WO1992017781A1

WO1992017781A1 - Agglutination assay

Info

Publication number: WO1992017781A1
Application number: PCT/GB1992/000615
Authority: WO
Inventors: Peter David Weston; James Francis Kelly
Original assignee: International Murex Technologies Corporation
Priority date: 1991-04-04
Filing date: 1992-04-06
Publication date: 1992-10-15
Also published as: IE921089A1; GB9107030D0; AU1462992A

Abstract

Improved direct and indirect agglutination assays are claimed, in which coated particles, after incubation with sample, are retrieved by the centrifugation or, in the case of magnetic particles, by the application of a magnetic field. The retrieval step increases the sensitivity of an assay. A further improvement is to accelerate agglutination by using magnetic particles and applying a magnetic field to the sample during agglutination.

Description

AGGLUTINATION ASSAY

The present invention relates to an agglutination test for the detection of a ligand.

Diagnostic test methods based upon the agglutination of antigens and antibodies, wherein either the antigen or the antibody is attached to a solid phase, are well known in the field of immunodiagnostic reagents. For example, US-A-3088875 describes a technique in which plastic microspheres coated with antigen are mixed with a test sample such that when the sample contains antibodies to the antigens, the antibodies attach themselves to the antigen causing visible agglutination or aggregation of the microspheres.

US-A-4419453 describes a latex agglutination test in which the test reagent comprises antigen- or antibody- coated latex particles of one colour and a water-soluble non-latex polymer particle absorbing dye of a different colour. When agglutination takes place, the contrast between the colour of the aggregate and the background colour of the solution assists visualisation.

EP-A-0174195 discloses agglutination assays, particularly latex agglutination assays, for simultaneous testing for a multiplicity of ligands. The reagents for use in the assays comprise two or more insoluble coloured substances, each substance being adapted to form a distinctively coloured agglutinate in the presence of a specific ligand or specific group of ligands.

A number of assays have been proposed which employ magnetic particles and labelled particles. US-A-4115535 describes an assay in which magnetic particles are used to facilitate separation of agglutinated particles from solution and a second kind of particles is present to facilitate detection of agglutination. Both types of particles are coated with a protein capable of inter- acting specifically with a protein whose presence it is wished to determine. Agglutination occurs when the latter protein is present in a test sample and is detected by observing, for example, a fluorescent label on the second kind of particles. Similar assays are disclosed in JP-A-177265/60 and JP-A-128168/61.

Two types of agglutination assay are direct assays and indirect assays. In a direct agglutination assay, for example, as described in US-A-3088875, particles are coated with a substance that binds to the ligand under investigation. When the coated particles are admixed with a liquid sample, ligand present in the sample combines with the binding substance coated on the surface of the particles, and agglutination occurs.

In an indirect assay, the particles are coated with the ligand under investigation, or with an analogue of the ligand, or with a conjugate comprising the ligand or an analogue of the ligand, generally with an inert protein. The coated particles, together with a substance that binds with the ligand under investigation, are incubated with the sample under investigation. The binding substance is not bound to particles as in a direct assay, but is free in the incubation mixture. Indirect assays are generally inhibition assays or competition assays. In both types of indirect assay ligand present in a sample under investigation combines with binding sites on the binding substance in the incubation mixture, so that fewer sites are available to combine with the particle-bound ligand, and so fewer particles are agglutinated. Accordingly, in contrast to a direct assay, in an indirect assay agglutination indicates the absence of ligand and lack of agglutination indicates the presence of ligand.

For a direct or an indirect assay, the substance that binds to the ligand is preferably a specific binding substance, especially an antibody to the ligand.

Direct assays may be used to investigate, for example, ligands that are antibodies or antigens, or ligands that are receptors or their target molecules. Direct assays are particularly useful in the case where a ligand is multi-epitopic, for example, in the case of antigens that present several different epitopes, or that present repeating epitopes.

Indirect assays may be used, for example, when the ligand is small and presents a single epitope, for example, when the ligand is a hapten. Examples of ligands that may be investigated using an indirect assay are therapeutic drugs, drugs of abuse and certain hormones. EP-A-0296883 describes an indirect agglutination assay for haptens.

The term "particles" is used herein to denote particles that are suitable for use in an agglutination assay. The term "coated particle" is used to denote a particle that is coated with a substance that binds to the ligand under investigation (for use in a direct agglutination assay), or a particle that is coated with the ligand itself, an analogue of the ligand, or a conjugate comprising the ligand or an analogue of the ligand (for use in an indirect agglutination assay). An improved assay has now been devised which employs a single type of particle and which permits enhanced detection of a ligand, for example, an antigen. Coated particles (and free binding substance for the ligand, where appropriate), are incubated with a liquid sample under investigation. After incubation, the particles are retrieved. Liquid is removed, preferably so that, at most, just enough liquid remains to suspend the particles, and the retrieved particles are suspended in the remaining liquid. Alternatively, the liquid is removed and the particles are suspended in a second liquid. After further treatment, agglutination is observed by eye. This assay simplifies previous assays and it is surprising that agglutination can still be observed after separation of the bulk of the test sample liquid.

