CA1305411C

CA1305411C - Method for the determination of anti-hiv, means therefore and theiruse in this method

Info

Publication number: CA1305411C
Application number: CA000550259A
Authority: CA
Inventors: Udo Krupka; Thomas Buck; Hans Erwin Pauly
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1986-10-27
Filing date: 1987-10-26
Publication date: 1992-07-21
Anticipated expiration: 2009-07-21
Also published as: EP0265851A2; NZ222282A; NO171754C; FI874676A; PT85992A; NO874453D0; ES2061468T3; ATE101725T1; DK559187A; EP0265851A3; NO171754B; DE3789087D1; FI874676A0; FI90801B; JPS63128260A; EP0265851B1; NO874453L; FI90801C; PT85992B; AU611102B2

Abstract

ABSTRACT OF THE INVENTION

An immunochemical competitive method for the detection and for the determination of antibodies against HIV using a solid phase consisting of a carrier and, irreversibly bound thereto, envelope and core proteins of HIV and labeled antibodies directed against envelope and core proteins of HIV is described, in which the antibodies compete for the binding site on the solid phase. Furthermore, assay kits for carrying out the method and their use are described.

Description

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, ~EHRINGWERKE AKTIENGESELLSCHAFT 86/~ 036 - Ma 603 Dr. Ha/~n A method for the determination of anti-HlV, means there-S fore and their use in this method The invention relates to a method for the imrunochemical determination of HIV-sPecific antibodies, which employs the competition principle, as well as to means suitable for this method and their use. The method is suitable for the highly sensitive and specific detection and det-ermination of HIV-specific antibodies of the IgG and IgM
classes in humans.

A virus (retrovirus) or a closely related group of v;rus-es, which have recently been designated as human immune deficiency virus (HIV) but are also referred to as T-lym-photropic virus type III (HTLV III) or lymphadenopathy-associated ~irus (LAY) is regarded as being the causative ; 20 a~ent of the recently recognized infectious disease AIDS
(Acquired I~mune Deficiency Syndrome), which shows a high mortality rate.

The virus can be transmitted by human blood and contamin-ated blood products and has been isolated from cerebro-- spinal fluid, seminal fluid, lacrimal fluid, and saliva.
The serological determination of HIV-specific antibodies is carried out to clarify whether an individual has had contact with the virus and is to be regarded as a potent-ial carrier of the infectious agent~ An essential signif-icance of the ant;body determination lies in the use as a diagnostic tool for AIDS (together with other investiga-tions and the case history) as well as in the lowering of ~ 35 the risk of transmission of the causatiue agent of the j~ disease via transfusion, organ transplantation, blood products or semen by excluding material donated by ~ antibody-positive subjects. Moreover, the HIV antibody ,~
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determination ;s of importance in members of so-called risk groups (homosexuals, drug addicts, hemophiliacs, children of infected mothers) as well as in epidemio-logical investigations.

The detection of anti-HIV using the Western blot tech-nique as is described for example in US Patent 4,520,113 is regarded as being the most sensitive detection method for HIV-specific antibodies at present. In this method, concentrated and inactivated HIV protein material is sep-arated according to molecular weights by means of elect-rophoresis and then transferred to nitrocellulose paper, preferably electrophoretically. As a modification of US
Patent 4,520,113, where an RIA is used, an antigen immob-ilized in such a way can be used for an indirect ELISAwithout loss of sensitivity, and in this the Jse of spar-ingly soluble dye systems in the enzyme reaction makes it possible to visualize the bands at which an immune reaction has taken place. Stained virus-specific bands can then be differentiated from non-virus antigen-specific and cross- or unspecific-reacting contaminants after cali-bration with suitable control material, and therefore a discrimination between genuine- and false-positive reac-tions can be accomplished.
~ 25 ; Furthermore a method is described which is a competitive ; ELISA and is said to fulfil the criteria o~ the Western blot technique with regard to specificity and sensitiv-ity (Arzte-Zeitung/No. 1,25./26.10.8S, p.2û).
In this method undiluted samples are used. It can be used for the simultaneous detection of HIV-specific an~i-bodies of the IgG and IgM classes. The method can be carried out using a Wellcome test kit, named We~lcozyme 3~5 Anti-HTLV III. Microassay plates onto which purified HIV
antibodies are bound, and onto the latter, in turn, HIV
antigens are immunochemically immobilized, are used for the solid phase. The samples are incubated in the wells .~
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together with horseradish peroxidase-labeled anti-HIV. In this, the antibodies against HIV from the sample compete with the labeled anti-HIV for the HIV binding sites on the solid phase. The amount of labeled antibody bound in the wells is inversely proportional to the anti-HlV concen-tration in the sample.

