groups capable of reacting with those materials to be coupled to it. Examples of preferred functional groups are: alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino-, dihalo triaziτiyl-. Typical examples of suitable fluorescer derivatives are: 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, teteramethylrhodamine isothiocyanate, amino-pyrene, amino-anthracene, and similar compounds as will be familiar to those skilled in the art.

It has been observed that on binding a fluorescent compound fluorescer, to a solid material, the wavelength, of emission of the bonded fluorescer shifts to either a longer or a shorter wavelength, depending on the specific fluorescer employed.

We have also found that the length, of "space arm", the ligand between the fluorescent compound and the material bonded to it, effects the emission wavelength of the bonded fluorescer.

The exact concentration of fluorescer derivative employed for binding is not critical providing that the immunological or enzymatic active conjugates produced therefrom have the desired activity, and that the intensity of light thus produced is visible, with or without the help of instruments, and may be differentiated from the background.

The intensity of the light generated by the coupled fluorescer depends upon the structure of the fluorescer, the type of linkage between the fluorescer and the bonded materials, and the available functional groups of the anchored substance. In general, the intensity of the light produced by a fluorescer is not as great after coupling as it is when in free solution. It is also important that the fluorescer conjugate be stable in the presence of the chemiluminescent reaction.

The following examples are given to illustrate the various ways the fluorescer may be attached to another moiety by covalently bonding using an inorganic support for convenience, which is in no way intended to limit the scope of the invention described herein.

Examples T-V

In each of the Examples I-V the linkage attached to a controlled pore glass surface was synthesized to imitate the representative chemically active sites on a typical protein or biological conjugate; For example, amino-, carboxyl-, mercapto-, or hydroxyl groups are representative of attachment sites.

A glass support is used so that the activity and specificity of the functional group is easily controlled, and to immobilize the fluorescer so that it may be readily separated from the free or unbound fluorescent compound in order that the fluorescent spectra may easily be recognized as distinct from the oxalate CL reagent.

The results of visual observation as to the color of the fluorescent glass, and color and intensity of emitted light for l-aminopyrene covalently bonded to porous glass (CPG) (500A pore size) fluorescer with various different linkages are set forth in the attached Table 7.

The methodology employed for preparing each fluorescer/glass sample was as follows:

Example I Ten grams of porous glass of 500 (A) (angstrom pore size) was treated with 100 ml 15% gamma-aminopropyltrimethoxysilane in toluene and refluxed for at least

16 hours, then removed. The unbound silane was thoroughly washed with methanol, filtered and the glass air dried before use. Approximately 25 milligrams l-amino-pyrene was dissolved in dioxane (20 millimeter). To this solution about 153 milligrams of succinic anhydride was added. After two hours, 10 millimeter of 5 m mole N,Ndicyclohexyl-carbodiimide dioxane solution was added. 500 mg of this gaπrma-aminopoply-trimethoxysilane treated glass (from here on, aminopropyl-glass) as prepared above was added to dioxane solution. The slurry was then stirred for one hour and let stand overnight at room temperature. Continuous stirring is preferable. The excess pyrene-dioxane solution was decanted and the glass washed exhaustively with dioxane, methanol and acetone (15 ml of each wash and three times for each solvent). The wet pyrene coupled glass was filtered and allowed to air dry.

Example II 500 mg of the aminopropyl-glass prepared as stated in Example I was added to 25 ml of 10% thiophosgene in chlorofrom and the slurry was refluxed for 4 hours . The chloroform was decanted and then washed with chloroform, methanol, acetone (25 ml of each, wash and three times for each, solvent). The slurry was filtered and air dried. 30 milligrams of l-aminopyrene was dissolved in 15 ml dioxane. To this solution, the dry isocyanatoglass was added and stirred for one hour and then allowed to stand at room temperature overnight. After the reaction was complete, aminopyrene dioxane solution was decanted and the pyrene coupled glass was washed in the same manner as stated in Example I. Example III 500 mg of aminopropyl-glass, prepared as stated in Example I, was added to 10 ml of dioxane dissolved with 50 mg succinic anhydride. The slurry was allowed to stand overnight at room temperature preferably with continuous stirring. After the reaction was complete, the aminopropyl-glass, being converted to carboxy-glass, was washed in the same manner as stated in Example I. Approximately 23 mg l-aminopyrene was dissolved in 1 ml of dioxane. To this solution, 58 mg of N-acetyhomocystein was dissolved. The solution was then kept 4 hours at room temperature. 50 mg of N,N-dicyclohexyl-carbodiimide was then added to it. At the same time, the prepared and dried carboxyl-glass was added to the solution for coupling. The reaction was allowed to stand at room temperature for 24 hours. Pyrene coupled glass was then washed and dried in the same manner as stated before.

