AU611102B2

AU611102B2 - A method for the determination of anti-hiv, means therefore and their use in this method

Info

Publication number: AU611102B2
Application number: AU80126/87A
Authority: AU
Inventors: Thomas Buck; Udo Krupka; 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: 1991-06-06
Anticipated expiration: 2007-10-26
Also published as: DK559187D0; EP0265851A2; PT85992A; AU8012687A; FI874676A; FI90801B; FI874676A0; EP0265851B1; ATE101725T1; DE3789087D1; NZ222282A; MX168853B; CA1305411C; JPS63128260A; EP0265851A3; FI90801C; PT85992B; NO171754B; NO171754C; NO874453D0

Abstract

An immunochemical competitive method for detecting and determining antibodies for HIV uses a solid phase consisting of a carrier and HIV envelope and core proteins irreversibly bound thereto and marked antibodies directed against HIV envelope and core proteins, the antibodies competing for the binding site on the solid phase. Test kits for carrying out the method and their use are also described.

Description

1, "6t;,"a ture of Applicant (s) or Seal of Company anI Signatures of its Officers as prescribed by Its Articles o Association.

James Murray Registere"Pfatent "Attorney T' FE STAMP TO VALUE OF TS. ATACHED I THE COMMISSIONER OF PATEN i COMMONWEALTH OF AUSTRALIA 6 1 I F2 PATENTS ACT 1952-69 COMPLETE

SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Prority: 0 005 0 00 0R *Ralated Art: 0 0 0 0 0 0 BO 0 0 00 go Name of Applicant: 0 BEHRINGWERKE AKTIENGESELLSCHAFT o Address of Applicant: D-3550 Marburg, Federal. Republic of Germany 000 o Actual Inventor: 0o oAddress for Service: p UDO KRUPKA, THOMAS BUCK, HANS ERWIN PAULY EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: A METHOD FOR THE DETERMINATION OF ANTI-HIV, MEANS THEREFORE AND THEIR USE IN THIS METHOD The following statement is a full description of this invention Including tha best method of performing it known to us To th- Commissioner of Patents BEHRINGERKE

AKTIENGESELLSCHAFT

Prokurist Prokust ppa. Stein ppa. B BEHRINGWERKE AKTIENGESELLSCHAFT 86/8 036 Ma 603 Dr. Ha/Bn A method for the determination of anti-HIV, means therefore and their use in this method The invention relates to a method for the immunochemical determination of HIV-specific antibodies, which employs the competition princ iple, as well as to means suitable for this method and their use. The method is suitable for the highly sensitive and specific detection and determination of HIV-specific antibodies of the IgG and IgM classes in humans.

S 15 A virus (retrovirus) or a closely related group of viruses, which have recently been designated as human immune deficiency virus (HIV) but are also referred to as T-lymphotropic virus type III (HTLV IIl) or lymphadenopathyassociated virus (LAV) is regarded as being the causative agent of the recently recognized infectious disease AIDS (Acquired Immune Deficiency Syndrome), which shows a high mortality rate.

I 'o "The virus can be t-ansmitted by human blood and contamin- 0too ated blood products and has been isolated fro cerebro- I spinal fluid, seminal fluid, lacrimal fluid, and saliva.

0 O The serological determination of HI\V-specific antibodies a o a is carried out to clarify whether an individual has had V, 30 contact with the virus and is to be regarded as a potential carrier of the infectious agent. An essential significance of the antibody determination lies in the use as a diagnostic tool for AIDS (together with other investigations and the case history) as well as in the lowering of the risk of transmission of the causative agent oT the disease via transfusion, organ transplantation, blood products or semen by exciuding material donated by antibody-positive subjects. Moreover, the HIV antibody t 2 determination is 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 technique 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 separated according to molecular weights by means of electrophoresis and then transferred to nitrocellulose paper, preferably electrophoretically. As a modification of US O Patent 4,520,113, where an RIA is used, an antigen immobo °o 15 ilized in such a way can be used for an indirect ELISA aooo.