Very frequently class II human leukocyte antigens tHLA Il, particularly DR4), which originate from the human lymph-ocyte cell lines usually used for HIV culture, and canlead to false-positive results particularly in polytrans-fused patients (anti-HLA DR4), are held responsible for false-positive results in the other presently used tests.

This type of unspecific reactions is excluded in the com-petitive test kit mentioned for the detection of HIV-specific antibodies (Walgate R., ~eardsley T~, (1986) Nature 320, 96) by the use of the human cell line CCRF-CEM to culture the virus, since these cells carry no HLA
of class II.

If, in the competitive ELISA described, use was made of HIV which was cultured in the H9 cell line which is known for better yields, it was found that extremely large amounts of virus starting material are necessary to purify the trapping antibodies for immobilization on the solicd phase, on the one hand, and to achieve the saturation necessary for maximum sensitivity of the trap-ping antibodies immobilized on the synthetic material with antiyen, on the other hand, and that furthermore the immunoadsorptive purification of human serum only proceeds unsatisfactorily with regard to an improvement of the specificity when HIV antigenic material cultured on H9 and thereby contaminated with nuclear, mitochondrial and leukocytic antigens is used. In addition, onty very low yields of specific human antibodies were obtained, which makes use as a screening test impossible on econ-omic grounds.

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It was found that a competitive method for the detection and for the determination of antibodies against HIV, both of the IgM and the IgG classes, is possible on use of a solid phase onto which HIV antigens, which may also be contaminated by antigens originating from the host cell, may be bound directly, i.e. without antibody-mediated binding, when labeled antibodies against HIV
are used, which react neither with antigens or the host cell nor with any antigens from human tissue.
The invention relates to an immunochemical competitive method for the detection and for the determination of antibodies against HIV using a solid phase consisting of a carrier and, bound thereto, envelope and core proteins of HIV and labeled antibodies, directed against envelope and core proteins of HIV, in which the antibodies comp-ete for the binding sites on the soLid phase, wherein the proteins are irreversibly bound directly or via a non-immunochemically binding spacer to the carrier.
The labeled antibodies used in the method are of a type not reacting with antigens of an HIV-free host cell or a medium for the host cell, or with human, HIV-free tissue.

The invention also relates to an assay kit for the detec-tion and for the determination of antibodies against HIV
in an immunochemical competitive method comprising a carrier to which envelope and core proteins of HIV are bound irreversibly either directly or through a non-;mmunochemically binding spacer, as well as labeledantibod;es against envelope and core proteins and, where appropriate, means for the detection of the labeling.

It is advantageous that the sensitivity of the method according to the invention corresponds to the sensitivity of the Western blot technique since even samples with low HIV antibody titers lead to a significant inhibition of the bind;ng of the labeled antibodies which a.lows 13~5;4~.1 visual evaluation of the color development when, for example, an enzyme marker is used, and makes possible a discrimination of positive and negative samples with ex-tremely high selectivity, that even early seroconversion, caused by HlY-specific IgM antibodies, can be detected reliably, that no interference and thus false-positive results occur through human leukocyte antigens (HLA), nuclear antigens or mitochondrial ant;gens, so that a trial, using conventionaL control antigens from the host cell, as to ~hether a positive result is specific is su-perfLuous, and that in sera, citrated, heparinized and EDTA-plasma and also in recalcified plasma of human ori-gin reliable determination of HIY-specific antibodies is guaranteed and inactivation of the samples for 30 minutes at 55C (HIV inactivation) and even for 10 hours at 60C (hepatitis ~ virus inactivation) does not lead to false-positive results.
Antigenic preparations of HIV suitable for the solid phase can be prepared by first obtaining HIV-positive permanent T cells a~ described in Canadian Patent 1,266,243 and obtaining an ~IV
preparation as de~cribed in U.S. Patent 4,520,113.
In th;s, the HIV is first propagated after transmission to the human T cell line H9 by co-cultivation ~ith lym-phocytes of an AIDS patient and then separated from thehost cell culture supernatant by ultracentrifugation.
After removal of cell debris and fat the virus material ;s purif;ed by sucrose density gradient centrifugation and finally inactivated by addition of Tr;ton~ and heat treatment.