Example IV 4 grams of aminopropyl-glass prepared from Example I was added to 10% p-nitrobenzoyl chloride with 1 ml of triethylamine in 50 ml chloroform solution. The slurry was stirred and refluxed for at least 8 hours. The resulting acylated glass was thoroughly washed with chloroform and let air dry. 0.1M of sodium dithionite (30 ml) was prepared and the acylated glass was added. The temperature was then raised to 40°C. The reaction was completed in one hour. The glass was washed thoroughly with warm water. The arylamino-glass thus prepared was ready to diazotize. 1 gm of arylamino-glass was added to 20 ml aqueous solution of 350 mg sodium nitrite and 0.2 ml IN hydrochloric acid. The temperature was brought down to 4°C using an ice bath. The reaction was allowed to continue for one hour. The acid solution was then decanted, the glass was thoroughly washed and the pH was adjusted to above 8.0. The filtered glass was then added to 10 ml of 20 mg aminopyrene dioxane solution. Reaction was complete in 8 hours at room temperature. The pyrene coupled glass was then washed in the same manner as in Example I.

Example V

One gram of 500 (Å) pore size porous glass treated with 10 ml 15% gamma-glycidoxypropyltrimethoxy- silane in toluene and refluxed for at least 16 hours, then washed the glass with acetone thoroughly and air dried. To 30 ml aqueous solution containing 1.5 mg/ml of m-sodium periodate, the silane treated glass (epoxyglass) was added. The reaction was allowed to go on for 2 hours. Then the glass was washed with water thoroughly. 25 mg of l-aminopyrene was dissolved in 30 ml dioxane. To this solution, the filtered wet cake glass was added. The slurry was stirred for one hour and then let stand overnight at room temperature. The pyrene coupled glass was washed in the same manner as stated in Example I.

I Ui

Example VI Different lengths of "space arm" for binding of the fluorescer were used to study the effect on the chemiluminescence quality of the resultant bonded fluorescer.

A long "space arm" of about 20 (Å) in length stretching out from a controlled glass pore surface was prepared as follows: 500 mg of carboxy-glass prepared as stated in Example III was activated by adding a 20 ml dioxane solution containing 200 mg of N,N-dicyclohexyl carbodiimide. The glass was stirred for 24 hours and then washed with, dioxane and methanol. 20 ml of 200 mg hexamethylene diamine aqueous solution was prepared and cooled beforehand. The activated carboxy-glass was added to the cooled solution and stirred for five hours, then allowed to stand for 24 hours at 4°C. The glass was then washed thoroughly with water, methanol and dioxane. 20 ml dioxane containing 50 mg succinic anhydride was then added to the glass. This reaction was completed in 24 hours. The glass was subsequently washed thoroughly with methanol. 25 mg l-aminopyrene was dissolved in 30 ml dioxane. To this solution 5 m mole N,N-dicylohexylcarbodiimide was added and dissolved prior to adding the prepared glass. The slurry was stirred for one hour and then let stand overnight at room temperature. After 24 hours reaction, pyrene coated glass was then washed in the same manner as in Example I.

Pyrene coated glass with a short "space arm" of about 10 (A) in length was prepared as stated in Example I, as the control. The results of these two glasses is set forth in attached Table 8.

Examples VII-IX Porous glass having various pore sizes were coated with l-aminopyrene to show the effect of pore size on the chemilouminescence. Threeodifferent poroous glasses having 170 (A) (angstrom), 500 (A) and 3000 (A) pore size, respectively, were coated with 1-aminopyrene in the same manner as stated in Example 1. The effect on the chemiluminescence is set forth in attached Table 9.

I σ-i o

Examples X-XV Several different fluorescers were coated on porous glass to study the effect of structure on color emission. 1-amino-pyrene and 2-amino-anthracene were coated on the porous glass (500 Å) in the same manner as described in Example I.

20 mg of 3,4,9,10-perylenetetracarboxylic dianhydride was added to 25 ml of dioxane, to this solution 25 mg of aminopropyl-glass was added and stirred for one hour before allowing to stand for another 6 hours at room temperature. The glass was then washed thoroughly with methanol or acetone, then filtered and air dried.

