AU645970B2 - A method for the determination of antibodies against organisms causing infectious diseases in body fluids, agents for this purpose and the use thereof in this method - Google Patents

Abstract

The invention relates to an immunochemical competitive method for the detection and determination of antibodies against bacteria, viruses, parasites and fungi. It is also possible, with a variant of the described method, to detect the causative agent in body fluids. Also described are assay kits for carrying out the method and the use thereof.

Description

64 5'970 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class In Application Number: Lodged: Form it. Ciass Complete Specification Lodged: Accepted: Published: 9 09*O** 9 0ees S S S. S

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0 Priority Related Art Name of Applicant: BEHRINGWERKE AKTIENGESELLSaIAET

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S. Address of Applicant: D-3550 Marburg, Federal Republic of Germany Actual Inventor: :Address for Service: HEUvIUT PETERS and UDO KRUPKA WATERMARK PATENT TRADEMARK ATTORNEYS.

LO(CKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: A METHOD FOR THE DETERMVINATION OF ANTIBODIES AGAINST ORGANISMS CAUSING INFECTIOUS DJ1SEASES IN BODY FLUIDS, AGENTS FOR THIS PURPOSE AND THE USE THEREOF IN THIS MET'HOD Thie following ,tatement is a full deicription of this invent~on, including the best method of performing it known to us BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 013 Ma 769 Dr. Pfe/Zi A method for the determination of antibodies against organisms causing infectious diseases in body fluids, agents for this purpose and the use thereof in this method The invention relates to an immunochemical, competitive method for the detection and determination of antibodies against bacteria, viruses, parasites and fungi. It is also possible, using a variant of the described method, to detect the causative organism in body fluids. Furthermore, test kits for carrying out the method and the use S•thereof are described.

15 A special embodiment permits the quantitative detection *1 of antibodies, especially in the case of syphilis.

The importance of reactivation of latent syphilis in HIV patients, who frequently have double infection, has recently been recognized. The diagnosis of neurosyphilis is particularly difficult because of the special immunological situation in such patients. There is a wide variety of detection methods for syphilis antibodies, reflecting the fact that diagnostic involvement started early (in 1906) in the case of this causative agent, which had enormous importance in health policies even at the start of this century. Three categories can be distinguished among the detection methods for syphilis antibodies: I. Tests which are not Treponema-specific. Examples: The Wassermann reaction or cardiolipin CFR, VDRL test or cardiolipin microflocculation test, RPR test: E.L. Reed, Public Health Lab. (1965) 23:96- 103, VDRL ELISA: N.S. Pedersen, 0. Orum and S.

Mouritsen, J. Clin. Microbiol. (1987) 25:1711-1716.

2 II. Treponema-specific tests, for example the Reiter CFR or the so-called flagellum ELISA Van Eijk, H.E. Menke, G.J. Tidemann and E. Stolz, Genitourin.

Med. (1988) 62:367-372).

III. T. pallidum-specific tests, for example the Nelson test pallidum immobilization test), the fluorescent treponemal antibody (FTA) test Hunter, W.E. Deacon and P.P. Meyer, Publ. Hlth. Rep. (1964) 79:410-412), the Treponema pallidum hemagglutination assay (TPHA: T. Tomizawa and S. Kasamatsu, Jap. J.

med. Sci. Biol. (1966) 19:303-308).

Indirect enzyme immunoassays for detecting IgG or IgM are based on the binding of specific antibodies to T. pallidum antigen preparations which are bound (asually by adsorption) to solid phases.

S" The bound antibodies are detected by means og enzyme-labeled second antibodies directed against human IgG or IgM. The indirect ELISA method for detecting syphilis IgM is described in US Patent No. 4,288,426.

In summary it is possible to say that none of the tests described in the literature or available commercially meet the requirements to be met by a screening test in terms of acceptable technical elaboration, objective and quantitative test evaluation, substantially automatable procedure and the necessary high degree of sensitivity and specificity.