0 without loss of sensitivity, and in this the Jse of spar- 0 0 0 o° 0 ingly soluble dye systems in the enzyme reaction makes ooo a o it possible to visualize the bands at which an immune 0 a o Sreaction has taken place. Stained virus-specific bands can then be differentiated from non-virus antigen-specific 0° and cross- or unspecific-reacting contaminants after cali- 0 0 0 o00 o bration with suitable control material, and therefore a o o discrimination between genuine- and false-positive reactions can be accomplished.

o 25 00000 o Furthermore a method is described which is a competitive So°°o ELISA and is said to fulfil the criteria of the Western o000 oo blot technique with regard to specificity and sensitiv- In this method undiluted samples are used. It can be used for the simultaneous detection of HIV-specific antibodies of the IgG and IgM classes. The method can be carried out using' a Wellcome test kit, named Wetlcozyme Anti-HTLV Ill. 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 l 7RA4, a f Ot a o a 4a4 to a t e a a a a o t 0 a a.

6t a 0 40 Q 0 3 together with horseradish peroxidase-labeled anti-HIV. Il 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-HIV concentration in the sample.

Very frequently class II human leukocyte antigens (HLA II, particularly DR4), which originate from the human lymphocyte cell lines usually used for HIV culture, and can lead to false-positive results particularly in polytransfused patients (anti-HLA DR4), are held responsible for false-positive results in the other presently used tests.

15 This type of unspecific reactions is excluded in the competitive test kit mentioned for the detection of HIVspecific antibodies (Walgate Beardsley (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 solid phase, on the one hand, and to achieve the cs turation necessary for maximum sensitivity of the trappinc antibodies immobilized on the synthetic material with antigen, 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, mitochendrial and leukocytia antigens is used. In addition, oiiy very low yields of specific human antibodies were obtained, which makes use as a screening test impossible on economic grounds.

i I 4 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 antibodymediated 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 0 00 oo a carrier and, bound thereto, envelope and core proteins 00 0 o° 15 of HIV and labeled antibodies, directed against envelope o o and core proteins of HIV, in which the antibodies comp- 0 0 o00 0 ete for the binding sites on the solid phase, wherein 0000 o oo the proteins are irreversibly bound directly or via a 0 0 0

O

0 non-immunochemicatly binding spacer to the carrier.

"o The labeled antibodies used in the method are of a type 0 009 o oOo not reacting witn antigens of an HIV-free host cell or a a 00 medium for the host ceLl, or with human, HIV-free tissue.

00oo 0 0 25 The invention also relates to an assay kit for the detec- 000000 o 0 tion and for the determination of antibodies against HIV 0 om in an immunochemical competitive method comprising a oo o carrier to which envelope and core proteins of HIV are S0 0 S o bound irreversibly either directly or through a nonimmunochemically binding spacer, as well as labeled antibodies against envelope and core proteins and, where approrriate, 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 binding of the labeled antibodies which allows 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 extremely high selectivity, that even early seroconversion, caused by HIV-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 antigens, so that a trial, using conventional control antigens fro the host cell, as to whether a positive result is specific is superfluous, and that in sera, citrated, heparinized and EDTA-plasma and also in rncalcified plasma of human ori- S O gin reliable determination of HIV-,pecific antibodies 0O 0 is guaranteed and inactivation of the samples for 00 0 °o 15 minutes at 55 C (HIV inactivation) and even for 10 hours o at 60 0 C (hepatitis B virus inactivation) does not lead to o 0 00 false-positive results.

ooo Antigenic preparations of HIV suitable for the solid phase can be prepared by first obtaining HIV-positive 00 °0 permanent T cells as described in W085/04897 and obtaino ooo 00o, ing an HIV preparation as described in US Patent 4,520,113.

In this, the HIV is first propagated after transmission to the human T cell line H9 by co-cultivation with lym- 4400 o..oo 25 phocytes of an AIDS patient and then separated from the 0 w host celt culture supernatant by ultracentrifugatiop.

o o' After removal of cell debris and fat the virus material ooo "o is purified by sucrose density gradient centrifugation and finally inactivated by addition of Triton® and heat treatment.

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

6 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.

