Agglutination method for detection of ligands and kit therefor

The present invention relates to an agglutination method for the detection of a ligand in a biological liquid medium and to a reagent for use in this method.

Diagnostic test methods based upon the agglutination of immunogens and antibodies, wherein either the immunogen or the antibody is attached to a solid phase, are well known in the field of immunodiagnostic reagents. For example, US Patent 3080875 describes a technique wherein 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 thereby causing visible agglutination or aggregation of the microspheres.

Coloured solid phases or particles have been used to aid visualisation of the agglutination process. For example, there are marketed test kits for the grouping of Beta Haemolytic Streptococci which include reagents in which the solid phase is a suspension of killed red-dyed or blue-dyed Staphylococcus aureus cells. There is also marketed a test kit containing *our separate reagents, each containing a different colour of latex particle, for the detection and grouping 0* streptococci A,B,C and G. By assigning a specific colour to each test reagent any confusion which could be caused by eg incorrect labelling is avoided.

US Patent 4419453 describes a latex agglutination test in which the test reagent comprises antigen or antibody coated latex particles of one colour and a watersoluble ηοπ-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.

A diagnostic technique such as a latex agglutination test is generally used to reinforce an initial diagnosis based upon the clinical symptoms exhibited by a patient suffering from a particular disease. In cases where a disease is characterised by very distinctive symptoms, confirmation of the presence 0* a causative agent (eg bacteria or viruses) could involve relatively few tests. However, with diseases in which the symptoms could be ascribed to any one of a large number of causative agents, it will be apparent that much time and effort

A718 could be expended in performing a test against each of the possible causative agents and that the size of sample required from the patient could be considerable. This could be a serious problem in instances (eg when the test fluid is neonatal cerebro-spinal fluid (CSF)) where it is only possible to take small samples of biological material from the patient.

Heterogeneous specific binding assays are known wherein a plurality of ligands can be determined simultaneously in the single test sample thereby reducing the number of tests needed. Such combined assays are considered (see UK Patent application 2 034 466A) to be of particular advantage where the assays are of a screening nature, for example in- the diagnosis of immunity to viruses and other antigens responsible for congenital malformations such as Rubella, Cytomegalovirus and Herpes simplex virus. A combined assay could indicate the patient’s immunity against two or more of these antigens, in a single test by detecting the presence of antibodies to each of the antigens. UK Patent application 2034466A describes such an assay wherein the ligands are differentiated by the use of a number of differentially separable solid phases. Such solid phases can be, for example, a plate coated with one immunologically active substance in a container the walls of which are coated with another immunologically active material. Following the appropriate series o* immunochemical reactions the ligand-immunochemical complexes can be separated simply by removing the plate from the container.

Further combined assays are known where, for example, different specific binding substances are linked to particles of different size (UK Patent 1561042), or where microscopically distinguishable rosettes are formed between ligand and binding substance (UK Patent Application 2122345), and where each specific binding substance is linked to a latex particle distinguishably labelled with a radioactive substance or an element detectable by eg X-ray fluorescence spectroscopy (US Patent 4436826).

The abovementioned methods for determining more than one ligand suffer from the disadvantages of either requiring complex instrumentation or requiring mechanical separation of the various types of solid phase or of being inapplicable as a general means of detecting immunogens, antibodies and other specifically bindable substances. The above methods in general also take longer to perform

MRH/OLM/24th July 1985

76307/2 than agglutination tests. Furthermore, those assays which employ radioactive or heavy metal elements have the additional disadvantage of requiring special safety and waste disposal procedures. It is thus apparent that there exists a need for a technique which is rapid, safe, simple and broadly applicable and which can significantly reduce the number of tests required to identify the causative agent of an infection or disease thereby reducing the volumes of biological material required to be taken from a patient in order to carry out those tests. There has now been discovered such a technique.

Accordingly, the present invention provides in a first aspect an agglutination method for the detection of a ligand in an aqueous biological liquid medium comprising the steps of:

A.

mixing a sample of the said medium with a reagent selected from the group consisting of

i) particles of a first colour having attached thereto polyclonal antibody bindable to the said ligand, in admixture with particles of a second colour having attached thereto immunoglobulin from a non-inoculated animal of the same species as that in which the said polyclonal antibody was raised, and ii) particles of a first colour having attached thereto a first monoclonal antibody bindable to the said ligand, in admixture with particles of a second colour having attached thereto a second monoclonal antibody derived from a non-inoculated animal of the same species as that from which was derived the said first monoclonal antibody.

whereby non-specific interaction between the said sample and the said reagent is indicated by agglutination of both the said

76307/2 particles of the first colour and the said particles of the second colour and the presence of the said ligand in the said sample, free from said non-specific interaction, is indicated by agglutination of the said particles of the first colour alone;

B. after an appropriate period of time, optically inspecting the mixture resulting from step A.; and

C. determining the test result from the appearance of the said mixture.

The present invention provides in its second aspect a test reagent useful for use in the said method.