500 mg of aminopropyl-glass was added to 30 ml dioxane containing 50 mg succinic anhydride and stirred for one hour before being allowed to stand overnight at room temperature. The glass was then washed thoroughly with acetone, filtered and air dried. One part of 250 mg of such glass (carhoxyl-glass) was added to 25 ml 0.01M potassium phosphate of pH=7.6 solution containing 20 mg of isothiocyanate fluorescein. Another part of 250 mg of carhoxyl-glass was added to acetone/dioxane (50/50 by volume) solution containing 20 mg of 3-amino-phthalhydrazide). The two glass slurries were stirred for one hour and then allowed to stand at room temperature overnight. After the reaction was completed, the glass was washed with deionized water and acetone, respectively. Finally, they both were washed with acetone, then filtered and air dried. 300 mg of aminopropyl-glass prepared as shown Example I was added to 50 ml 0.01M potassium phosphate of pH=7.6 solution containing 25 mg of O-phthalicdicar- boxaldehyde. The glass slurry was stirred for one hour, then allowed to stand at room temperature for another 24 hours. The glass was then washed thoroughly with deionized water, acetone, then filtered and air dried.

The attached Table 10 sets forth the observed chemiluminescence characteristics of different fluorescers bonded to porous glass in an oxalate ester/peroxide system.

1

Co

1

Example XVI Aminopyrene conjugate with antibody to Hepatitis B Surface Antigen coated on porous glass

30 mg commercially available antibody to Hepatitis B Surface Antigen coated porous glass was added to 5 ml of 0.01 m potassium phosphate of pH=7.6. 24 mg of 1-aminopyrene was dissolved in 2 ml dioxane. To this solution 45 mg of succinic anhydride was added and mixed for two hours. Approximately 95 mg of N,N-dicylohexyl- carbodiimide was dissolved in 1 ml of dioxane. The latter two solutions were mixed together and stirred for 30 minutes. Then 250 lamda of pyrene solution was transferred to the glass slurry solution. The slurry was stirred for two hours at room temperature and then allowed to stand at 4°C overnight. The glass was washed four times with 10 ml phosphate buffer (pH=7.6) each wash, and was given two additional t-butanol washes with 10 ml phosphate buffer each time before testing. If necessary, the slurry was washed until no light could be detected from the supernate of the slurry. Then the 1-aminopyrene-antibody conjugate coated on the porous glass was tested by reacting with oxalate and peroxide. It was found that only the glass particle glowed in faint blue color.

Example XVII

Fluorescein isothiocyanate anti-human gamma-globulin conjugate was prepared as follows: 4 mg of fluorescein isothiocyanate thoroughly mixed in 10 ml 0.1 M potassium phosphate buffer of pH=9.0. 4 ml of anti-human gamma-globulin (protein concentration of 20 mg/ml) was then added to the fluorescein phosphate solution. The mixture was continuously stirred for one hour at 4°C and allowed to stand at the same temperature for another 24 hours. Excess fluorescein was removed by extensive dialysis against 0.1 M potassium phosphate buffer of pH=7.2. During dialysis, 100 ml of buffer each time was used, and the buffer was changed every 2 hours for 5 times.

Gamma-globulin coated porous glass was prepared as follows: 50 mg of epoxy-glass (3000 (Å) pore size) was prepared in the same way as described in Example IV. 2.5 mg m-sodium periodate was dissolved in 5 ml of deionized water. Glass was then added to this solution and stirred at room temperature for 2 hours. The glass was washed thoroughly with deionized water and then with 10 ml 0.1 M potassium phosphate pH=9.0 buffer and kept for one hour. The glass was then filtered and was ready for coupling. 5 τnl human gamma-globulin (protein concentration of 30 mg/ml) was diluted with 5 ml of 0.1 M, pH=9.0 phosphate buffer. The activated glass was then added to this solution and was stirred at 4°C for 2 hours before being allowed to stand overnight at the same temperature. After reaction was completed, the glass was washed extensively with 0.1 M potassium phosphate buffer of pH=7.2 and then filtered for immediate use.

30 mg of human gamma-globulin coated porous glass was added to 0.5 ml of fluorescein-antihuman gamma- globulin conjugate. The slurry was incubated on 24 cycles of agitation/settling (60/90 seconds ratio). Excess antibody solution was decanted and the glass was washed with 0.01 M potassium phosphate buffer of pH=7.2 until no light was detected by testing the decanted buffer in oxalate/ peroxide system.

The glass was then washed with 5 ml t-butanol and excess butanol was withdrawn. Green color light was observed on glass particles upon addition of oxalate and peroxide.

Although the above examples illustrate various modifications of the present invention, other variations will suggest themselves to those skilled in the art in the light of the above disclosure. It is to be understood, therefore, that changes may be made in the particular embodiments described above which are within the full intended scope of the invention as defined in appended claims.