S On 0o 0 Irreversible binding in the sense of the invention is 0 0 0 o oo 15 present, for example, in the case of 0 0000oooooo o 0 o°O°°o 1) adsorptive binding, which is not split by immuno- 0. o chemical agents, such as high-affinity antibodies or Q00 by the agents, such as labeled antibodies, dilution and buffer solutions, used in the method, 0 000 2) bioaffinity binding mediated by a non-immunochemically 00 binding spacer, in which the spacer may consist of 0oO°°° btotin and avidin or of other pairs of receptors and 00 0 00oe00 25 ligands, O 0 0 o a 3) direct covalent binding, 000 0 0 0 0 4) covalent binding brought about through a chemically bifunctional spacer Covalent binding is preferred when water-permeable carriers are used, and adsorptive binding is preferred when water-permeable or water-impermeable carriers are used.

Direct adsorptive binding of HIV antigen preparations to gamma ray-treated polystyrene as a carrier is particularly preferred.

-a- 7 Antibodies intended for labeLing are selected on the basis of the results from the Western bLot anaLysis of sera from AIDS patients.

In order to minimize the risk of false-negative diagnosis, 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 0000 00 a °o °o 15 1. the core protein designated p24 and the envelope o protein designated gp41 or 0 0 00Jooo 00 0 0 20 The detection of further antibody specificities, namely o o d Oooo those for the core proteins designated p18, p30, and o o and the HIV polymerases designated ?s p53 and p65 means an additional safety measure in the detection of anti-HIV- 00000 positive samples.

0oo0 0 o In order to exclude a false-positive diagnosis the antioO°o bodies intended for labeling may Iiot have any specificitoo ies which detect constitutents of human tissue such as, for example, leukocyte antigens, nuclear and mytochondrial proteins.

Antibodies of AIDS patients can be used for labeling provided thi'y fulfil the previously described criteria as ascertained in the Western blot technique.

Furthermore, polyclonal antibodies obtained from sera of animals immunized with HIV can be used provided these, after absorption of the specificities which recognize 8 8 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 combinations of specificities, i.e. for example in the combination anti,-p24 and anti-gp41.

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

I Labeling is performed by methods which are described as S 15 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 according to Ishikawa et al., 1983, J. Immunoassay 4, 209-327, in which the partntrs are linked with a heterobifunctional reagent.

The method according to the invention can be performed, 25 for example, by, into the wells of a microassay plate which contains bound a previously described HIV antigen preparation, a) simultaneously adding the sample and a solution of the labeled antibody, optionally adding the sample Sfirst 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 salution containing the labeled antii. i.; 9 body to the solid-phase bound antigen, the labeled antigen being directed against said bound antigen; removing the solutions after certain incubation time and washing the wells with buffer resulting in an antigen-antibody complex bound to a solid phase. (This "new" solid phase may be optionally dried or used directly.) Then adding the test sample to the "new" solid phase whereby competitive binding takes place and incubating for a certain time, then removing the sample and measuring the amount of 44 o0 10 label in the sample or on the solid phase.

o.o4 oo a The method according to the invention can also be o performed in the case the use of a diagnostic element which oooo0 contai,,is the solid phase and some or all the necessary 0 a 040 0 0 reaq(ents in dry form.

o 15 The following example represents an embodiment of 0 so o ao the invention, without, however, limiting it thereto.

Example 1. BINDING OF HIV ANTIGEN ONTO MICROASSAY PLATES An HIV antigen preparation prepared according to 0 P 20 U.S. Patent 4,520,113, purified by sucrose density gradient o centrifugation and heat-inactivated was prediluted to 0 0 o 50 pg/ml protein with 100 mmol/l sodium bicarbonate of co 0 pH 9.6. From this dilution a 2-fold dilution series was set 4 00 up with 10 dilutions in the same buffer, i.e. a series with the concentrations 50, 25, 12.5, 6,25, 3.12, 1.56, 0.78, oo 0.39, 0.2 and 0.1 pg/ml was obtained. Sach 100 /il portion of each dilution was placed in 16 wells of type B microassay plates from NUnc, Roskilde, Denmark. The assay plates filled with the dilutions were left at 200C for 18 hours, the solutions in the wells were then aspirated and the wells were washed 3-4 times with 200 #i of a solution of 10 g/l of bovine serum albumin in phosphate-buffered physiological saline, pH 7.4 (PBS) by filling and aspiration, and the assay plates were then dried over silica gel at 200C.