The colour of the agglutinate formed in the presence of one particular ligand is different from that of any agglutinates formed in the presence of the other ligand and that it is distinguishable from the background colour, i.e. the colour due to any unagglutinated particles. The distinctive colour of the agglutinate is due to each insoluble substance being different in colour from the other insoluble substances. The agglutinate is preferably visible to the naked eye.

The term ligand, as used in the context of the present invention includes antigens, haptens, monoclonal and polyclonal antibodies and other substances capable of being bound by a specific binding substance. Such other substances include avidin, biotin, lectins, carbohydrates specifically bindable to lectins, Protein A and the FC fragment of igG.

The biological liquid medium may be a biological liquid sample such as a body fluid taken from an animal, human or otherwise, or it may be any other type of liquid medium in which a ligand may be found. Such media can include for example culture broths, suspensions from liquid or solid growth media, culture

76307/2 supernatants, tissue culture supernatants, enzyme or chemically extracted material from bacteria and viruses (e.g. Lancefield extracts for serological grouping of Streptococci), foodstuffs or environmental samples (e.g. water samples from the public supply).

Biological samples which can be taken from animals include cerebrospinal fluid, blood, urine, sputum, tissue extracts, sweat, tears, secretions, faeces, mucus and synovial fluid.

The above list is not intended to be exhaustive and the skilled man will appreciate that other types of biological samples may be taken and tested by the method of the present invention.

The colour particle is preferably of a microscopic size. Particulate materials which are coloured, or can be dyed, and are suitable for use in the abovementioned assay include non-viable bacterial cells, alginate particles, Sepharose beads, silica, alumina, erythrocytes, polymer latexes such as polystyrene latexes, styrene-glycidyl methacrylate latex and other polymer latexes such those described in US Patent 4419453. Coloured particles may be prepared or dyed according to standard methods, see for example US Patent 4419453 and German published Patent Application DT-3000-483, or they may be purchased from an appropriate source. Particularly suitable colours include red, yellow, blue, green, black, cyan, magenta and white.

The antibody is linked to the particle by adsorption, by chemical coupling, by incorporation into the particle or by any other method known in the art.

Adsorbing or coating the antibodies onto the colour particles is typically achieved by incubation of the particles with a suitably buffered solution of the antibodies. Chemical coupling can be achieved for example by the method described in US Patent

76307/2

4140662.

The present invention is especially useful for the detection of bacterial, viral or parasitic infections and the identification of antigen or antibody in biological fluids. It is particularly useful in the analysis of spinal fluid (e.g. neonatal spinal fluid) for such species as Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. An important advantage of the present invention is the reduced volume of spinal fluid required for analysis compared with more conventional agglutination test methods.

The present invention is also useful for the identification of serologically distinct strains e.g. Streptococcal serogroups A, B. C, D, F and G, Salmonella 0 or H antigens and Meningococci serogroups A, B, C, Y, 29E and z.

In one particularly preferred embodiment of the present invention, the reagent comprises a suspension of antibody coated latex particles of two colours, one bound to said first antibody and the other to said second antibody, the colour of each particle indicating the particular specific antibody with which it is coated. The overall appearance of the reagent before reaction is a dull or greyish colour. Following admixture of the reagent with a medium containing an antigen capable of being bound to the said first antibody, the said antigen will react with the appropriate antibody causing formation of an agglutinate. It will be apparent from the colour of the agglutinate which antibody has taken part in the reaction and hence the identity of the antigen will be revealed. Visualisation of the agglutinate is enhanced by the colour contrast with the background colour provided by the non-agglutinated particles.

Non-specific interactions, such as those due to the presence of interfering substances such as Rheumatoid Factor (RF) or Protein

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A (found on most S. aureus bacteria) and Protein A-like substances (found on some streptococci) will tend to show up as dark clumps on a lighter background of similar hue.

