FI90801B - Method for the determination of anti-HIV, supplies therefor and their use in this method - Google Patents

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

90801

Method for the determination of anti-HIV, supplies therefor and their use in this method

The invention relates to a method for the immunochemical determination of HIV-specific antibodies using the principle of competitiveness, as well as to means suitable for the method and their use. The method is suitable for the highly sensitive and specific detection and determination of HIV-specific antibodies of the human IgG and IgM classes.

The short-term infectious disease, AIDS (Acquired Immune Deficiency Syndrome), which has a high mortality rate, is thought to be caused by a virus (retrovirus) or a closely related group of viruses, recently described as Human Immune Deficiency Virus (HIV), but also by the terms T -lymphotropic Virus Type III (HTLV III) or Lymphadenopathy Associated Virus (LAV) has been used.

The virus can be transmitted through human blood and contaminated blood products and has been isolated from cerebrospinal fluid, semen, tear fluid and saliva.

Serological testing for HIV-specific antibodies is performed to determine whether the individual has been in contact with the virus and should be considered a potential carrier for the infectious agent. In addition to being used as a diagnostic tool for AIDS (along with other studies and history), the essential role of an antibody assay is to reduce the risk of transmission of a pathogen through transfusion, transplantation, blood products, or sperm by rejecting donations from antibody-positive individuals. In addition, HIV antibody testing is relevant for relatives of so-called risk groups (homosexuals, drug addicts, children with bleeding disorders, children of infected mothers) as well as in 35 epidemiological studies.

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Detection of anti-HIV using Western Blot, as described, for example, in U.S. Patent 4,520,113, is currently considered the most sensitive method for detecting HIV-specific antibodies. In this case, the enriched and inactivated HIV protein material is separated by electrophoresis on the basis of molecular weights and then transferred to nitrocellulose paper, preferably electrophoretically. In contrast to U.S. Patent 4,520,113, which uses 10 RIAs, the antigen immobilized in this way can be subjected to an indirect ELISA without loss of sensitivity, making it possible to visualize the lines in which the immune reaction has occurred in the enzyme reaction using sparingly soluble dye systems. The distinguished virus-15-specific lines can then be distinguished from the corresponding control material by comparison with non-viral antigen-specific and cross- and non-specifically reacted contaminations, and thus a distinction can be made between true and false positive reactions.

Further described is a method which is a competitive ELISA and which, in terms of specificity and sensitivity, presumably meets the criteria of the Western Blot method (Ärzte-Zeitung / No. 1, 25 / 26.10.85, p. 20).

Undiluted samples are used in this method. It can be used to detect HIV-specific antibodies of IgG and IgM classes simultaneously. The method can be performed using the Wellcome test kit Wellcozyme Anti-HTLV III. As a solid phase, microtest plates are used to which purified HIV antibodies are bound and to which HIV antigens are immunochemically immobilized. Samples are incubated in wells with horseradish peroxidase-labeled anti-HIV. In this case, the HIV antibodies in the sample compete with the labeled anti-HIV for binding sites in the solid phase. The amount of labeled antibodies bound to the wells is inversely proportional to the anti-HIV concentration in the sample.

li 90801 3

Very often, false positive results can be attributed to other tests at the time. Human class II leukocyte antigens (HLA II, especially DR4) derived from human lymphocyte cell lines commonly used for HIV culture and which may lead to anti-HLA DR4 false positive results.

This type of “non-specific” reactions using said competitive test kit for the detection of HIV-specific antibodies (Walgate R., Beardsley T., (1968) Nature 320, 96) can be ruled out by using the human cell line CCRF-CEM for virus culture, as these the cells do not have class II HLA.

When HIV was cultured in the described competitive ELI-15 SA cultured in the H9 cell line, which is known to give a better yield, it was observed that very large amounts of viral starting material were needed to purify the antibodies to be immobilized on the solid phase on the one hand and in order to achieve the maximum sensitivity required for the antigen saturation of immuno-20-bilized antibodies, antigenic material. Furthermore, only very small yields of specific human antibodies were obtained, making use in a screening test impossible for economic reasons.