A competitive ELISA for the quantitative detection of antibodies against HIV is described in European Patent Application EP-A 0,265,851.

A specific embodiment of the competitive ELISA for quantification of rubella virus in cell culture is described by Y. Varela, E. Ortega and B. Gomez, J. Virol.

3 Meth. (1988) 19:79-87. These authors do not, however, use directly-labeled rubella-specific antibodies. WPI 86-130689/20 describes the use of the competitive assay principle for detecting rubella antibodies.

The object on which the invention is based comprised the development of a highly specific and highly sensitive detection for antibodies against bacteria, viruses and parasites. This test was intended to permit small and large series of tests to be carried out both manually and substantially automated, for example with the aid of the Behring ELISA Processor II. As a screening test, it vws especially intended to make possible, taking account of the low prevalence of the diseases, a reliable positive/negative discrimination. It is essential in this *:15 connection that such a test alo detects with high reliability the particularly infectious early primary stage when the IgG titer is usually low. On the other hand, the test to be developed had to have a high S* specificity in order to minimize the rate of falsepositive results, which can be verified only using more elaborate tests. It has now been found that a competitive method for the detection and determination of antibodies against the organisms causing syphilis, toxoplasmosis, rubella, cytomegaly, amebiasis, echinococcosis, tetanus and whooping cough, both of the IgM and of the IgG class, is possible, on use of a solid phase to which the antigens, which can also be contaminated to a certain extent, are bound directly, that is to say without antibody-mediated binding, if an appropriately selected conjugate antibody is used.

It was possible to show in experiments with pure IgG-free IgM preparations that specific IgM antibodies also significantly reduce the measured signal (Fig. 1; samples 1, 2 and 3).

The invention relates to an immunochemical competitive method for the detection and determination of antibodies 4 against the organisms causing syphilis, toxoplasmosis, rubella, cytomegaly, amebiasis, ochinococcosis, tetanus and whooping cough by use of a solid phase composed of a carrier and, irreversibly bound thereto, antigens of the particular causative organism and labeled antibodies directed against the essential antigens of the particular causative organism, there being competition between the antibodies for the binding site on the solid phase, wherein the proteins are irreversibly bound to the carrier directly or via a non-immunochemically binding spacer.

A method for the detection and determination of antibodies against the organisms causing syphilis, toxoplasmosis and rubella is preferred.

:.15 The invention also relates to a test kit for the detection aid determination of antibodies in an immunochemical 0e competitive method as described above, containing a carrier to which the antigens of the particular causative agent are bound directly or via a non-immunochemically binding spacer, labeled antibodies against the said membrane proteins and, where appropriate, reagents for detecting the labeling.

The invention furthermore relates to the use of the abovementioned test kit in a method for the detection and determination of antibodies against the organisms causing syphilis, toxoplasmosis, rubella, cytomegaly, amebiasis, echinococcosis, tetanus and whooping cough.

The invention also relates to a method, as described above, in which the signal reduction is linearly related to the logarithm of the titer.

The invention furthermore relates to a method for the detection and determination of the organisms causing syphilis, toxoplasmosis, rubella, cytotiLegaly, amebiasis, echinococcosis, tetanus and whooping cough, where the 5 sample is initially preincubated with the conjugate antibody in an uncoated vessel and then transferred into the measuring vessel coated with antigens of the particular causative organism.

The titration plots for syphilis-positive sera are linear over a wide range of titers and are parallel to one another (Fig. 1).

Titration plots are known to have this property in indirect ELISAs.

Hyperimmune sera from animals show a comparable linearity and parallelism and are suitable as a basis for •international standards. For quantitative comparison of the antibody content of various samples, the so-called 50 titer was determined, for example, i.e. that serum :15 dilution at which 50 of the signal of the negative go control is reached (Fig. The signal reduction on •testing the concentrated serum sample correlates with its specific antibody titer. It has emerged, surprisingly, that when the method according to the invention is used, 20 the signal reduction (as of the negative control) is ooo* linearly related to the logarithm of the titer (Fig. 2).