Accordingly, the present invention provides a method of determining a ligand in a liquid sample, which method comprises:

(a) incubating a first liquid medium comprising the sample and having dispersed therein either particles coated with a substance capable of binding to the ligand or particles coated with the ligand itself, an analogue of the ligand, or a conjugate comprising the ligand or an analogue of the ligand, the first liquid medium also comprising a substance capable of binding to the ligand, the sole particles in the medium that are coated being the said particles;

(b) retrieving the thus-incubated particles;

(c) suspending the retrieved particles in a portion of the first liquid medium or in a second liquid medium;

(d) allowing the suspension or a sample thereof to agglutinate or treating the suspension or a sample thereof to encourage agglutination to occur; and

(e) observing by eye whether the suspension has agglutinated.

The particles used in the present invention may be magnetic particles. (The term "magnetic particles" is used herein to denote particles having ferromagnetic properties. ) If the particles are magnetic then, after the incubation step (a), the particles may be retrieved by applying a magnetic field to the dispersion. For example, using a permanent magnet or an electromagnet, the particles are pulled to one surface of the container in which the sample and the particles have been incubated. Alternatively, the particles may be retrieved in step (b) by centrifugation, for example in the case of non-magnetic particles, for example coloured and white particles.

On retrieving the particles from the first liquid medium in step (b), ligand (or binding partner for the ligand in the case of an indirect assay) is thus rapidly and effectively concentrated from the bulk of the dispersion onto the surface of the particles. Preferably, most of the liquid is removed and discarded, and in step (c), the particles are then suspended in the small volume of liquid that remains. Alternatively, the particles may be suspended in a second liquid medium.

Step (d) may be carried out according to conventional procedures, for example, a sample of the suspension from step (c) may be placed on a suitable support, for example, a card or a piece of glass, tile or plastics material. The suspension is generally spread out in a thin layer, and the support may be moved in a horizontal plane or rocked to encourage agglutination. In a direct assay, in the absence of ligand in the original sample, the mixture remains unagglutinated after step (d) and presents a smooth appearance in step (e) . In the presence of ligand, agglutinates form and the sample has a granular appearance. In an indirect assay, the presence of ligand is indicated by lack of agglutination.

The retrieval step (b) in the method of the invention enables a ligand to be detected sensitively in samples in which the ligand is present at a low concentration.

Agglutination requires an initial incubation of coated particles with ligand, and it is generally considered to be necessary that free ligand is present during the agglutination. It has now been found, surprisingly, that after the initial incubation, the particles can be washed yet will still agglutinate. Hence it appears that, using the method of the present invention, the presence of free ligand is not required during the agglutination process itself.

A further surprising observation is that, if a suspension of magnetic particles resulting from step (c) above is subjected to the influence of an applied magnetic field during the agglutination step (d), agglutination occurs more rapidly than it does in the absence of an applied magnetic field. For example, if a dispersion of magnetic particles resulting from step (c) above is transferred to a suitable support, for example, to a card or piece of glass or tile, and spread into a thin layer, and is then subjected to the influence of a magnetic field, preferably from beneath the support, for example, a permanent magnet rotated or held under the support, or an electromagnet beneath the support, the mixture will agglutinate more rapidly than it would without the applied magnetic field. This unexpected observation may also allow detection of ligands to a greater sensitivity than hitherto, using manipulations which are simple, rapid and reliable.

The concept of applying a magnetic field to enhance the rapidity of an agglutination assay has general applicability. For example, in some cases the retrieval step (b) described above may not be necessary, for example, if the ligand is present in high concentrations. In such a case, steps (b) and (c) may be omitted and a magnetic field may be applied to the dispersion resulting from step (a) above to accelerate agglutination.

In other cases, for example, when the ligand is present in low concentrations, it will be advantageous to carry out the retrieval step (b) according to the method of the invention. Accordingly, the present invention also provides a method of determining a ligand in a liquid sample, which method comprises:

(i) incubating a liquid medium comprising the sample and having dispersed therein magnetic particles coated with a substance capable of binding to the ligand or magnetic particles coated with the ligand, an analogue of the ligand, or a conjugate comprising the ligand or an analogue of the ligand, the first liquid medium also comprising a substance capable of binding to the ligand, the sole particles in the medium that are coated with the said substance being the said particles; (ii) applying a magnetic field to the incubated medium or to a sample of the incubated medium; and

(iii) thereafter determining agglutination by eye. In step (i) above, the mixture of the sample under investigation and the appropriately coated particles may be incubated in a container and then a portion of the mixture may be removed for step (ii). Alternatively, the sample under investigation and the coated magnetic particles may be admixed directly on the support to be used for agglutination. In both cases a magnetic field is applied to cause the agglutination to occur more rapidly.

Considering the invention in more detail, the present methods are concerned with determining a ligand in a sample. The ligand may be any substance to be investigated for which there is a specific binding substance.