30 It was found that the competitive method as well

For the detection and determination of IgM and IgG HIV antibodies, it is possible to use a solid phase in which HIV antigens, which may also contain host cell-derived antigens as an impurity, are bound directly, i.e. without antibody-mediated 4 binding. , when using labeled HIV antibodies that do not react with host cell antigens or other human tissue antigens.

The present invention relates to an immunochemical, competitive method for the detection and determination of HIV antibodies using a solid phase consisting of a carrier and bound HIV virus proteins obtained from HIV cultures and a labeled antibody preparation, wherein the labeled and unlabeled antibodies compete for solid-phase binding gaps and the solid-phase proteins are bound to the support directly or via a non-immunochemically binding spacer structure, the method being characterized in that the antibody preparation comprises a combination of at least two antibody specificities, of which one of the specificities is directed against the p24 core protein and the other against at least one envelope protein which is gp41 or gp120, and the labeled antibodies do not react with the HIV-free host cell or the HIV-free medium or the HIV-free medium; with antigens from human tissue.

The invention further relates to a test kit for the detection and determination of HIV antibodies by an immunochemical, competitive method, the test kit comprising a carrier and bound HIV viral proteins obtained from HIV cultures, and a labeled antibody preparation, wherein the solid phase proteins are bound to the support directly or via a non-immunochemically binding spacer structure, and optionally 30 reagents to demonstrate labeling, wherein the antibody preparation comprises a combination of at least two antibody specificities, one directed against the p24 core protein and the other against at least one shell protein; , which is gp41 or gp120, 35 and the labeled antibodies do not react with non-HIV-containing

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90801 with antigens from 5 host cells or non-HIV medium or non-HIV human tissue.

It is preferred that the sensitivity of the method 5 according to the invention corresponds to the sensitivity of the Western Blot method, whereby even in samples with low HIV antibody titers, the binding of labeled antibodies is significantly inhibited, which allows, for example, the use of an enzyme label. color development measurement (Bewer-10 Tung) and allows the separation of positive and negative samples with unusually good resolution, that early serological changes caused by HIV-specific IgM antibodies are also detected with certainty that no interactions and thus human leukocyte antigen (HLA) genes, false-positive results due to nuclear antigens or mitochondrial antigens do not occur, so that testing for a positive result using specific control antigens from host cells is unnecessary and that sera of human origin, citrate, heparin and EDTA in lasas and recalculated plasmas, a reliable determination of HIV-specific antibodies is guaranteed, and inactivation of samples for more than 30 min at 55 ° C (HIV inactivation) and even more than 10 h at 60 ° C (hepatitis B virus inactivation) 25 lead to false positive results.

Solid phase HIV antigen preparations can be prepared by first preparing HIV-positive persistent T cells as described in WO85 / 04897 and then HIV antigen preparations as described in U.S. Patent 4,520,113. using transmission to the human T cell line H9 by co-culturing it with AIDS patient lymphocytes and then separating the host cell culture supernatant by ultracentrifugation. After removal of cell debris and fat, the viral material is purified by sucrose density gradient centrifugation and then inactivated by the addition of Triton and heat treatment.

Suitable carriers for the solid phase are suitable excipients, such as polystyrene, polyvinyl chloride, poly-amide and other synthetic polymers, natural polymers, such as cellulose, and derivatized natural polymers, such as cellulose acetate and nitrocellulose, as well as glass, in particular as glass fiber.

Carriers may be in the form of balls, rods, tubes, and microtest plates. Flat surface formations such as paper strips, sheets and membranes are also suitable. The surface of the carriers can be both permeable and impermeable to aqueous solutions.

Preferred supports are balls, tubes, paper strips and membranes. Particularly preferred carriers are microtest plates.

The solid phase is prepared by irreversibly attaching the HIV antigen preparation to the carrier.