This property is made use of in a specific embodiment (see Example 2) for quantification of syphilis antibodies. Since the only sera to display a 50 titer are those which reduce the measured signal to less than 50 of the negative control on testing of the concentrated samples, the ordinate (signal reduction as of the negative control) shows only a measurement range from 50 to 100 signal reduction. More than 95 of the samples hitherto investigated fall within this measurement range.

The linear relation between the signal reduction and the logarithm of the titer no longer applies to serum samples which display a signal reduction o;E more than 90 (see also Fig. After the screening test (with undiluted -6 serum), such samples must be retested once again after prior dilution (for example 1%20). If at least 2 standard sera (or 2 different dilutions of a standard serum) are included when the techniqwo is carried out quantitatively, it is possible to determine the titer or antibody content in international units by linear interpolation as shown in Annex 1. This linear interpolation can also be carried out by computation and, in the preferred mode of evaluation, with the aid of a computer program.

It has furthermore been observed on titration of serum samples and testing by the described method that the titers from the competitive ELISA are linearly related to those from the TPHA (Fig. 3).

The high sensitivity of the test was demronstrated by 'e~g :testing a large number of clinicall~y defined serum samples from patients in all stages of Otyphilis (Fig. 4).

The high specificity is demonstrated by the negative reaction of samples from autoiznmune patients, from borreliosis patients and from a large number of healthy blood donors (Fig. 4).

It has furthermore been observed, surprisingly, that in a special embodiment, namely by preincubation of the sample with the conjugate antibody in an uncoated vessel and subsequent transfer into the vessel, it is possible to detect antigen. It has been possible to verify by experiments that soluble bacterial equivalents significantly reduce the measured signal in this test mixture (Fig. The qualitative version of the competitive ELISA makes it possible, due to its simple technical procedure in particular, but not exclusively, to test large amounts of samples within about 3 hours. Because of the additional detection of specific IgM, even in the absence of specific IgG, very sensitive detection is possible even in 7 very early stages of infection. In a special embodiment, i.e. after preincubation of the sample with the PODlabeled antibody and subsequent transfer into the measuring vessel, it is also possible to detect antigens.

The quantitative version of the competitive ELISA provides data which are important, for example in syphilis diagnosis, for assessing the stage of the disease or assessing the result of antibiotic therapy (besides the IgM-specific tests). The good correlation with the TPHA must be emphasized in this connection.

The utility of this principle has furthermore been demonstrated for the detection of antibodies againt Toxoplasma gondii and against rubella virus. It is regarded as being particularly advantageous that it is O* :15 possible to improve the specificity of the test by the choice of the conjugate antibody and, for this reason, to dispense with elaborate extensive purification of antigens in certain circumstances.

Antigen preparations suitable for the solid phase can be obtained by methods known per se, for example Treponema pallidum according to WO 83/02886. This entails the soO. Treponema being obtained from the testes of infected rabbits and extracted with the non-ionic detergent noctyl glucoside.

Suitable carrier materials for the solid phase are o0.0 plastics such as polystyrene, polyvinyl chloride, polyamide and other synthetic polymers, natural polymers such as cellulose, as well as derivatized natural polymers such as cellulose acetate and nitrocellulose, also glass, especially glass fiber.

The carriers can be in the form of microparticles (0.01 to 1 pm), beads, rods, tubes and microassay plates.

Sheet-like structures such as paper strips, small plates and membranes are likewise suitable. The surface of the carriers can be both permeable and impermeable to aqueous 8solutions.

Preferred carriers are beads, microparticles, tubes, paper strips and membranes. Particularly preferred carriers are microassay plates.

The solid phase is produced by irreversibly binding an antigen preparation to the carrier.

An irreversible binding within the meaning of the invention is present, for example, in the case of 1) binding by adsorption which is not broken by immunochemical agents such as high-affinity antibodies nor by the agents used in the method, such as labeled

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antibodies, and dilution and buffer solutions, 2) bioaffinity binding mediated by a non-immunochemically binding spacer, it being possible for the spacer to be composed of biotin and avidin or from other conjugates of receptors and ligands, 3) a direct covalent bond *SO: I 0* 9 4) a covalent bond mediated by a bifunctional chemical spacer.