In many cases the ligand under investigation is an antigen, for example, a protein antigen or a poly- saccharide antigen. The ligand is typically a multivalent immuno-reactive species such as a multi-epitope antigen. The antigen may be derived from a human or animal pathogen, for example, it may be an antigen associated with, or characteristic of, a bacterium, virus, fungus or parasite. The method may therefore be used to detect a target antigen derived from bacteria, for example, selected from Streptococcus spp. , Streptococus pneumoniae, Haemophilus influenza b, Neisseria meninqitidis spp. , Neisseria gonorrhea and Gardnarella vaqinalis; a viral antigen, for example, derived from a human immunodeficiency virus, for example, HIV-1 or HIV- 2, or from a hepatitis virus, for example, from hepatitis A virus, hepatitis B virus or hepatitis C virus; derived from a fungal species for example, Candida albicans; derived from a parasite for example, Chla ydia spp. ; or the antigen may be a human or animal host-derived protein, for example, human chorionic gonadotrophin, luteinizing hormone, prostatic acid phosphatase, carcinoembryonic antigen or creatine kinase. Alternatively, the ligand may be an antibody, for example, an antibody to an infectious agent, for example, to a human immunodeficiency virus, for example, Hiv-i or HIV-2, or to a hepatitis virus, for example hepatitis A virus, hepatitis B virus, or hepatitis C virus. If an antibody is to be detected, the particles will be coated with an appropriate antigen. Further classes of substances that can be investigated are, for example, receptors and the corresponding target molecules, and carbohydrates and lectins. As indicated above, ligands that are haptens, for example, therapeutic drugs, drugs of abuse and certain hormones, may be investigated according to the methods of the present invention. It may be desired to detect the presence of such a substance in a sample of body fluid from a human or animal, for example, in the case of a drug of abuse, for example, cocaine, heroin or morphine, or in the case of an antibiotic, for example, in the milk of a cow being treated for an infection. It may be desired to monitor the level of a substance, for example, a therapeutic drug, particularly in the case of drugs the level of which should be maintained within relatively narrow therapeutic limits to obtain the most advantageous therapeutic effects and/or to minimise adverse reactions, for example, theophylline and phenytoin. Such substances may be assayed according to the present invention using an indirect assay.

The ligand under investigation may be obtained directly in the form of a liquid sample, or a solid or semi-solid sample comprising the ligand may be dissolved, suspended or extracted into a suitable liquid, generally an aqueous liquid. The sample may be a biological sample derived from a human or animal. Examples of liquid samples are blood, serum, plasma, sputum, milk, urine or cerebrospinal fluid. The sample may be a suspension of whole organisms or an extract of organisms originating from a human or animal source such as a suspension of bacteria obtained from a culture plate or an extract of a swab taken from a site of suspected infection.

The sample may be diluted with or extracted into a buffer so that the liquid medium incubated in step (a) has a pH which is appropriate, for example from pH 4 to pH 9 and preferably about neutral pH.

Particles suitable for use in agglutination assays are well known and many types are available commercially. The particles that are dispersed in the first liquid medium in step (a) are, for example, polystyrene latex particles, styrene-glycidyl methacrylate latex particles - or other polymer latexes-, for example, those described in US-A-4419453. As indicated above, the particles may be magnetic particles or non-magnetic, and may be white or coloured, for example red, blue or green. Non-magnetic particles are available commercially in a range of colours. Particles of different colours may be employed in the same test, the different colours identifying the specificity of the respective particles. Mixed coloured magnetic particles may be used therefore in which the different coloured particles are coated with substances able to bind to different ligands respectively.

The size of particles used in agglutination assays is typically less than 2μm in diameter, for example from 0.1 to l.Oμrn diameter, and smaller particles smaller than O.lμm in diameter may be used. Particles are available commercially in a wide range of suitable sizes. The size may be selected so that, in assessing the results of an assay, an agglutinated positive can easily be distinguished by eye from an unagglutinated negative. The size of the particles preferably therefore is selected so that a negative has a smooth appearance or at least an appearance which is not too granular.

The particles are coated with the ligand or with a substance capable of binding to the ligand it is wished to detect. Adsorption, chemical coupling or any other appropriate method may be used to link the coating substance to the particles. The sole particles in the medium in step (a) may be the coated particles.

As described above, the binding substance may be an antibody, antigen or any other substance that binds, preferably specifically, to a particular target ligand. An antibody used for coating may be a monoclonal antibody or a polyclonal antibody and may be of any class, for example, IgA, IgM or IgG. An antibody may therefore be employed which is capable of binding to an antigen characteristic of one of the organisms listed above or from other organisms of .interest or to host-derived antigens such as those listed above. The specific binding substance may be an antigen derived for example from a virus, bacterium, fungus or parasite. The antigen may be obtained using the techniques of molecular biology or it may be an extract or purified extract from the organism in question.