Claims

1. A system for the detection of a biological analyte of interest which comprises contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an energy source which is capable of activating the fluorescer.

2. A system for the detection of a biological analyte of interest which comprises contacting a sample with a fluorescer which has been conjugated to an immunological specie specific to the biological analyte of interest, in the presence of an excess of an energy source which is capable of activating the fluorescer.

3. A method for the qualitative detection of a biological analyte of interest comprising:

(b) contacting the fluorescer labeled specie and the biological of interest;

(c) separating the fluorescer labeled specie/ biological complex; (d) contacting the separated fluorescer labeled specie/biological complex of (c) with an energy source which is capable of activating the fluorescer label; and

(e) determining the presence or absence of chemiluminescent light emitted from the activated fluorescer.

4. A quantitative method for measuring the amount of a biological analyte of interest comprising;

(a) labeling an immunological specie specific to the analyte of. interest with a fluorescer material which is biologically compatible with such specie;

(b) contacting the fluorescer labeled specie and the biological of interest;

(c) separating the fluorescer labeled specie/ biological complex;

5. The method of claim 3 wherein the fluorescer of (a) is chemically conjugated to the immunological specie specific to the biological of interest.

6. The method of claim 5 wherein the chemical conjugation of the fluorescer material to the immunological specie specific to the biological of interest is carried out using known techniques in such a way as to prevent substantial biological damage to the attached specie.

7. The method of claim 3 wherein the fluorescer material utilized has a spectral emission of from about 260 millimicrons to about 1000 millimicrons.

8. The method of claim 3 wherein the fluorescer material utilized has a spectral emission above the light absorption wavelength of either the immunological specie specific to the biological of interest or the biological of interest and below the light absorption wavelength of any solvent system utilized.

9. The method of claim 3 wherein the fluorescer material utilized has a structure which possesses one or more functional groups capable of reacting with the immuno- logical specie specific to the biological of interest without adversely affecting such specie.

10. The method of claim 3 wherein the fluorescer material utilized has a structure which possesses one or more functional groups selected from the group comprising alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carhαxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino- and dihalo triazinyl-.

11. The method of claim 3 wherein the fluorescer material utilized is selected from the group comprising 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothio-cyanate, teteramethyl- rhodamine isothiocyanate, amino-pyrene, amino-anthracene, and the like.

12. The method of claim 3 wherein the energy source of (d) which is contacted with the separated fluorescer labeled specie/biological complex is present in excess of the amount required to activate all of the fluorescer labeled specie.

13. The method of claim 3 wherein the energy source of (d) which is contacted with the separated fluorescer labeled specie/biological complex is any source which is capable of activating the particular flourescer selected to be compatible with the labeled specie.

14. . The method of claim 3 wherein the energy source of (d) which is contacted with the separated fluorescer labeled specie/biological complex is the peroxyoxylate reaction.

15. A method according to claim 13 wherein the energy source is a reaction selected from the group comprising 2-napthol-3,6,8-trisulfonic acid, 2-carhoxyphenyl, 2-carboxy-6-hydroxyphenol, l,4-dihydroxy-9, 10-diphenyl- anthracene, 2-napthol., luminol, lophine, pyrogallol and luciferin reactions.

16. A method according to claim 13 wherein the energy source is derived from ozone, an electrogenerated species, or a mechanically generated species.

17. A method according to claim 3 which is carried out utilizing solid phase analytical techniques.

18. A method according to claim 3 which is carried out utilizing a sandwich technique.

19. A method according to claim 3 which is carried out utilizing homogeneous analytical techniques.

20. A method according to claim 3 which is carried out utilizing heterogeneous analytical techniques.

21. A method according to claim 3 which is carried out utilizing competitive binding techniques.

22. A method according to claim 3 which is carried out utilizing quenching techniques.

23. A method according to claim 3 which is carried out utilizing immuno-precipitant reaction techniques.

24. A method according to claim 3 which is carried out utilizing ion exchange techniques.

25. A method according to claim 3 which is carried out utilizing ion exclusion techniques.

26. A method according to claim 3 which is carried out utilizing masking techniques.

27. The method of claim 4 wherein the fluorescer of (a) is chemically conjugated to the immunological specie specific to the biological of interest.

28. The method of claim 4 wherein the chemical conjugation of the fluorescer material to the immuno- logical specie specific to the biological of interest is carried out using known techniques in such a way as to prevent substantial biological damage to the attached specie.

29. The method of claim 4 wherein the fluorescer material utilized has a spectral emission of from about 260 millimicrons to about 1000 millimicrons.