Covalent bonding is preferred in the case when water-permeable carriers are used, and binding by adsorption is preferred in the case when water-permeable and water-impermeable carriers are used.

Direct binding by adsorption of antigen preparations onto polystyrene treated with gamma rays as carrier is particularly preferred.

The starting material used for the conjugate antibody is a serum pool from donors with positive serology for the particular causative organism. Monoclonal antibodies are 9 likewise suitable.

It is possible to use as markers radioactive isotopes, fluorescent and chemiluminescent dyes, and enzymes which can be detected by chromogenic, luminogenic or fluorogenic substrate systems.

The labeling is effected by methods described as state of the art for the said markers.

In the case where the antibodies are labeled with peroxidase it is possible to use the periodate technique of Nakane et al., i974, J. Histochem. Cytochem. 22, 1084-1090, or a method described by Ishikawa et al., 1983, J. Immunoassay 4, 209-327, in which the partners are linked by a heterobifunctional reagent.

The method according to the invention can be carried out, .15 for example, by introducing into the cavities (wells) of a microassay plate which contains a bound antigen preparation described above a) simultaneously the sample and a solution of the labelled antibody where appropriate first the sample and after a certain time a solution of the labeled antibody, removing the solution after a defined incubation time, and washing the wells with a buffer and subsequently measuring the label in the wells, or b) first a solution containing the labelled antibody, removing the solution after a defined incubation time, and washing the wells with a buffer, where appropriate drying the microassay plate and subsequently adding the diluted sample and incubating for a defined time, then removing the sample from the wells and measuring the label in the sample.

10 The method according to the invention can also be carried out using a diagnostic element which contains the solid phase and, in dry form, a part or else all the reagents required.

In a preferred embodiment, the method according to the invention is employed for the detection and determination of causative organisms. This entails, for example, first the sample and a solution of the labeled antibody being incubated in an uncoated cavity of an microassay plate.

After a defined time, the preincubated solution is transferred into a cavity coated with antigen. After a defined incubation time, the solution is removed, the cavity is washed with a buffer where appropriate, and subsequently the label in the cavity is measured.

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,15 The examples which follow are intended to illustrate the Sinvention and do not represent a restriction to the embodiments specified in the examples.

8* 9 Examples: 1. Binding of T. pallidum antigens to microassay plates: Treponema pallidum spirochaetes are obtained from rabbit testes which have been infected with live causative organisms 10 to 15 days beforehand. The Treponemas are washed three times with 0.1 M phos- 25 phate buffer, pH 8.0, by centrifugation at 48,000 x g, adjusted to an organism count of 100/ml and extracted with the non-ionic detergent n-octyl glucoside as described in WO 83/0289 The antigen solution obtained in this way with a protein content of 0.2 mg/ml is diluted in the different dilutions of 1:50, 100, 150, 200, 300, 400, 600 and 800. Preferred as assay plate are 7, divisible polystyrene microtitration plates from SC. Nune (Roskilde, Denmark). One assay plate with 11 100 pl in each well is made up for each different dilution.

The assay plates charged with the antigen solutions are left at 20*C for 18 h and then the solutions in the wells are aspirated out, and the wells are washed three times by filling with and aspirating out tris/citric acid solution (0.05 M, pH 7.4) containing 0.2 bovine serum albumin, and the assay plates are subsequently dried over silica gel at 20 0

C.

a 2. Preparation of a peroxidase-labeled antibody against T. pallidum: 0 *0 as The starting material used for the conjugate antibody is a serum pool from donors with positive syphilis serology. The requirements are a minimum titer of 1:10,000 in the TPHA and positive VDRL and cardiolipin CFR titers. Such sera show complex Se patterns with a plurality of reactive bands in the T. pallidum Western blot.