Su;table carrier materials for the solid phase include plastics such as polystyrene, polyvinyl chloride, nylon :, and other synthetic polymers, natural polymers ~uch as cellulose~ as well as derivatized natural polymers such as cellulose acetate and nitrocellulose as well as glass, particularly as glass fibers.

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,, The carriers can be in the form of spheres, rods, tubes, and microassay plates. Sheet-like structures such as paper strips, platelets and membranes are also suitable.
The surface of the carriers can be permeable as well as impermeable to aqueous solutions.

Spheres, tubes, paper strips and membranes are preferred carriers. Microassay plates are particularly preferred carriers~
The solid phase is prepared by irreversibly binding an antigen preparation of HIV to the carrier.

Irreversible binding in the sense of the invention is present, for example, in tne case of 1) adsorptive binding, which is not split by immuno-chemical agents, such as high-affinity antibodies or by the agents, such as labeled antibodies, dilution and buffer solutions, used in the method, 2) bioaffinity binding mediated by a non-immunochemically binding spacer~ in which the spacer may consist of biotin and avidin or of other pairs of receptors and ligands, 3) direct covalent binding, 4) covalent binding brought about through a chemically bifunctional spacer Covalent binding is preferred when water-permeable car-riers are used, and adsorptive binding is preferred when water-permeable or ~ater-impermeable carriers are used.
Direct adsorptive binding of HIV antigen preparations to gamma ray-treated polystyrene as a carrier is particular-ly preferred.

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_ 7 -Antibodies intended for labeling are selected on the basis of the results from the Western blot analysis of sera from AIDS patients.

5 In order to minimize the risk of false-negative diagno-sis, in a method for the detection of pre-AIDS and AIDS
the possibility of detection of at least two antibody specificities, namely those for a core protein and an envelope protein, must be given in accordance with the present state of knowledge.

Suitable combinations of specificities are, for example, those which recognize 1. the core protein designated p24 and the envelope protein designated gp41 or 2. p24 and the envelope protein designated as gp120.

The detection of further antibody specificities, namely those for the core proteins designated p18, p30, and p55 and the HIV polymerases designated as p53 and p65 means an additional safety measure in the detection of anti-HIV-positive samples.
In order to exclude a false-positive diagnosis the anti-bodies intended for labeling may not have any specificit-ies which detect constitutents of human tissue such as, for example, leukocyte antigens, nuclear and mytochond-rial proteins.

Antibodies of AIDS patients can be used for labeling pro-vided they fulfil the previously described criteria as ascertained in the Western blot technique.
Furthermore, polyclonal antiboclies obtained from sera of animals immunized with HI~ can be used provided these, after absorption of the specificities which recognize .

constituents of the host cell and of the culture medium, fulfil the above criteria after testing in the Western blot.

Monoclonal antibodies are also suitable. In this case, at least two must be used in one of the above combinat-ions of specificities, i.e. for example in the combin-ation anti-p24 and anti-gp41.

Radioactive isotopes, fluorescent and chemiluminescent dyes as ~elL as enzymes, whose detection is possible by chromogenic, luminogenic or fluorogenic substrate systems, can be used as markers.

Labeling is performed by methods which are described as the state of the art for the above markers.

Where the antibodies are labeled with peroxidase the periodate technique of Nakane et al., 1974, J. Histochem.
Cytochem. 22, 1084-1090, can be used or a method accord-ing to Ishikawa et al.~ 1983, J. Immunoassay 4, 209-327, ;n which the partners are linked with a heterobifunctional reagent.

The method according to the invention can be performed, for example, by, into the wells of a microassay plate which contains bound a previously described HIV antigen preparation, a3 simultaneously adding the sample and a solut;on of the labeled antibody, optionally adding the sample first and a solution of the labeled antibody after a certain time, removing the solution after a certain incubation time and washing the wells with a buffer and then measuring the labeling in the wells or b) first adding a solution containing the labeled anti-~3~S4~

body, removing the solution after a certa;n incubat-ion ti-e and washing the wells with a buffer, opt-ionally drying the microassay plate, then adding the diluted sample and incubating for a certain time, then removing the sample from the wells and measur-ing the labeling in the sample.

The method according to the invention can also be per-formed in the case the use of a diagnostic element which contains the solid phase and some or all the necessary reagents ;n dry form.