In carrying out the present method, the sample under investigation is contacted with a dispersion of the appropriately coated particles. In the case of an indirect assay, the mixture also contains a substance that binds to the ligand. under investigation. The first liquid medium, generally an aqueous medium, comprising the sample together with the particles and any free binding substance required, is incubated in step (a) preferably for from 30 seconds to 10 minutes, for example from 1 to 3 minutes. Incubation may take place at room temperature or at an elevated temperature, for example up to 50°C. After the incubation step (a), the particles are retrieved in step (b) . The particles are concentrated in this step. Where the particles are magnetic, that may be achieved by applying a magnetic field. The container in which step (a) has been carried out is typically placed alongside a magnet to which the particles are attracted. After a short period of time, for example 1 or 2 minutes, substantially all the particles have accumulated at the side of the container adjacent to the magnet. Liquid free of particles is removed and discarded. Where the particles are not magnetic particles, retrieval may be achieved by centrifugation and supernatant removed and discarded.

If, in step (c), the particles are to be suspended in the first liquid medium, the volume of liquid left, whether after magnetic retrieval or after centrifugation, is preferably about the minimum required for suspension of the particles such that any subsequent agglutination can be observed.

Alternatively, the retrieved particles may be suspended in a second liquid medium in step (c). The particles are suspended in a sufficient volume of the second liquid medium to permit observation of any subsequent agglutination. The second liquid medium is typically an aqueous medium and may be an aqueous buffer medium.

If desired, the retrieved particles may be washed after step (b) . Where the particles are magnetic particles, washing solution is added and, after the particles have been dispersed therein, a magnetic field may be reapplied. The particles are attracted to the magnet and washing solution is removed and discarded. In the case of particles which are non-magnetic, they are suspended in the washing solution and retrieved by centrifugation. The supernatant consisting of the used washing solution is removed and discarded. After washing, the particles are generally suspended in a second liquid medium but they may be suspended in a portion of the first medium that had been removed before washing and retained for further use. Washing is an optional step, however, and is not often required. In step (d), the suspension, or a sample of the suspension, is allowed to agglutinate or is treated to encourage agglutination. The treatment generally comprises spreading an aliquot of the suspension of particles on a suitable support, for example, a card or a piece of glass, and manually rotating and tipping the support or agitating the support in a single plane. Spreading the suspension may be done first, or may be accomplished by the rotating or shaking. In a preferred embodiment, the suspension from step (c) is transferred onto a card suitable for agglutination testing and the suspension is spread, typically with a small wooden or plastic spatula up to the edge of a ring printed on the card. The card may be rotated to allow the formation of aggregates which are then observed by eye. Alter- natively, a piece of glass, tile or plastics material may be used instead of a piece of card.

The suspended particles are simply observed by eye in step (e) to see if agglutination of the particles has taken place. Initially, the dispersion has a smooth appearance. If agglutination occurs, aggregates form and the dispersion becomes more granular. An advantage of the assay of the present invention is that it does not depend upon assessing agglutination indirectly by detecting a label carried by the particles, such as a fluorescent label. As indicated above, it has surprisingly been found that, when magnetic particles are employed, the rapidity of agglutination can be increased by applying a magnetic field to a sample of the particles after incubation with the sample. The particles may be in a dispersion from step (a), a suspension from step (c), or the liquid comprising the coated particles and the sample comprising the ligand may be admixed in situ on the support on which agglutination is to occur. As described above, the agglutination is generally carried out on a suitable support, for example, a card, a piece of glass or tile, or a similar flat support. The dispersion or suspension is preferably spread in a layer on the support. The support may be placed over a magnet, either a permanent magnet or an electromagnet. If desired, the support and/or the magnet may be moved relative to one another, or pulses of a magnetic field may be applied. Application of a magnetic field may be for from 5 to 20 seconds, depending upon the strength of the field. After removal of the magnetic field, agglutination may be observed. The applied magnetic field may serve, in effect, to pull the particles down onto the support and to aid agglutination.

As indicated above, all steps of an assay involving the application of a magnetic field to accelerate agglutination may be carried out in situ on the support on which agglutination is to be observed.

As indicated above, when the coated particles used in a direct or indirect assay of the invention are magnetic, the use of an applied magnetic field to accelerate agglutination may be carried out independently of or in conjunction with a particle retrieval step assisted by the application of a magnetic field. Assays according to the invention have wide applicability. They may be qualitative or semi- quantitative. They can be used to detect the presence or otherwise of a ligand in a sample. In cases where the ligand is present at only low concentrations where conventional agglutination tests would have failed to show agglutination, they can detect the ligand successfully. This allows detection and identification of an infectious agent in cases where the infection level is low or of antibody where the level of antibodies is low at the time of sampling. The assays of the invention also enable detection and/or monitoring of levels of haptens, for example, therapeutic drugs, drugs of abuse and certain hormones. There are certain therapeutic agents,, for example phenytoin, the level of which should be maintained within narrow limits. An indirect assay of the invention may be used to monitor the level of such agents.

Each of the assays according to the invention is typically run with a positive control, in which there is used a sample known to contain the ligand it is wished to determine. A positive control is used in order to ensure that the assay is functioning correctly and that the test components have not deteriorated during storage. It is an advantage of the methods, of the present invention that the agglutination may be determined by eye. It will be appreciated, however, that the methods of the present invention are not limited to detection of agglutination by eye, and that they may also be used in an agglutination assay where the particles carry a detectable label, and the label is detected by the appropriate method for that label. Suitable labels are well known, and include fluorescent, enzyme and radio- isotope labels. Detection of agglutination may be carried out by other known methods, for example, measurement of turbidity. The following Examples illustrate the invention.