0 The serum is dialyzed against 30 mM Na 2

HPO

4 /NaH 2

PO

4 SpH 7.2, buffer and applied to a column packed with DEAE-cellulose DE 32 (Whatman) and equilibrated with the same buffer. The IgG fraction contained in the S* flow-through is dialyzed against PBS and adjusted to 10 mg/ml. It was verified that this method does not result in any loss of particular antibody specificities and in no significant decrease in titer. The coupling of the peroxidase label to the antibody through GMBS is described in detail in EP-A-O 265 851.

3. Qualitative examination of human antibodies against T. Pallidum Serum, plasma or CSF (25 pj of each) were introduced into wells of anti-T.pallidum assay plates and incubated at 37 0 C for 1 h. The plates were covered with a wrapper-film. Subsequently 100 pl anti-T.pallidum/ POD were added to each well, and the assay plate was incu- 12 bated at 370C for a further hour. The contents of the wells were removed by aspiration, and the wells were washed four times with washing buffer.

100 yl of TMB substrate preparation were introduced into each well and incubated at 20-22 0 C for 30 min, and incubation was stopped by adding 100 pl of 1 N sulfuric acid. The OD 450 of the coloured solution was measured with PBS as reference. Samples which had an OD of less than 60 of that of the negative control serum were categorized as anti-T.pallidum positive, samples whose OD 450 was in the range of •go..60-70 of the negative control were categorized as anti-T. pallidum marginal, and samples which had an *OD 450 of greater than 70 of the negative sampleG 15 were categorized as anti-T.pallidum negative. Fig. 4 S0.shows the frequency distribution (histogram) of 128 .0 •syphilis-positive samples, of 80 serum samples from autoimmune patients, of 53 samples from borreliosis patients and of 71 samples from clinically healthy 20 blood donors.

It is evident from the figure that the positive group is distinctly separate from the negative "00 group, while one of the sera of the autoimmune patients and of normal donors is in the positive 25 range. On the other hand, all the samples which gave 0* "a positive signal in this test were confirmed by comparison tests in other techniques.

4. Quantitative determination of human antibodies against T. pallidum The test procedure corresponds to that described in Example 3. A high-titer serum pool (TPHA titer 1:10000) was first diluted in the stages 1:1, 1:4, 1:16, 1:64 and 1:256 in the negative serum. These diluted serum samples represent the range of titers to be expected from natural sera. The samples were then titrated in each case in PBS, pH 7.2, in 13 different dilutions by a factor of 3. In the same test design, the standard serum L3 was titrated in 8 stages (factor 3) and additionally (in duplicate determination) included in the different dilutions of 1:20 and 1:500. The titration plots showed courses comparable to the plots depicted in Fig. 1.

The 50 titers of the 5 diluted serum samples were first determined as shown in Fig. 1 and related to the 50 titer of the standard serum L3 (reference value 40 IU). Finally, the content (in IU) of the diluted serum samples in each different dilution was determined by computation and plotted against the figures determined graphically (by the 50 method).

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5. Detection of soluble T. pallidum antigen o 3 In this test design, 60 pl of an antigen solution, in place of a serum sample, was diluted in PBS corresponding to the description in Section 4 (from 10 bacterial equivalents per ml, dilution factor 3) 20 and incubated with the conjugate dilution determined to be optimal (60 pl) in an uncoated microtiter plate at 37"C for 1 h. After 100 l of this mixture had been transferred into the assay plate, it was incubated at 37 0 C for a further hour and, after 4 25 washes, the test was carried out by addition of substrate and evaluation as described in Example 3.

FIa. 5 shows that organism counts of more than 106 to 107 bacterial equivalents per ml, corresponding to 0.1-1 pg/ml, can be detected with this method.

6. Competitive ELISA for detection of serum antibodies against Toxoplasma aondii: The cavities of a microtitration plate are each charged with 100 pl of a solution of Toxoplasma gondii ult asonic antigen (of the RH strain) in 0.01 M bicarbonate buffer, pH 9.5, and left at room 14 temperature for 20 h. The plates are washed and dried as described in Example i..