Irreversible binding within the meaning of the invention is, for example, 1) adsorptive binding which is not broken by immunochemical means, such as by high affinity antibodies, nor by dilution and dilution of substances used in the method, such as labeled antibodies. by buffer solutions, 2) non-immunochemically binding spacer-mediated bioaffin binding, where the spacer may be composed of biotin and avidin or other receptor-ligand pairs, 3) covalent direct binding, 4) covalent, chemically bifunctional spacer-mediated binding.

Covalent bonding with water-permeable carriers is preferred, as well as adsorptive bonding with both water-permeable and water-impermeable carriers.

Il 90801 7

Particularly preferred is the direct adsorptive binding of HIV antigen preparations to a gamma-treated polystyrene support.

Antibodies for labeling are selected based on the results of Western Blot analysis of sera from AIDS-5 patients.

To minimize the risk of a false negative diagnosis, to the best of our knowledge, the method must have the ability to detect at least two specific antibodies for the identification of preAIDS and AIDS, namely for the core protein and for the coat protein.

Suitable combinations of specificities include, for example, those that recognize the core protein described as 1. p24 and the envelope protein described as gp41 or the 2. envelope protein described as pp1 and gp120.

Demonstration of other antibody specificities, namely, against the core proteins described as p18, p30, p55, and against the HIV polymerases described as p53 and p65, means anti-HIV additional verification of the detection of positive samples.

To rule out a false-positive diagnosis, antibodies for labeling must not have any specificities that recognize human tissue components, such as leukocyte antigens, nuclear and mitochondrial proteins.

Antibodies from AIDS patients can be used for labeling when they meet the criteria described above by Western Blot.

Furthermore, polyclonal antibodies obtained from sera from animals immunized with HIV can be used if they meet the above criteria when tested by Western blot after adsorption of specificities recognizing the host cells and the culture medium component.

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Monoclonal antibodies are also suitable. In that case, at least two of some of these combinations of specificities must be used, i.e., for example, a combination of anti-p24 and anti-gp41.

Radioactive isotopes, fluorescent and chemiluminescent dyes, as well as enzymes that can be detected using chromogenic, luminogenic or fluorogenic substrate systems, can be used as labels.

Labeling is performed using methods described in the prior art for said markers.

When labeling antibodies with peroxidase, the periodate method of Nakane et al., 1974, 15 J. Histochem. Cytochem. 22, 1084-1090, or the method of Ishikawa et al., 1983, J. Immunoassay 4, 209-327, wherein the parties are bound using a heterobifunctional reagent.

The method according to the invention can be carried out, for example, by simultaneously placing in the wells of a microtest plate bound to the HIV antigen preparation described above a solution and a solution containing a labeled antibody, optionally first a sample and after a certain time a labeled antibody-containing solution. solution, after a certain incubation time the solution is removed and the wells are washed with buffer and then the labeling in the wells is measured, or b) first the solution containing the labeled antibody is placed, after a certain incubation time the solution is removed and 30 wells are washed with buffer, then the microtest plate is dried, then diluted sample and incubate for a period of time, then the sample is removed from the wells and labeling in the sample is measured.

The method according to the invention can also be carried out using a diagnostic element comprising a solid phase and in dry form some or all of the necessary reagents.

The following example illustrates, but does not limit, the practice of the invention.

Example 1. Binding of HIV antigen to microtest plates A preparation of HIV antigen prepared according to U.S. Patent 4,520,113, purified by sucrose density gradient centrifugation and heat-inactivated, was first diluted with sodium bicarbonate, 100 mmol / l, pH 9.6, to give a protein content of 50 pg / ml. From this dilution, parallel dilution series of 10 dilutions were made in the same buffer, i.e. a series of concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 15 0 was obtained. , 2 and 0.1 pg / ml. 100 μΐ of each dilution was placed in a well of 16 Nuncin Typ B microtest plates, Nunc, Roskilde, Denmark. Test plates filled with dilutions were allowed to stand for 18 hours at 20 ° C, then the solutions were aspirated off and the wells were washed 3-4 times with 200 of a solution of 10 g / l bovine serum albumin in phosphate buffered saline, pH 7.4 ( PBS), filling and aspirating off and then the test plates were dried over silica gel at 20 ° C.