Reference Example 1: Magnetisable latex

Rhone-Poulenc supplies polystyrene particles (called polystyrene latex) containing magnetic pigment and five preparations have been used in this work. (In the context of agglutination assays, the term "latex" is often used to denote particles suitable for carrying out an agglutination assay or, particularly, a dispersion or suspension of such particles in a liquid medium. The term "latex" will be used in the sense of a suspension of particles in the following Examples. )

10% w/v suspensions of magnetic particles were washed by the addition of glycine/saline buffer, which was 0.1M glycine in 0.15M saline with 0.05% v/v Bronidox and 0.01% w/v sodium dodecyl sulphate. The particles were separated from the buffer by attracting them to the side of the vessel using a strong magnet as supplied by Dynal U.K. (Magnetic Particle Concentrator MPC-1). The buffer was then removed using a disposable pipette and the latex suspended in fresh buffer to a final concentration of 1% w/v.

Example 1: Absorption of antibody to magnetic latex The latex was sensitised with antibody (i.e., the particles were coated with antibody) by methods commonly employed. Purified immunoglobulin from rabbit anti- streptococcus B was mixed with the latex suspension at two levels, 300μg IgG/ml latex and 450μg IgG/ml latex. The mixture was heated at 56°C for 30 mins. and then bovine serum albumin solution added at the rate of 33μl, 30% per ml latex suspension. After standing for 1 hour the latex was washed once by attraction to a magnet, removal of liquid and suspension to the same volume in glycine saline buffer.

Use of the magnetic latex

A pronase extract of a suspension of Streptococcus B organisms was diluted in glycine saline buffer containing 0.1% v/v bovine serum albumin and this provided the antigen to be detected. An aliquot of the antigen solution (1ml) was mixed with the magnetic latex with antibody attached (50μl) and left to stand (10 mins.). The test tube containing the suspension was inserted in the magnetic particle concentrator and the brown coloured latex migrated to the side of the tube. After 1 min. all the liquid was removed using a disposable pipette and the tube taken out of the magnetic device.

The magnetic latex was suspended by gentle agitation in fresh buffer (200μl) and then an aliquot of the suspension (50μl) transferred to one of the printed rings (2.5 cm diameter) .on a white card used for latex agglutination testing. The drop was spread on the card to cover the area within the ring using a wooden cocktail stick. The card was then rocked manually or rotated at 150 rpm for 2 minutes on a horizontal flat bed rotator. The card was then allowed to stand 1 minute before the pattern of the latex particles was observed. Agglutination of the brown particles was readily visible and was scored on a 1+ to 3+ scale, see Table 1 below. TABLE 1: DILUTIONS OF STREPTOCOCCUS B ANTIGEN DETECTED WITH WHITE LATEXES AND MAGNETIC LATEX

Dilution of Streptococcus B Antigen 5K 10K 20K 40K 80K 160K

White latex K 595310 2+ White latex K 9525 1+/2 White latex K 9208 2+

Magnetic latex D

Neg with no antigen concentration Magnetic latex D 2+ 2+ 2+ 2+ 1+/2 tr with antigen concentration using the new method

NOTE 1. All K numbers designate the lot numbers of production batches of white latex suspensions issued by Wellcome Diagnostics, Dartford, Kent; they were used within their useful life. Antigen dilution (40μl) was mixed with latex suspension (20μl) and spread on a black printed area (2.8 cm diameter) on a card, then rocked by hand (2 min) before observing for agglutination. Magnetic latex D with no antigen concentration was done in the same way except that a white printed area (2.5 cm diameter) on a card was used. K9208 white latex used the same antibody preparation as was used for sensitisation of the magnetic latex.

NOTE 2. With antigen concentration according to the present method, magnetic latex D gave the most clearly visible results; E was the next best giving clearly visible agglutination for a positive sample but a more granular negative than D. When magnetic latexes were used they were always applied to a white printed area (2.5 cm diameter) on a card.

NOTE 3. No advantage was obtained using the higher level (450 μg/ml) of antibody/latex suggesting that tne magnetisable latex surface had been saturated with antibody even at the lower level (300 μg/ml). NOTE 4. Neg = negative; tr = trace NOTE 5. 5K means a 5000 fold dilution of a pronase extract of a suspension of Streptococcus B organisms. Dilutions were made with 0.1% bovine serum albumin in glycine/saline buffer pH 8.2.

Time Study Very dilute antigen solutions were used to determine the time necessary for latex-antibody to combine with antigen in the immune incubation. Aliquots (1ml) of Strep B antigen (Streptococcus B antigen extract) at 80K and 160K dilution were distributed into tubes and magnetic latex D antibody (50μl) was added at time intervals to allow combination times of 1 ' , 2', 4' and 10'. After magnetic separation all the supernatant liquid was removed, the particles were suspended in fresh buffer (200μl), then spread onto a card which was rotated (2 min. ) and stood (l min. ) before reading and scoring. Allowing only 1 min. immune incubation gave a readily visible result and thereafter the agglutination became only slightly clearer with increased incubation times.