M

10 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 specificities for the core proteins p18, p24, p30 and for the envelope proteins gp41 and gp120 as well as for the HIV polymerases p 5 3 and p 6 5 was fractionated by chromatography on DEAE cellulose. For this, the serum was dialyzed against a buffer of mmol/l Na 2 HPO4/Nal 2 P0 4 pH 7.0, and added onto a column filled with PEAE cellulose DE 32 (Whatman) and equilibrated with the same buffer. The IgG frai 0° ction obtained in the forerun was dialyzed against 0° 15 PBS and adjusted to 4 mg/m protein.

0 0000G0 0 0 0o o A further test with the Western blot technique show- 000 o o ed that all the above antibody specificities were 0 0 0 0 00 recovered in the IgG fraction.

0o The IgG fraction was then reacted with N-gamma-malei- 0 000 O°00 midobutyloxysuccinimide (GMBS), obtained from Behring Diagnostics as described by Tanamori et at., 1983 o°0000 in J. Immunol. Meth. 62, 123-131. 2-Iminothiolanhydo 25 rochloride (Sigma, catalog no. I 6256) was reacted with horseradish peroxidase (POD), obtained from o Boehringer Mannheim, catalog no. 413470, as described o"Oo by King et al. 1978 in Biochem. 17, 1499-1506. An IgG-POD conjugate was prepared from the GMBS-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 ug/mt. 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 mi/I of fetal calf serum, 5 g/l sorbitan monolaurate (Tween 20 R) in PBS and was -11 designated as anti-HIV-POD.

3. Obtaining a TMB substrate preparation A substrate system or a substrate preparation containing hydrogen peroxide and tetramethyltbenzidine (TMB), which was prepared from two stock solutions, was used for the detection of anti-HIV-POD.

Stock solution 1: TMB dihydrochLoride was dissoLved with stirring at a concentration of 5 g/l, i.e. 16 mmol/l, in twice-distilled water and adjusted to pH with 5 N hydrochloric acid. Penicillin G was 0 00 Io added to this solution with stirring in a final con- 00 0 0 g 15 centration of 200 mg/L, i.e. 0.56 mmol/Lt, 00 0 00 C, 0 0 00o0 Stock solution 2: 1.4 mL glacial acetic acid, 1.5 mL S0 0101 N NaOH and 250 mg, i.e. 3 mmol H202 as a urea- 0 00 P 0o 0f 000 hydrogen peroxide adduct were added to 900 ml of twice-distiL led water, After dissolution was complete it was made up to 1 L with twice-distilLed aw water.

O09 0 00 o TMB substrate preparatQni qne part by volume of stock solution I and 10 parts by volume of stock 0 a solution 2 were mixed with one another.

0 '0O 00 0 a n 4. Optimization of the method on0 16 sera, which were confirmed as being HtV negative by the Western blot technique, were diluted 1:5$ with anti-HIV-POD (1 part by volume serutm plus 4 Arts by volume anti-H IV-PO).

1Z5 tL of the dilution was added to each of fhe wetko of the microessay pLates treated with HIV antigen preparation and incubated for I h at A C6 The dilutions were aspiratod anid the wells washed four 12 times with a solution of 1 g/L Tween 20d in PBS by filling and aspiration. 100 ul of the TMB substrate preparation was then added to each well and incubated for 30 min at 20 to 22 C. The incubation was ended by addition of 100 pl 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 pg/ml HIV antigen showed an E 4 50 between 1.68 and 1.74. The solutions from the wells loaded with 1.56 pg/ml HIV antigen showed an E 4 50 between 1.38 and 1.45.

Wells of microassay plates were then treated with an HIV antigen preparation of exclusively 2.5 pg/ml in the previousLy described manner. The evaluation of the 16 sera in the wells treated with 2.5 pg/ml HIV antigen preparation gave a mean E 45 0 of 1.56 0.05 with the same test procedure.

Since in the determination of anti-HIV a limiting value fixing of E greater than 0.8 for anti-HIV-negative 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 samples confirmed to be negative), a concentration of pg/ml HIV antigen preparation was selected for the preparation of microassay plates for the determination of anti-HIV (anti-HIV assay plates) in the treatment of the wells which is also designated as loading.

In all the determinations of anti-HIV described in the following text, the reagents used in the hitherto described preliminary tests, namely the anti-HIV-POD, the TMB substrate preparation and the solution of 1 g/L Tween 2 0 R in PBS designated as washing buffer l~ce F I i' 13 below, we,'e furthermore used.