In the methods according to the present invention, it is desirable in certain instances to pre-treat the medium before testing; such methods of pre-treatment include treatment with acids, or enzyme extractions, and also filtering, centrifuging, diluting, concentrating and heating. By heating, for example, it is possible to deactivate or significantly reduce the activity of the abovementioned interfering substances.

In conventional agglutination techniques, it is usual to employ a control latex which is a suspension of latex particles coated with the immunoglobulin fraction (hereinafter referred to as the control antiserum) of an animal that has not been inoculated with the antigen under test, or a monoclonal antibody of the same class as that used on the test latex but having a different specificity. Agglutination of the control latex in the presence of a test sample indicates a non-specific interaction.

The drawback to using a control latex reagent in conventional procedures is that at least one additional aliquot of biological test fluid is required for each test or series of tests, this additional aliquot subsequently yielding no useful information as to the identity of the infecting agent. The disadvantageous requirement for a separate control latex reagent has been overcane by the present invention.

For example, in a typical test procedure, the test sample is mixed with a reagent containing blue latex particles coated with antibody to the antigen to be detected and red latex particles coated with a control serum. If the suspected antigen is present and there are no non-specific interactions, a blue agglutination will be formed against a red background. If non-specific

76307/2 interactions do occur, both red and blue particles will agglutinate giving rise to clumps of a purplish colour. It will be apparent to the skilled man that although red and blue particles are described by way of example, any two contrasting colours could be used in place thereof.

The reagent of the invention may be provided in the form of a kit. Other items which can usefully be included in the kit include spotting cards, mixing sticks, a positive control antigen for the antigen being tested, negative control antigen (e.g. saline solution) and a set of instructions for use of the test kit.

The present invention will now be illustrated by means of examples. The examples should not be construed as imposing a limitation on the scope of the

EXAMPLE Preparation of Sensitised Latex

I Preparation of antibody.

Immunoglobulin G was obtained partially purified from immune rabbit sera by treatment with octanoic acid (BDH Chemicals Ltd.) using the method of Steinbach and Audran (Arch. Biochem. and. Biophys, 134, 279284, 1969).

Π. Binding of antibody to coloured latex.

To 5.0mg of Coloured latex (Estapor, K58, 0.2p, polystyrene, black, red, blue, yellow or green, EP-A 0 085 016)was added 600pg. of antibody in 1ml. of glycine .saline pH 8.2 (0.1M glycine in 0.85% NaCl, pH adjusted to 8.2 with NaOH). Latex and antibody were heated at 56°C for 30 mins. After cooling to room temperature bovine albumin (Miles Laboratories Ltd.) was added to give a l%(w/v) concentration.

Ill, Preparation of polyvalent latex.

Three different coloured latexes each one sensitised with antibody of a different specificity eg. red latex coated with antibody to Salmonella serogroup A, blue latex coated with antibody to serogroup B and green latex coated with antibody to serogroup C, were mixed together in equal proportions. The resultant latex was brown in colour.

EXAMPLE 2 Use of the polyvalent Latex.

Latex agglutination tests were performed on white cards (Syfacard -R, Wellcome Diagnostics Ltd.). Equal volumes of the test sample and the polyvalent latex (usually 20yl) were mixed together on a circle, diameter 2cm., on the white card. The card was rocked for 3 mins after which the sample was examined for agglutination. The colour of the agglutinate was identified eg. red, blue or green and at the same time the colour of the unagglutinated latex changed from brown to a combination of the colours of the two particles remaining unagglutinated in suspension.

(a) Bacterial colony, identification.

A single colony of bacteria was removed from a solid growth medium and emulsified in 200pl of 0.85% saline. The bacterial suspensions were mixed with the polyvalent latex as described above.

ANTIGEN

1. Saline only.

2. Salmonella Serogroup A eq. S.paratyphi A

3. Salmonella Serogroup B eg. S.typhimurium

RESULT

Brown homogenous solution.

red agglutinate in a turquoise solution blue agglutinate in an orange solution.

4. Salmonella Serogroup C eg. S.newport green agglutinate in a purple solution.

(b) Antigen detection in biological fluids.

’ Spinal fluid taken from a patient with meningitis was tested, as described above, against the Salmonella polyvalent latex. Agglutination of the red latex particles occurred indicating the presence of antigen from Salmonella serogroup B organisms in the spinal fluid. Spinal fluid from an uninfected person did not cause agglutination of the latex.