Rapid agglutination of magnetic particles As an alternative to hand rocking or machine rotation to see the agglutination of magnetic particles mixed with antigen on a card, it was surprisingly found that a magnet held under the card would speed the formation of an agglutination pattern. The procedures used were as follows:

(a) 30μl of magnetic latex D coated with the anti- Streptococcus B antibody described above were premixed in a test tube with 40μl of Streptococcus B extract des¬ cribed above and a sample was removed and applied to an agglutination card.

(b) 30μl of magnetic latex D were applied directly to an agglutination card and mixed with 30μl of the Streptococcus B extract in situ on an agglutination card.

In both cases, after spreading the mixture within the printed circle on a card the individual reaction areas were held over a strong magnet for approximately 10 sees, then removed and tipped gently then returned to the magnet.

On removal from the magnet, for the negative the aligned pattern relaxed to show a smooth suspension whereas for the positive agglutination was visible. Alternate gentle rocking of the card followed by application to the magnet gave rapid formation of an agglutination pattern for positive samples. When compared with machine rotation of cards for 2 mins at 150rpm in a horizontal plane this new method gave more rapid results with more easily read agglutination patterns. A rotating magnet gave no advantages over a stationary magnet.

The technique of using a magnet under the card on which the agglutination test is carried out was combined with the magnetic particle concentration method described above: 1 ml samples of the Streptococcus B antigen extract described above were mixed in a test tube with 30μl of magnetic latex D coated with of the anti- Streptococcus B antibody described above, incubated for 4 minutes at room temperature, and then put over strong magnet as described above. Most of the supernatant (830μl) was removed; the tube was taken away from the magnet and the particles suspended in the residual supernatant. An aliquot of the suspension (50μl) at the several dilutions of the antigen was transferred to two cards and agglutination induced either by rotation in a flat plane (150 rpm, 2 minutes) or by holding over a magnet as described above. Both of the methods showed comparable sensitivity, but the magnetic-assisted assay was more rapid.

Increasing volume of sample Using a very dilute solution of the Streptococcus B antigen extract at 160K, increasing volumes of sample (1ml, 2ml, 4ml and 6ml) were mixed with a fixed volume of magnetic latex antibody (50μl). After 2 min. the suspensions were put over a magnet; after separation and suspension in buffer (200μl) an aliquot of each was spread on a reaction card and then rocked (2 min.) and stood (1 min.). As the volume of sample increased the sensitivity improved until by 4ml the maximum had been reached.

Investigation of the dependence of agglutination on the presence of free antigen

Dilutions of the Streptococcus B antigen extract

(lml) were mixed with magnetic latex D antibody (50μl).

After incubating a maximum of 6 min. the tubes were put into the Magnetic Particle Concentrator to take the latex particles to the side. 3 different techniques were then used;

1. All supernatant was removed and discarded and the particles suspended in fresh buffer (200μl).

2. All supernatant was removed, the particles were suspended in fresh buffer lml and the tubes put into the Magnetic Particle Concentrator again. After 1 minute all the liquid was removed and the particles suspended in fresh buffer (200μl).

3. The majority of the supernatant (850μl) was removed and the particles suspended in the residual liquid. An aliquot (50μl) of the suspensions from each of the three techniques was transferred to and spread on a card which was then rotated (2 mins), stood (1 min) and read. When the three methods were run simultaneously the antigen gave the same titre. This suggested that agglutination on the card occurred independently of the presence of free antigen.

Example 2: Extension of the method to other antigen/- antibody systems Magnetic latex was sensitised with purified immunoglobulin with a high titre against meningo A,C,Y and W135 antigens using 680μg IgG/ml latex, heating at 56°C, 40 mins. then adding bovine serum albumin 33μl, 30% per ml latex. After standing 35 mins. the particles were washed twice with a large volume of glycine buffered saline and then taken up to give a suspension of 1% w/v latex in buffer.

The magnetic latex suspension was used in the same way as previously described mixing an aliquot (lml) of dilutions of the meningo A,C,Y and W135 positive control antigen in buffer with 0.1% BSA together with an aliquot (50μl) of the 1% magnetic latex antibody suspension. The mixture was stood for 2 to 3 minutes then the tubes put into the magnetic particle concentrator. After standing (1 min.) the liquid was removed and the solids suspended in buffer (200μl); an aliquot taken onto a latex card spread and then rocked for 2 mins. The card was then stood for 1-2 mins. before the agglutination was observed and the result recorded, see Table 2 below. TABLE 2: MENINGO A.Cϊ, W135 ANTIGEN DETECTED WITH WHITE

LATEXES AND MAGNETIC LATEX

Dilution of meningo A,C,Y, W135 positive control

512 White latex K8362 White latex K9214 Magnetic latex D

with no antigen concentration Magnetic latex D 3+ 3+ 2+ 1+ using the new method

NOTE: White latex K8362 used the same preparation of antibody as was used to sensitise the magnetic latex. Also see Notes to Table 1 for a description of the methods used for comparative testing.