Determination of human antibodies against HIV a) 25 pl serum or plasma and 100 pl anti-HIV-POD were added to the wells of anti-HIV assay plates and incubated for 1 h at 370C. The content of the wells was removed by aspiration and the wells were washed four times with washing buffer. 100 pl TMB substrate preparation was added to each well, incubated for 30 min at 20-22 0 C and the incubation ended by the addition of 100 pl of 1 N sulfuric acid. E450 of the colored solution was measured against a blank PBS.

S' 0 0Samples which produced an E 4 5 0 of less than 0.30 o were graded as anti-HIV positive, those whose o gE 450 was in the range of 0.31 to 0.80 as antio HIV borderline, and those which produced an E450 above 0.81 as anti-HIV-negative.

00 0 006 o Of 420 samples which were anti-HIV-positive in 0 40 the Western blot technique, 419 samples were likewise found to be positive.

00 40 o a 0 On 1896 plasma or serum samples of healthy sub- So°° jects or of subjects showing other pathological 0000 00o a conditions, the results shown in Table 1 were obtaned.

obtained.

TabLe 1n Positive or SampLes positive boderLine samples on repetition Normal group 1150 0 0 SampLes from risk groups 329 5 2 Lupus erythematos-us 30 0 0 Sera with antibodies against Leukocytic antigens 29 0 0 NucLear antigens 5 0 0 Mitochondrial antigens 5 0 0 HBsAg-positive sera 16 0 0 Anti EBV positive sera 17 0 0 Sera with raised vaLues of Triglycerides 5 0 0 Bi lirubin 5 0 0 HemoLytic sera 5 0 0 SampLes from the same subjects Ser-a 100 0 0 Citrated plasma 100 0 0 Heparinized plasma 100 0 0 antibody-positive subjects. Moreover, the HIV antibody 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 sampIes were pos itive 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 ul whole blood were diluted with pi distilled water containing 20 g/l Triton X-100

R

V tr nd 0.2 mol/l trisodium citrate, and 50 uI of 15 this lysate was added to wells of anti-HIV assay plates. After an incubation of 1 h at 370C 100 pl

I

of anti-HIV-POD was added and then incubated again 0 B 0 0 for 1 h at 37'C. The content of the wells was 0 0 1 q0 removed by aspiration and the wells washed five times with warhing buffer. 100 pl TMB substrate "o preparation was added to each well, incubated for 0 oo 30 min at 20-25°C and the reaction ended by °o addition of 100 pl 1 N sulfuric acid. The exo°°0o tinction of the solutions was measured at 450 nm 25 -gainst a blank of PBS (E 450 0 0000 0 0 Sa° 0 The evaluation of the samples is as described oooo oO o under 5 Of 51 and 5 blood samples which, as 0 0 0 Soo serum, were positive and weakly positive, respectively, in the Western blot technique, all the samples were likewise found to be positive.

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 according to the invention is in good agreement with the Western blot technique regarded as a reference method.

the solid phase. The samples are incubated in the wells ccrrs-l

I.,

4F C 4

CS

16 c) Samples of 50 il serum or plasma or of 50 pl Lysate of whole blood obtained as described in 5 b) were added to the wells of the microassay plates with 100 ul of a solution of 5 pg/ml IgG-POD, prepared by dilution of the 440 pg/ml IgG-POD conjugate described in Example 2, to 5 pg/ml with the already described solution containing fetal calf serum and Tween 20- in PBS and incubated for 10 min at 450C.

The wells were then washed 5 times with washing buffer. 100 pL TMB substrate preparation was then added to each well and incubated for 5 min at 20-22°C. The incubation was ended by addition 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-HIV-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 agreement with the Western blot technique regarded as being a reference method, with regard to sensitivity and specificity.

d) 100 pl of anti-HIV-POD was added to the wells of anti-HIV assay plates and incubated for 1 h at 37 C, 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 20-22 0

C.

omic grounds.

0,

I

17 pl sample and 100 pl of a solution of 10 g/L bovine serum albumin in 50 mmol/l Tris, adjusted to pH 7.4 with HCL, were then added to the wells.

After incubation at 370C for 1 h the content of the wellS was removed and added to weLLs of an unloaded assay plate, mixed there with 100 pL TMB substrate preparation, the mixture was left for 30 min at 20-22 0 C and 100 pU 1 N sulfuric acid was added. E 4 50 was then measured against PBS.