25 2. Preparation of peroxidase-labeled HIV antibody

Western blot-selected serum from an AIDS patient containing antibody specificities for core proteins p18, p24, p30 and p55, envelope proteins 30 pg41 and gp120, and HIV polymerases p53 and p65 was fractionated by chromatography using DEAE cellulose. .

For this, serum was dialyzed against Na 2 HPO 4 / NaH 2 PO 4 buffer, 30 mmol / l, pH 7.0, and applied to a column packed with DEAE Cellulose DE 32 packing (Whatman) and ta-35 weighted with the same buffer. The IgG fraction obtained in the eluate was dialyzed against PBS and adjusted to contain 4 mg / ml protein.

In the next test, the Western Blot method showed that all of the above antibody specificities were still detectable in the IgG fraction.

The IgG fraction was then reacted with Ν-γ-maleimidobutylyloxysuccinimide (Behring Diagnostics) as described by Tanamori et al., 1983, J. Immunol. Meth. 62, 123-131, have described. 2-Iminothiolane anhydride 10 (Sigma, Cat. No. I 6256) was reacted with horseradish peroxidase (POD) (Boehringer Mannheim, Cat. No. 413470) as described by King et al. 1978, Biochem. 17, 1499-1506, have described. An IgG-POD conjugate was prepared from the GMBS-IgG conjugate and the iminothiolane-POD conjugate as described by Tanamori.

The resulting IgG-POD conjugate solution had a protein content of 440 pg / ml. The ratio of POD to IgGr was determined to be 2.5. The solution was then diluted to 500 ng / ml IgGPO 4 with a solution of 50 ml / l bovine 20 fetal serum and 5 g / l polyoxyethylene (20) sorbitan monolaureate (RTween 20) in PBS and labeled anti -HIV-POD.

3. Preparation of a TMB substrate preparation

A substrate system containing hydrogen peroxide and tetramethylbenzidine (TMB) or a substrate preparation prepared from two stock solutions was used to detect anti-HIV-POD.

Stock solution 1: TMB dihydrochloride was dissolved with stirring to a concentration of 5 g / l, i.e. 16 mmol / l in doubly distilled water, and the pH was adjusted to 1.5 with 5N hydrochloric acid. To this solution was added with stirring penicillin G to a final concentration of 200 mg / l, i.e. 0.56 mmol / l.

Stock solution 2: To 900 ml of doubly distilled water 35 was added 1.4 ml of glacial acetic acid, 1.5 ml of 1N NaOH

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90801 11 and 250 mg, i.e. 3 mmol H 2 O 2 as urea hydrogen peroxide adduct. After complete dissolution, it was made up to 1 liter with distilled water.

TMB substrate preparation: One volume of stock solution 1 and 10 volumes of stock solution 2 were mixed together.

4. Optimization of the method 16 sera confirmed to be negative by Western Blot were diluted 1: 5 with anti-HIV POD (1 volume of serum and 4 volumes of anti-HIV POD).

125 μΐ dilution was placed in all wells of microtest plates treated with HIV antigen preparation and incubated for 1 h at 27 ° C. The dilutions were aspirated off and the wells were washed four times with a solution of 1 g / l RTween 20 in PBS, filling and aspirating. 100 μΐ of TMB substrate preparation was then placed in each well and incubated for 30 min at 20-22 ° C. The incubation was stopped by adding 100 μΐ of 1-normal 20 sulfuric acid in each case. The extinction of the solutions was measured against PBS at 450 nm.

Solutions from wells coated with HIV antigen at a concentration of 3.12 pg / ml gave E450 values between 1.68 and 1.74. Solutions of coated wells with HIV-an-25 tigen at a concentration of 1.56 pg / ml gave E450 values between 1.38 and 1.45.