Example 3: Use of magnetic latex for the detection of antigens from meningo B, streptococcus pneumoniae and haemophilus influenzae B

Magnetic latex D at 1% w/v was coupled to immunoglobulin preparations from sera specific to Neisseria meningitidis group B, Streptococcus pneumoniae and Haemophilus influenzae B. Antibody was offered at 450μg/ml latex and heated at 56"C for 30 minutes in the presence of 0.1M glycine buffered saline pH 8.2. Bovine serum albumen 30% (33μl/ml) was added and the mixture stood 30 min.; the particles were washed twice by attraction to the side of the vessel by magnet, removal of the liquid and suspension in glycine buffered saline containing bovine serum albumen 0.1%. The magnetic latex-antibody particles were finally taken to 1% w/v suspensions and tested with their homologous antigens. The sensitivities achieved are detailed in Tables 3, 4 and 5 below. TABLE 3: MENINGO B ANTIGEN DETECTED WITH WHITE LATEXES AND MAGNET LATEX

Dilution of positive control antigen 16 32 64 128 256 ZL24 White Latex K8221 1+ tr Neg ZL24 White Latex K8686 2+ 1+ tr Magnetic latex using the new method 2+ 1+ tr Neg

TABLE 4: STREPTOCOCCUS PNEUMONIAE ANTIGEN DETECTED WITH WHITE LATEXES AND MAGNETIC LATEX

Dilution of positive control antigen

256 ZL22 White Latex K9212 ZL22 White Latex K8223

Magnetic latex using the new method 3+ 3+ 1+ tr

TABLE 5: HAEMOPHILUS INFLUENZAE B ANTIGEN DETECTED WITH WHITE LATEXES AND MAGNETIC LATEX

Dilution of positive control antigen 16 32 64 128 256

ZL21 White Latex K8681 2+ 1+ tr Neg ZL21 White Latex K8216 2+/3 1+/2 tr tr Magnetic latex using the new method 2+ 2+ 1+ tr

Example 4: Use of a 3 colour latex product to give sensitive assays, concentrating by centrifugation

The salmonella latex product Wellcolex (Trade Mark) containing red, blue and green coloured latexes sensitised individually with three different antibodies, was used to discover whether concentration by centri¬ fugation would give a sensitive test. All three of the antigens were diluted in 2 fold dilutions using glycine buffered saline containing bovine serum albumen 0.1%. For direct agglutination, product latex (1 drop, 30μl) was added to dilutions of the antigens (40μl) on the white card printed with circles (2.5 cm diameter). The pairs of drops were mixed with a wooden cocktail stick, the mixture was spread to the edge of the printed circle and the card rotated in a horizontal plane by machine at 150rpm for 2 min. Agglutination was scored on a 1+ to 3+ scale. For the centrifugation method, latex (1 or 2 drops, 30 or 60μl) was added to dilutions of antigen (lml) distributed in Sarsted Centrifugable Reagent tubes; Cat No. 72.690/478 and mixed thoroughly. After standing (1 min) the tubes were centrifuged at 6,500 rpm for 2.5 min. in an MSE Micro Centaur centrifuge.

The majority of the supernatant (920μl) was carefully removed and discarded whilst the latex was suspended in the remaining drops of liquid by vibration on a Gallenkamp Spinmix (Trade Mark). An aliquot (70μl) of the suspension was transferred to the reaction card and spread with a wooden cocktail stick; the card was rotated at 150 r.p.m. for 2 min. by machine and then the agglutinated colour patch scored; see Table 6 below.

TABLE 6: SALMONELLA Vi ANTIGENS DETECTED BY THE 3 COLOUR LATEX WITH OR WITHOUT CONCENTRATION BY CENTRIFUGATION

Dilutions of Vi antigen 32 64 128 256 512 1024 Centrifugation 2+ 2+ 2+ 1+ Control 2+ 2+ 1+ tr Dilutions of G antigen

4 8 16 32 64 128 256 Centrifugation 2+/3 2+ 1+/2 1+ tr

Control 2+ 1+ tr Dilutions of A antigen

4 8 16 32 64

Centrifugation 2+ 2+ 1+

Control 2+ 1+ Neg

Result

The scores in Table 6 show that an 8 fold increase in sensitivity is attained for all 3 antigens tested using concentration by centrifugation from a 1 ml sample.

Example 5: Identification of bacterial antigen in a clinical sample with increased sensitivity using centri¬ fugation of white latex

Two small samples of cerebrospinal fluid (CSF) were received from Great Ormond Street Hospital where laboratory staff were fairly certain that the child had meningitis caused by Streptococcus pneumoniae or possibly by Haemophilus influenzae, but they were unable to demonstrate antigen with a Wellcogen (Trade Mark) Kit. Gram film showed very scanty Gram negative diplococci. We were asked if we could concentrate the CSF samples and retest them. A sample in a glass bottle had been boiled and that in a plastic bottle was- untreated. Both samples were supernatants.