30. The method of claim 4 wherein the fluorescer material utilized has a spectral emission above the light absorption wavelength of either the immunological specie specific to the biological of interest or the biological of interest and below the light absorption wavelength of any solvent system utilized.

31. The method of claim 4 wherein the fluorescer material utilized has a structure which possesses one or more functional groups capable of reacting with the immunological specie specific to the biological of interest without adversely affecting such specie.

32. The method of claim 4 wherein the fluorescer material utilized has a structure which possesses one or more functional groups selected from the group comprising alkylamino-, arylamlno-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino- and dihalo triazinyl-.

33. The method of claim 4 wherein the fluorescer material utilized is selected from the group comprising 3,4,9,10 perylene tetracarboxylic dianhydride, amino- chrysene, fluorescein isothiocyanate, teteramethylrho- damine isothiocyanate, amino-pyrene, amino-anthracene, and the like.

34. The method of claim 4 wherein the energy source of (d) which is contacted with the separated fluorescer labeled specie/biological complex is present in excess of the amount required to activate all of the fluorescer labeled specie.

35. The method of claim 4 wherein the energy source of (d) which is contacted with the separated fluorescer labeled specie/biological complex is any source which, is capable of activating the particular fluorescer selected to be compatible with the labeled specie.

36. The method of claim 4 wherein the energy source of (d) which is contacted with the separated fluorescer labeled specie/biological complex is the peroxyoxylate reaction.

37. A method according to claim 35 wherein the energy source is a reaction selected from the group comprising 2-napthol-3,6,8-trisulfonic acid, 2-carboxy- phenyl, 2-carboxy-6-hydroxyphenol, l,4-dihydroxy-9,10- diphenylanthracene, 2-napthol, luminol, lophine, pyrogallol, luciferin reactions.

38. A method according to claim 35 wherein the energy source is derived from ozone, an electrogenerated species or a mechanically generated species.

39. A method according to claim 4 which is carried out utilizing solid phase analytical techniques.

40. A method according to claim 4 which is carried out utilizing homogeneous analytical assay techniques.

41. A method according to claim.4 which is carried out utilizing heterogeneous analytical assay techniques.

42. A method according to claim 4 which is carried out utilizing competitive binding techniques.

43. A method according to claim 4 which is carried out utilizing quenching analyses techniques.

44. A method according to claim 4 which is carried out utilizing immuno-precipitant reaction techniques.

45. A method according to claim 4 which is carried out utilizing ion exchange techniques.

46. A method according to claim 4 which is carried out utilizing ion exclusion techniques.

47. A method according to claim 4 which is carried out utilizing masking techniques.

48. A fluorescer composition useful in the labeling of an immunological specie specific to and for the detection of a biological of interest, such fluorescer having a chemical structure which possesses one or more functional groups capable of chemical reaction with the immunological specie without the adverse effect on the specificity of such specie to the biological of interest.

49. A fluorescer composition useful in the labeling of an immunological specie specific to and for the detection of a biological of interest, such, fluorescer having a chemical structure which posseses one or more functional groups selected from the group comprising alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole-, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide-, nitrobenzoyl halide-, carbonyl halide, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino- and dihalo triazinyl.

50. A fluorescer composition useful in the labeling of an immunological specie specific to and for the detection of a biological of interest, such fluorescer selected from the group comprising 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, teteramethylrhodamine isothiocyanate, amino-pyrene, amino-anthracene, and the like.

51. A conjugated fluorescer/immunological specie composition useful in the detection of a biological of interest which has been formed via reacting an immunological specie with a fluorescer having a chemical structure which possesses one or more functional groups capable of chemical reaction with the immunological specie without adverse effect on the specificity of such specie to the biological of interest.

52. A conjugated fluorescer/immunological specie composition useful in the detection of a biological of interest which has been formed via reacting an immunological specie with a fluorescer having a chemical structure which possesses one or more functional groups selected from the group comprising/ alkylamino-, arylamino-, isocyano-, cyano-, isothiocyano-, thiocyano-, carboxy-, hydroxy-, mercapto-, phenol-, imidiazole, aldehyde-, epoxy-, thionyl halide-, sulfonyl halide- , nitrobenzoyl halide-, carbonyl halide-, triazo-, succinimido-, anhydride-, haloacetate-, hydrazino and dihalo triazinyl-.

53. A conjugated fluorescer/immunological specie composition useful in the detection of a biological of interest which has been formed via reacting an immunological specie with a fluorescer selected from the group comprising 3,4,9,10 perylene tetracarboxylic dianhydride, amino-chrysene, fluorescein isothiocyanate, tetramethyl- rhodamine isothiocyanate, amino-pyrene, amino-anthracene, and the like.