The following example represents an embodiment of the invention, without, however, limiting it thereto.
Example . . . _ 1. Binding of HIV antigen onto microassay plates An HIV antigen preparation prepared according to US
Patent 4,5Z0,113~ pur;fied by sucrose density gradi-ent centrifugation and heat-inactivated was predilu-ted to 50 ~g/ml protein w;th 100 mmol/l sod;um bicar-bonate of pH 9.6. From this dilution a 2-fold dilution series was set up with 10 dilutions in the same buf-fer, i.e. a series with the concentrations 50, 25, 1Z.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.2 and 0.1 ~g/ml was obtained. Each 100 ~l portion of each dilution was placed in 16 wells of type ~ microassay plates from Nunc, Roskilde, Denmark. The assay plates filled with the dilutions were left at Z0C for 18 hours, the sol-ut;ons in the wells were then aspirated and the wells were washed 3-4 times with 200 ~l of a solution of 10 g/l of bovine serum albumin in phosphate-buffered physiological saline, pH 7.4 (PEIS) by filling and aspiration, and the assay plates were then dried over silica gel at 20C.

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2. Preparation of a peroxidase-labeled antibody against HIV

Serus from an AIDS patient which was selected by the Western blot technique and contained antibody spec-ificities for the core proteins p18, p24, p30 and p55, for the envelope proteins gp41 and gp120 as well as for the HIV polymerases p53 and p65 was frac-tionated by chromatography on DEAE cellulose. For this, the serum was dialyzed against a buffer of 30 mmol/l Na2HP04/NaH2P04, pH 7.0, and added onto a column filled with DEAE cellulose DE 32 (Whatman) and equilibrated with the same buffer. The IgG fra-ction obtained in the forerun was dialyzed against PBS and adjusted to 4 mg/ml protein.

A further test with the Western blot technique show-ed that all the above antibody specificities were recovered in the IgG fraction.
The IgG fraction was then reacted with N-gamma-malei-midobutyloxysuccinimide (GM~S), obtained from æehring Diagnostics, as described by Tanamori et al., 1983 in J. Immunol. Meth. 62, 123-131. 2-Iminothiolanhyd-rochloride (Sigma, catalog no. I 6256) was reacted with horseradish peroxidase (POD), obtained from Boehringer Mannheim, catalog no. 413470, as described by King et al. 1978 in ~iochem. 17, 1499-1506. An IgG-POD conjugate was prepared from the GM~S-IgG
conjugate and the iminothiolane-POD conjugate, as described by Tanamori.

The solution of the IgG-POD conjugate obtained had a protein content of 440 ~g/ml. The ratio of POD to IgG was determined to be 2.5. The solution was then diluted to 500 ng/ml IgG-POD with a solution of 50 ml/l of fetal calf serum, 5 g/l polyoxyethylene-(20)-sorbitan monolaurate (Tween 20R) in PBS and was ~: .
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designated as ant;-HIV-POD.

3. Obtaining a TM~ substrate preparation A substrate system or a substrate preparation con-taining hydrogen peroxide and tetramethylbenzidine (TM~), which was prepared from two stock solutions, was used for the detection of anti-HIV-POD.

Stock solution 1: TMB dihydrochloride was dissolved w;th stirring at a concentration of 5 g/l, i.e. 16 mmol/l, in twice-distilled water and adjusted to pH
1.5 with 5 N hydrochloric acid. Penicillin G was added to this solution with stirring in a final con-centration of 200 mg/l, i.e. 0.56 mmol/l.

Stock solution 2: 1.4 ml glacial acetic acid, 1.5 ml 1 N NaOH and 250 mg, i.e. 3 mmol H22 as a urea-hydrogen peroxide adduct were added to 900 ml of twice-distilled water. After dissolution was com-plete it was made up to 1 l with twice-distilled water.

TM~ substrate preparation: one part by volume of stock solution 1 and 10 parts by volume of stock solution ~ were mixed with one another.

4. Optimization of the method 3û 16 sera, which were confirmed as being HIV negative by the ~estern blot technique, were diluted 1:5 with anti-HIV-POD (1 part by volume serum plus 4 parts by volume anti-HlV-POD).

125 ~l of the dilution was added to each of ~e wells of the microassay plates treated with HIV antigen preparation and incubated for 1 h at 27C. The dilutions were aspirated and the wells washed four ~3~S~
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times with a solution of 1 g/l Tween 2 ~ in PBS by filling and aspiration. 100 ~l of the TMB substrate preparation was then added to each well and incubated for 30 min at 20 to 22C. The incubation was ended by addition of 100 ~l portions of 1 N sulfuric acid.
The extinction of the solutions was measured against PBS at 450 nm.