Instead of concentrating the sample by ultra- filtration, the new method was employed. The boiled sample (200μl) was mixed with Streptococcus pneumoniae latex (30μl) and left 10 min., centrifuged 6,500 rp for 2.5 min in a MSE Micro Centaur Centrifuge and the supernatant taken on for a further manipulation adding Haemophilus influenzae latex (30μl) waiting 10 min. then centrifuging and removing the supernatant. The latex pellets were suspended in saline (60μl), transferred to a card and spread with a wooden cocktail stick. The card was hand rotated for 3 min. then the agglutination pattern was scored as follows:- 1. S. Pneumoniae tr/l+

2. H. Influenza Neg

The unboiled sample was tested for non-specific agglutination with a control latex before and after boiling.

Before boiling + after boiling

The freshly boiled sample was then taken through the above procedure but with the test latex suspensions used in reverse order. After rotation on the card the result was:-

1• H. Influenza Neg 2. S.Pneumoniae 1+

Result The agglutination patterns suggested that the child's meningitis was associated with the presence of antigen from Streptococcus pneumoniae. The efficacy of the new procedure was amply demonstrated when previously undetectable antigen was concentrated and detected in a clinical sample.

Claims

1. A method of determining a ligand in a liquid sample, which method comprises:

(a) incubating a first liquid medium comprising the sample and having dispersed therein either particles coated with a substance capable of binding to the ligand or particles coated with the ligand, an analogue of the ligand, or a conjugate comprising the ligand or an analogue of the ligand, the first liquid medium also comprising a substance capable of binding to the ligand, the sole particles in the medium that are coated being the said particles;

(b) retrieving the thus-incubated particles; (c) suspending the retrieved particles in a portion of the first liquid medium or in a second liquid medium;

(e) observing by eye whether the treated particles have agglutinated.

2. A method according to claim 1, wherein the particles are magnetic particles.

3. A method according to claim 2, wherein a magnetic field is applied in step (d) and thereafter the particles are inspected for agglutination.

4. A method according to claim 2 or 3 , wherein the particles are retrieved in step (b) by applying a magnetic field.

5. A method according to claim 1, wherein the particles are not magnetic.

6. A method according to any one of claims 1 to 3 or to claim 5, wherein the particles are retrieved in step (b) by centrifugation and the supernatant resulting — Δ x5 — from the centrifugation is wholly or partially removed.

7. A method according to any one of the preceding claims, wherein the particles are polystyrene particles.

8. A method according to any one of the preceding claims, wherein the ligand is an antigen and the substance capable of binding thereto is an antibody.

9. A method according to claim 8, wherein the antigen is a viral, bacterial, fungal, or parasitic antigen, or is a naturally occurring human or animal host protein antigen.

10. A method according to claim 9, wherein the antibody is capable of binding to an antigen characteristic of Streptococcus spp. , Neisseria spp. , Streptococcus pneumoniae, Haemophilus influenza B or Salmonella, human immunodeficiency virus-1, human immunodeficiency virus-2, hepatitis A, hepatitis B or hepatitis C.

11. A method according to any one of claims l to 7, where the ligand is an antibody and the substance capable of binding thereto is an antigen, or the ligand is a target molecule for a receptor and the substance capable of binding thereto is the receptor or a part thereof.

12. A method according to claim 11, wherein the antibody is specific for a human immunodeficiency virus or for hepatitis A, hepatitis B or hepatitis C.

13. A method according to any one of the preceding claims, wherein the suspension from step (c) is transferred onto a moveable flat support suitable for agglutination testing and a layer of the suspension is formed on the surface of the support.

14. A method according to any one of claims 1 to 7, wherein the assay is an indirect assay and the ligand is a therapeutic drug, a drug of abuse, or a hormone.

15. A method of determining a ligand in a liquid sample, which method comprises:

(i) incubating a liquid medium comprising the sample and having dispersed therein magnetic particles coated with a substance capable of binding to the ligand or magnetic particles coated with the ligand, an analogue of the ligand, or a conjugate comprising the ligand or an analogue of the ligand, the liquid medium also comprising a substance capable of binding to the ligand, the sole particles in the medium that are coated with the said substance being the said particles;

(ii) applying a magnetic field to the incubated medium or to a sample of the incubated medium; and , (iϋ) thereafter determining agglutination by eye.

16. A method as claimed in claim 15, wherein the ligand is as defined in any of one of claims 8 to 12.

17. A method as claimed in any one of claims 14 to 16, wherein the assay is an indirect assay and the ligand is a therapeutic drug, a drug of abuse, or a hormone.

18. A method as claimed in any one of claims 14 to 17, wherein the liquid medium and the sample are admixed and incubated and agglutination is observed in situ.

19. A method as claimed in any one of claims l to 17 wherein, instead of or in addition to determining agglutination by eye, agglutination is determined by another detection method.

20. A method as claimed in claim 19, wherein agglutination is determined by measurement of turbidity, or wherein the particles are labelled and the label is determined.