The mean E 4 50 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 borderline and anti-HIV-positive.

TABLE 2 Anti-HIV-negative samples otto t t 0ob tptOtt( 1 0.140 2 0.218 3 0.267 4 0.275 5 0.609 6 0.308

E

450 0.148 0.206 0.279 0.274 0.454 0.316 0.136 0.205 0.270 0.272 0.457 0.351 25 Anti-HIV borderline samples I O t ft i Anti-HIV-positive samples 7 0.769 0.674 0.773 8 0.891 0.865 0.820 9 1.202 0.953 0.863 1.678 2.020 2.202 Table 2 shows that in contrast with embodiments a) to c) the extinction of anti-HIV-negative samples is low and of anti-HIV-positive samples is high here. The Border- Line value for E 4 5 0 for anti-HTV-negative samples is also to be set at 0.30 here.

Claims

18- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An immunochemical competitive method for the detection and determination of antibodies directed against HIV comprising the steps of binding envelope and core proteins of HIV irreversibly to a solid-phase, directly or via a non-immunochemically binding spacer, 0 0 (ii) incubating said bound HIV proteins with 0000oooo oo 0 a sample or a biological fluid suspected 0 Oo to contain HIV antibodies and 00oo. labeled homologous antibodies directed 0 o 000 against envelope and core proteins of HIV which labeled ooo antibodies are non-reactive with antigen from an HIV-free o00 host cell and HIV-free medium or with HIV-free human tissue, 00000 conMp e~ta- °o °(iii) having said labeled antibodies with said unlabeled antibodies from the test sample for said immobilized antigen oaOs (iv) determining the amount of said labeled on.0 antibodies which is bound to said proteins or which is not 00 0 bound. o 0 0 0 0 2. The method as claimed in claim 1, wherein the labeled antibody does not react with antigens from an oa O HIV-free host cell and an HIV-free medium or with HIV-free 0 0 0 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 p 2 4 and the envelope protein designated as gp 120. 1. 27/P SK 156/C.C. i 1 i Ly preferred. ii mmm iim i II .mmmmmmem a f 19 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 contain antibodies which are directed against either I) the p24 core and the gp4l envelope proteins or II) the p24 core and the gpl20 envelope proteins. 5. An assay kit for the detection and for the 0 °o determination of antibodies against HIV in an immunochemical o0 0 7on competitive method containing 0, a) a solid phase to which envelope eind core proteins of HIV are irreversible bound directly or via a non-immunochemically binding spacer, b) 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. 6. The assay kit as claimed in claim 5, wherein the labeled antibody is present as a constituent separated from C the solid phase. It 6 7. The assay kit as claimed in claim 5, wherein the labeled antibody is present as a constitutent immunochemically bound to the solid phase. 8. The assay kit as claimed in claim 5, which consists of a diagnostic element which contains, completely or i partially, the solid phase and the reagents necessary for the detection and the determination, in dry form. 1.27/DISK 156/C.C. S 1 6 after absorption of the specificities which recognize ,I C 00 00 0 S O 0 O000 o 0 0 0 0 0 S4t O 0( 6 C1 t a6 0 i 0 6 O 20 9. The assay kit as claimed in claim 7, which consists of a diagnostic element in which the solid phase and the labeled antibody in dry form are present as a constitutent separated from the solid phase. The assay kit as claimed in claim 8, wherein the labeled antibody is as a constitutent immunochemically bound on the solid phase and which contains further reagents optionally in dry .orm. 11. The use of an assay kit as claimed ir claim 5 in a method for the detection and for the determination of antibody against HIV. 12. The use of an assay kit as claimed in claim 6 or claim 7 in a method for detecting antibodies against HIV. 13. Method of claim 1 wherein said labeled homologous antibodies are contacted with said solid phase such that an antibody-antigen complex is formed and said solid-phase is cleared of any unbound labeled homologous antibodies before said sample is added to the solid phase. DATED this 28th day of January, 1991. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADE MARK ATTORNEYS 'THE ATRIUM' 2ND FLOOR, 290 BURWOOD ROAD HAWTHORN VIC. 3122 AUSTRALIA. 1.27/DISK 156/C.C.