The solutions from the wells which had been loaded with 3.12 ~g/ml HIV antigen showed an E4so between 1.6~ and 1.74. The solutions from the wells loaded with 1.56 ~g/ml HIV antigen showed an E4so between 1.38 and 1.45.

Wells of microassay plates were then treated with an HIV antigen preparation of exclusively 2.5 ~g/ml in the previously described manner. The evaluation of the 16 sera in the wells treated ~ith 2.5 ~g/ml HIV
antigen preparation gave a mean E4so of 1.56 + 0.05 w;th the same test procedure.

Since in the determination of anti-HIV a limiting value fixing of E greater than 0.8 for anti-HIV-neg-ative and of E less than 0.8 for anti-HIV-positive samples is regarded as being favorable (the limiting value is defined as half the mean extinction of sam-ples confirmed to be negative), a concentration of 2.5 ~g/ml HIV antigen preparation was selected for the preparation of microassay plates for the deter-mination of anti-HIV (anti-HIY assay plates) in the treatment of the wells which is also designated as loading.

In all the cdeterminat;ons of anti-HIV described in the ~ollowin~ text, the reagents used in the hitherto described preliminary tests, namely the anti-HIV-POD, the TM~ substrate preparation and the solution of 1 g/l Tween 20 in PBS designated as washing buffer .

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5. Determination of human antibodies against HIV

a) 25 ~l serum or plasma and 100 ~l anti-HIV-POD
were added to the wells of anti-HIV assay plates and incubated for 1 h at 37C~ The content of the wells was removed by aspiration and the wells were washed four times with washing buffer. 100 ~l TM~ substrate preparation was added to each well, incubated for 30 min at 20-22C and the incu-bation ended by the addition of 100 ~l of 1 N
sulfuric acid. E450 of the colored solution was measured against a blank PEIS.
Samples which produced an E4so of less than 0.30 were graded as anti-HIV positive, those whose E4so was in the range of 0.31 to 0.80 as anti-HIV borderline, and those which produced an E450 above 0.81 as anti-HIV-negative.

Of 420 samples which were anti-HIV-positive in the Western blot technique, 419 samples were like-wise found to be positive.
On 1a96 plasma or serum samples of healthy sub-jects or of subjects showing other pathological conditions, the results shown in Table 1 were obtained.

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The 5 samples from subjects from risk groups which initially gave a positive or borderline reaction were aditionally investigated with the Western blot technique. Of these, only the 2 sam-ples were positive which according to the method of the invention were repeatedly found to be positive.

The findings thus show that the method according to the invention is in good agreement with the Western blot technique.

b) Samples of 50 ~L whole blood were diluted with 50 ~l distilled water containing 20 g/l Triton X-100R
and 0.2 mol/l trisodium citrate, and SO ~l of this lysate was added to wells of anti-HIV assay plates. After an incubation of 1 h at 37C 100 ~l of anti-HIV-POD was added and then incubated again for 1 h at 37C. The content of the wells was removed by aspiration and the wells washed five times with washing buffer. 100 ~l TMB substrate preparation was added to each well, incubated for 30 min at 20-25C and the reaction ended by addition of 100 ~l 1 N sulfuric acid. The ex-; tinction of the solutions was measured at 450 nm Z5 against a blank of PBS ~E4so).

The evaLuation of the samples is as described under 5 a). Of 51 and 5 blood samples which, as serum, were positive and weakly positive, res-pectively, in the Western blot technique, all the samples were likewise found to be positive.
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Of 226 blood samples which reacted as serum anti-HIV-negative, all the samples were likewise found to be negative. This shows that the method accord-ing to the invention is in good agreement with the Western blot technique regarded as a reference method.

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c) Samples of 50 ~l serum or plasma or of 50 ~l lysate of whole blood obtained as described ;n 5 b) were added to the wells of the microassay plates with 100 ~l of a solution of 5 ~g/ml IgG-POD, pre-pared by dilution of the ~t40 ~g/ml IgG-POD con-jugate described in ~xample 2, to 5 ~g/ml with the already described solution containing fetal calf serum and Tween 2 ~ in P~S and incubated for 10 min at 45C.
The wells were then washed 5 times with washing buffer. 100 ~l TM~ substrate preparation was then added to each well and incubated for 5 min at 20-22C. The incubation was ended by addit-ion of 1 N normal sulfuric acid. The evaluation was as described in 5 a)~

Of 51 and 5 samples which, as serum, were positive and weakly positive, respectively, in the Western blot technique, all the s3mples were likewise found to be positive.