The wells of the microtest plates were then treated as described above with HIV antigen preparation alone at a concentration of 2.5 pg / ml. 16 sera were obtained with an HIV antigen preparation at a concentration of 2.5 pg / ml in the treated wells in a similar experimental procedure with a mean E450 value of 1.56 ± 0.05.

35 As the preferred cut-off value in the anti-HIV test is E> 0.8 for anti-HIV negative and 12 E <0.8 for anti-HIV positive samples (the cut-off value is defined as half of the average extinction), a concentration of 2.5 pg / ml of the HIV antigen preparation was selected for the preparation of microtest plates for the determination of anti-HIV (anti-5 HIV test plate) for the treatment of wells, also called coating.

Also used in all of the anti-HIV assays described below were the reagents used in the primers described so far, namely anti-HIV-POD, TMB substrate preparation and the following solution as wash buffer containing 1 g / l RTween 20 in PBS. .

5. Determination of human HIV antibodies (a) 25 μ 25 serum or plasma and 100 μ 100 anti-HIV-15 PDT were placed in the wells of anti-HIV test plates and incubated for 1 h at 37 ° C. The contents of the wells were removed by suction and the wells were washed four times with wash buffer. 100 μΐ of TMB substrate preparation was placed in each well, incubated for 30 min at 20-22 eC, and the incubation was stopped by the addition of 100 μΐ of 1N sulfuric acid. The E450 of the stained solutions was measured against zero in PBS.

Samples that gave an E450 of less than 0.30 were considered anti-HIV positive, those with an E450 in the range of 0.31 to 0.80 were considered anti-HIV, and those that gave an E450 in the range of 0.31 to 0.80 were considered anti-HIV. which gave an E450 value greater than 0.81.

Of the 420 samples that were positive by Western Blot, 419 samples were also found to be positive.

30 Based on 1896 plasma or serum samples from healthy subjects or subjects with other pathological conditions, the test result shown in Table 1 was obtained.

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Five samples from at-risk subjects who initially responded positively or were at the border were further examined using Western Blot. Of these, only the two samples that were found to be positive when repeated using the method of the invention were also positive.

The results thus show a good agreement between the method according to the invention and the Western Blot method.

10 b) 50 μl of whole blood samples were diluted in 50 μl of distilled water containing 20 g / l Triton x 100 and 0.2 mol / l trisodium citrate, and 50 μl of this lysate was placed in the wells of anti-HIV test plates. After incubation for 1 hour at 37 ° C, 15 100 μΐ of anti-HIV-POD was added, followed by another incubation for 1 hour at 37 ° C. The contents of the wells were aspirated and the wells were washed five times with wash buffer. TMB substrate preparation, incubated for 30 minutes at 20-25 ° C, and the reaction was stopped by the addition of 100 μapp of 1 N sulfuric acid.The extinction of the solutions was measured at 450 nm relative to the zero value of PBS (E450). All 51 and 5 serum samples positive or weakly positive by Western blot, respectively, were found to be positive. anti-HIV negative were also found to be negative. This shows a good agreement between the method according to the invention and the Western Blot method 30 used as a comparison.

c) 50 μ1: η serum or plasma samples or 50 μ1: η whole blood lysate samples prepared as described in 5b) above and 100 μΐ of a solution containing 5 pg / ml IgG-POD prepared by diluting 35 in Example 2 IgG-POD conjugate concentration

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90801 15 440 pg / ml to a concentration of 5 pg / ml with the above-described solution containing fetal bovine serum and RTween 20 in PBS was placed in the wells of microtest plates and incubated for 10 minutes at 45 ° C. The wells 5 were then washed five times with wash buffer. 100 μ 100 of TMB substrate preparation was then added to each well and incubated for five minutes at 20-22 ° C. Incubation was stopped by the addition of 1 N sulfuric acid. The evaluation was performed as described in 5a) above. All 51 10 resp. 5 samples that were positive or weakly positive by serum by Western Blot were found to be positive. All 225 samples that reacted anti-HIV negative were also found to be negative.