To obtain a high-titer antiserum, rabbits are immunized with the same antigen by standard methods over 3 weeks. The method of DE fractionation and GMBS conjugation to horseradish peroxidase is likewise described in Example 2. When carrying out the competitive ELISA, first 25 pl portions of the serum sample are introduced into the assay plate and, within 15 min, 100 Al portions of the conjugate antibody in PBS are added. After incubation at 37°C for 1 hour, the test mixture is washed 4 times as described in 3. After the chromogen TMB has been added the test is carried out and evaluated as S"5 described.

o For comparison, a group of 80 blood donor sera were also tested in a serum dilution of 1:231 in an indirect IgG ELISA using an anti-human IgG/POD *conjugate from rabbits (Enzygnost T.oxoplasmosis/ :20 IgG). Fig. 6 shows a very good agreement of the methods, while the competitive ELISA provides better positive/negative discrimination and a technically simpler procedure. The comparable sensitivity is evident from the result of the reaction of the titration series of a positive standard serum (see Fig. 6).

7. Competitive ELISA for detection of serum antibodies against rubella virus: The coating of the microassay plates and the test procedure corresponded to the methods described in 6. Rubella virus (strain RA27/3) was cultured in BHK cells, precipitated from the cell culture supernatant with polyethylene glycol (PEG) 6000 and subjected to a Tween 80/diethyl ether treatment as described by Laufs and Thomssen (Arch. Ges. Virus- I I I 15 forschung (1968) 24:164-180). The soluble antigen was dissolved in PBS, pH 7.2, for the coating of the plates. To prepare the antibody-POD conjugate, a high-titer serum pool from more than 5 donors (ELISA IgG titer greater than 1:10000 in each case) was fractionated by the method described in EP 0,265,851, and the pure IgG fraction was coupled by the GMBS method to horseradish peroxidase.

Care was taken in the selection of the sera that the reactivity thereof to control antigen in an indirect IgG ELISA was as low as possible. A group of blood donor sera was tested comparatively in an indirect IgG ELISA (serum dilution 1:231; anti-human IgG from rabbits) and in a competitive ELISA in 5 analogy to the details in Example 6.

8. Linearization of the method: At individual bars of each different antigen dilution, first a conjugate titration in the presence of a syphilis-negative donor serum is carried out. For this, first 25 pl portions of the serum sample are incubated at 37°C for 1 h and subsequently 100 pl portions of the anti-T. pallidum/POD conjugate are added in a 1:50, 100, 150, 200, 300, 400, 600 and 800 dilution series (in tris-HCl, 0.3 M, pH 7.2) and incubated at 37 0 C for a further hour, After 4 washes with PBS, pH 7.2, containing 0.1 Tween 20, subsequently 100 1l of TMB substrate preparation are added to each well and incubated at 20 to 22*C for min. The incubation is stopped by adding 100 pl of 1 N sulfuric acid to each. The extinction of the solutions at 450 rnm is measured with PBS as reference. The antigen/conjugate combinations which have extinctions (OD 450) between 1.5 and 1.8 are select ed for subsequent optimization experiments.

In another test design, serum samples whose content 16 of T. pallidum-specific antibodies in conventional tests was known were tested with antigen/conjugate concentrations mutually adjusted in each case. The antigen/conjugate combination selected as suitable is that for which the signal reduction is linearly related to the logarithm of the titer. Optimal results are obtained, for example, with an antigen dilution of 1:300 (corresponds to about 0.7 pg/ml) and a conjugate dilution of 1:200 Since it is regarded as beneficial for determination of anti- T. pallidum to establish limits of OD 450 70 of the extinction of the negative control for negative and of OD 450 70 for syphilis-positive, the o* concentration of about 1 pg/ml is selected for the 15 treatment of the wells, which is also called coating, for the production of microassay plates for the Sdetermination of anti-T. pallidum.