Of 225 samples which gave an anti-HIY-negative reaction, all the samples were likewise found to be negative.

Thus it is evident that the variant, according to the invention, of a rapid method is in good agree-ment with the Western blot technique regarded as being a reference method, with regard to sensit-;vity and specificity.

d) 100 ~l of anti-HIV-POD was added to the wells of anti-HIV assay plates and incubated for 1 h at 37C~ the anti-HIV-POD wash aspirated and the wells were washed four times with washing buffer.
The anti-HIV assay plates treated in this way were dried over silica gel at 20-22C.

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25 ~l sample and 100 ~l of a solution of 10 g/l bovine serum alburnin in 50 mmol/l Tris, adjusted to pH 7.4 with HCl, were then added to the wells.
After incubation at 37C for 1 h the content of the wells was removed and added tc, wells of an unloaded assay plate, mixed there with 100 ~l TM8 substrate preparation, the mixture was left for 30 min at 20-22C and 100 ~l 1 N sulfuric acid was added. E4so was then measured against PBS.
The mean E4so values from triple determination per sample are given in Table 2 for samples which were graded according to the method described under a~ as anti-HIV-negative, anti-HIV border-line and anti-HIV-positive.

TA~LE 2 Anti-HIV-negative samples 1 0.140 0.148 0.136 2 0.218 0.206 0.205 3 0.267 0.279 0.270 4 0.275 0.274 0.272 Anti-HIV borderline samples 5 0.609 0.454 0.457 6 0.308 0.316 0.351 Anti-HlV-positive samples 7 0.769 0.674 0.773 8 0.891 0.865 0.820 9 1.202 0.953 0.863 - 10 1.678 2~020 2.202 Table 2 shows that in contrast with embodiments a) ~o c) the extinction of anti-HIV-negative samples is low and of anti-HIV-positive samples is high here. The border-line value for E4so for anti-HIV-negative samples is also to be set at 0.30 here.

Claims

1. An immunochemical competitive method for the detect-ion for the determination of antibodies against HIV
using a solid phase consisting of a carrier and, bound thereto, envelope and core proteins of HIV and labeled antibodies, directed against envelope and core proteins of HIV, in which the antibodies compete for the binding site on the solid phase, wherein the proteins are irreversibly bound directly or via a non-immunochemically binding spacer to the carrier.

2. The method as claimed in claim 1, wherein the labeled antibody does not react with antigens from an HIV-free host cell and an HIV-free medium or with HIV-free human tissue.

3. The method as claimed in claim 1, wherein the labeled antibody at least reacts either I) with the core protein designated as p24 and the envelope protein designated as gp41 or II) with the core protein designated as p24 and the envelope protein designated as gp120.

4. The method as claimed in claim 1, wherein the labeled antibody consists of a mixture of 2 or more labeled monoclonal antibodies in which the mixture must con-tain antibodies which are directed against either I) the p24 core and the gp41 envelope proteins or II) the p24 core and the gp120 envelope proteins.

5. An assay kit for the detection and for the determin-ation of antibodies against HIV in an immunochemical competitive method containing a carrier to which env-elope and core proteins of HIV are irreversible bound directly or via a non-immunochemically binding spacer, labeled antibodies against envelope and core proteins of HIV which do not react with antigens of an HIV-free host cell or with an HIV-free medium or with human tissue and, where appropriate, reagents for the detection of the labeling.

6. The assay kit as claimed in claim S, wherein the lab-eled antibody is present either a) as a constituent separated from the solid phase or b) as a constitutent immunochemically bound to the solid phase.

7. The assay kit as claimed in claim 5, which consists of an element which contains, completely or partially, the solid phase and the reagents necessary for the detect-ion and the determination, in dry form.

8. The assay kit as claimed in claim 5, which consists of an element in which the solid phase and the labeled antibody in dry form are present either a) as a constitutent separated from the solid phase or b) as a constitutent immunochemically bound on the solid phase and which contains further reagents optionally in dry form.

9. A method for the detection and for the determination of antibody against HIV, using the assay kit as claimed in claim 5.

10. A method for detecting antibodies against HIV, using the assay kit as claimed in claim 6.

11. The use of an assay kit as claimed in claim 5 in a method for the detection and for the determination of antibodies against HIV.

12. The use of an assay kit as claimed in claim 6 in a method for detecting antibodies against HIV.