This indicates that the rapid method of the invention is consistent in sensitivity and specificity with the Western Blot method used as a reference method.

d) Wells of anti-HIV test plates were loaded with 100 μΐ anti-HIV-POD and incubated for 1 h at 37 eC, anti-HIV-POD was aspirated and the wells were washed four times with wash bag-20 discipline. Anti-HIV test plates treated in this way were dried on silica gel at 20-22 ° C.

25 μΐ of sample and 100 μΐ of a solution of 10 g / l bovine serum albumin in Tris, 50 mmol / l, pH adjusted to 7.4 with HCl, were then placed in the wells. After 1 h of in-25 incubation at 37 ° C, the contents of the well were taken and placed in the well of an uncoated test plate, 100 μΐ of TMB substrate preparation was added, the mixture was allowed to stand for 30 min at 20-22 ° C, and 100 μΐ of 1N sulfuric acid was added. E450 was then measured against PBS.

The E450 averages per sample for the three parallel assays are shown in Table 2 for samples that were considered anti-HIV negative, anti-HIV borderline cases, and anti-HIV positive 35 by the method described in (a).

16 TABLE 2 E450 anti-HIV negative samples 1 0.140 0.148 0.136 5 2 0.218 0.206 0.205 3 0.267 0.279 0.270 4 0.275 0.274 0.272 anti-HIV borderline cases 5 0.609 0.454 0.457 10 6 0.308 0.316 0.351 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 15 10 1.678 2.020 2.202

Table 2 shows that, in contrast to method a) to c), the extinction of anti-HIV negative samples is low and that of anti-HIV positive ones is high. In this exemplary embodiment of Method 20, the cut-off value for E450 for anti-HIV negative samples is similarly set at 0.30.

Claims (10)

90801 An immunochemical, competitive method for the detection and determination of HIV antibodies using a solid phase consisting of a carrier and bound HIV virus proteins obtained from HIV cultures and a labeled antibody preparation, wherein the labeled and unlabeled the antibodies compete for solid-phase binding compulsions and the solid-phase proteins are bound to the support directly or via a non-immunochemically binding spacer structure, characterized in that the antibody preparation contains a combination of at least two antibody specificities, one of which is oriented p24 and one against at least one coat protein, gp41 or gp120, and the labeled antibodies do not react with antigens from an HIV-free host cell or HIV-free medium or HIV-free human tissue. with them. Method according to Claim 1, characterized in that the antibodies to be determined and the labeled antibodies are introduced onto the solid phase simultaneously. A method according to claim 1, characterized in that the labeled antibodies are first applied to proteins bound to a solid support and after an incubation time and optionally removal of the liquid phase and washing of the solid phase, a sample containing the antibodies to be detected is applied to the solid phase. . A method according to claim 1, characterized in that the solid phase is first incubated with a sample containing the antibodies to be determined and then, optionally after removal of the liquid phase and washing of the solid phase, labeled antibodies are added. 35 The method of claim 1, characterized in that the labeled antibodies are monoclonal antibodies. 6. A test kit for detecting and determining HIV antibodies by an immunochemical, competitive method, the test kit comprising a carrier and bound HIV virus proteins obtained from HIV cultures and a labeled antibody preparation, wherein the solid phase proteins are bound to the support 10 directly or via a non-immunochemically binding spacer structure, and optionally reagents for detecting labeling, characterized in that the antibody preparation contains a combination of at least two antibody specificities, one of which is directed against the p24 core protein, and the other against at least one envelope protein, gp41 or gp120, and the labeled antibodies do not react with antigens from an HIV-free host cell or HIV-free medium or HIV-free human tissue. Test kit according to claim 6, characterized in that the labeled antibody is separate from the solid phase. Test kit according to Claim 6, characterized in that the labeled antibody is part immunochemically bound to the solid phase. Test apparatus according to one of Claims 6 to 8, characterized in that it consists of an element which contains, in solid form and in whole form, all or part of the reagents required for detection and determination. Use of a test kit according to any one of claims 6 to 9 in a method for detecting and determining HIV antibodies. Il 90801