Legend Fig. 1: Titration plots typical of the competitive '0o syphilis ELISA (serum samples 1, 2 and 3 represent pure IqM fractions taken up in negative serum).

Fig. 2: Correlation of the signal reduction (in with the logarithm of the 50 titer (for 48 syphilis-positive serum samples).

Fig. 3: Correlation of the competitive ELISA (Enzygnost syphilis) with the TPHA (Cellognost Syphilis, H).

Fig. 4: Frequency distribution (histogram) of syphilispositive 60 signal of the negative control) and syphilis-negative 70 signal of the negative control) serum samples over the entire measurement range, Fig. 5: Correlation of the titers (in IU) calculated by linear interpolation with those determined graphically.

The measurements for 5 serum samples of different 17 strengths are depicted as a function of the sample dilution.

Fig. 6: Dependence of the signal reduction in the competitive ELISA (modified version) on the antigen concentration.

Fig. 7: Comparison of the measurements in the indirect (IgG) ELISA (Enzygnost Toxoplasmosis/IgG) and the competitive toxoplasma antibody ELISA (80 sera from healthy asymptomatic blood donors). The figure shows that the '*"0120 competitive ELISA permits a more distinct positive/negative discrimination than the indirect ELISA.

be e so S o* 5 SOS.* I -17a- Annex 1 Quantifications of the competitive ELISA by linear interpolation (for explanation, see Figures 1 and 2) Basis: Signal reduction (in log titer (IU) (Signal reduction in

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100 (sample measurement) y cut-off SX X 3 .x log Titer 3 2 (I) Measurement range Definitions: Reference value of the negative control (0D450)- 1.5-2.0 S0 signal reduction Y signal reduction for standard 1 Y signal reduction for standard 2 X log reference titer for standard 1 (IU) X log reference titer for standard 2 (IU) a gradient b axis intercept Y3 signal reudction for sample X1 log titer for sample (IU)

Claims (6)

1. An immunochemical competitive method of detection and determination of antibodies against an organism selected from the group consisting of T. pallidum, Toxoplasma gondli and rubella virus which comprises the step of contacting said antibody and a solid phase composed of a carrier and, irreversibly bound thereto, an antigen of said organism and a labelled antibody directed against said antigen, there being competition between said anti-bodies and said labelled antibody for the binding site on the solid phase, wherein the antigen is irreversibly bound to the carrier directly or via a non-immunochemically binding spacer.

2. The method as claimed in claim 1, wherein the antigen/antibody conjugate combination is adjusted so that the signal reduction is linearly related *to the logarithm of the titer.

3. The method as claimed in claim 2, wherein the evaluation is by computation. V o

4. The use of a test kit containing a carrier to which an antigen of said organism is bound directly or via a inon-immunochemically binding spacer, and a labelled antibody against the said antigen for the detection and determination S. in an immunochemically competitive assay of antibodies against the organisms causing syphilis, toxoplasmosis and rubella.

The use of a test kit as defined in claim 4 and which is composed of an element in which the solid phase, and, in dry form, the reagents required for the detection and determination, In whole or in part are contained for the detection and determination in an immunochemically competitive assay of antibodies against the organisms causing syphills, toxoplasmosis and rubella. 19

6. An immunochemical competitive method of detection and determination of antibodies against an organism selected from the group consisting of T. pallidum, Toxoplasma gondii and rubella virus causing syphilis, toxoplasmosis or rubella, which comprises the step of contacting in a test vessel said antibody and a solid phase composed of a carrier and, irreversibly bound thereto, an antigen of said organism and a labelled antibody directed against said antigen, a fluid containing said antibody being initially preincubated with the labelled antibody outside the test vessel and then added to the solid phase, there being competition between said antibody and said labelled antibody for the binding site on the solid phase, wherein the antigen is irreversibly bound to the carrier directly of via non- immunochemically binding spacer. DATED this 25th day of November, 1993 BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA DO.44 AU5299190.WPC [DBM/KJS/SDW]