WO1991019011A1

WO1991019011A1 - Immunoassays for anti-hiv-1 antibodies

Info

Publication number: WO1991019011A1
Application number: PCT/US1991/004021
Authority: WO
Inventors: James R. Rusche; Albert T. Profy
Original assignee: Repligen Corp
Current assignee: Repligen Corp
Priority date: 1990-06-08
Filing date: 1991-06-07
Publication date: 1991-12-12
Anticipated expiration: 1992-12-08
Also published as: CA2082806A1; EP0532673A4; EP0532673A1; JPH05507559A

Abstract

A method of diagnosing an HIV-1 infection in an individual comprising contacting a biological sample from the individual with a first antigen capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in an individual of HIV-neutralizing antibodies, formation of immune complexes with the first antigen being diagnostic of an HIV infection in the individual.

Description

IMMUNOASSAYS FOR ANTI-HIV-1 ANTIBODIES

pac σround of the Invention The invention relates generally to immunoassays in which antibodies specific for human immunodeficiency virus type 1 (HIV-1) are detected.

HIV-1 is the proposed causative agent of Acquired Immune Deficiency Syndrome (AIDS) . Individuals infected by HIV-1 produce serum antibodies which bind various viral proteins, particularly the core, poly erase, and envelope proteins (Allan et al., 1985, Science 228:1091; Chang et al., 1985, Science 228:93, and 1985, Nature 315:151; Crowl et al., 1985, Cell 41:979; Veronese et al., 1985, Science 229:1402, and 1986, Science 231:1289). Immunoassays which detect these antibodies are used to diagnose HIV-1 infection and to screen donated blood for HIV-1 contamination. For example, enzyme-linked im unosorbant assays (ELISAs) and radioimmunoassays (RIAs) of various types are used for rapid screening and initial diagnosis. Whole viral lysates, which contain both HIV and non-HIV proteins, and purified viral components, including recombinant proteins and synthetic peptides, have been used as antibody adsorbants. Western blots have been used to detect antibodies which recognize HIV proteins that have been separated by size.

Summary of theT Invention The invention provides diagnostic and detection methods which can be used to distinguish between persons who are infected with HIV-1 and persons who have been vaccinated against HIV-1 with peptides corresponding to all or a portion of the principal neutralizing determinant (PND) , and also offers a more sensitive and less expensive diagnostic method, e.g., for detecting seroconversion. Accordingly, in one aspect, the invention features a method of diagnosing HIV-1 infection in a human which includes contacting a biological sample from the human with a first antigen which is capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in an human of HIV-neutralizing antibodies, formation of immune complexes with the first antigen being diagnostic of an HIV infection in the individual. (As used herein,

'neutralization* refers to the ability of the antibody to inhibit HIV infection of cells by cell-free virions, or fusion of infected and uninfected cells, or both; 'immune complex' refers to a non-covalently bound antibody/antigen pair; 'specific for' and 'reacting with' mean capable of specifically binding to an antigen; an antibody that is 'substantially incapable of forming immune complexes' means -that there will be less than a three-fold difference between the binding of the antibody to a specified antigen and the binding of normal human serum to the same antigen.)

In preferred embodiments, the biological sample may be a bodily fluid, e.g., serum; and the first antigen is immobilized on a solid support, although any standard immunoassay format can be used. To determine if a person has been vaccinated, the method may further include the steps of contacting the sample with a second antigen which is immobilized on a second solid support, wherein the second antigen is the immunogen. (As used herein, "first" and "second" do not imply the order in which the screening steps are performed.)

Preferably, the first antigen is HIV-1 gpl60 envelope protein which lacks a portion of the principal neutralizing determinant (PND) or a portion of the PND to which neutralizing antibodies can be raised, i.e., a neutralizing portion thereof. (As used herein, the 'principal neutralizing determinant' encompasses the region of gpl60 located between amino acids 297 and 331, inclusive, according to the number convention of Ratner et al., 1985, Nature 313:277, hereby incorporated by reference.) Most preferably, this protein is gpl60Δ135. (As used herein, gpl60Δ135 means any gpl60 protein that lacks the PND; gpl60Δ135 can be expressed from a recombinant gene that has been engineered to delete the DNA coding for the PND or a portion of the PND to which neutralizing anitbodies are raised, or may be a protein which has been chemically altered to lack the PND.)

Preferably, the second antigen includes the PND of gpiβO; more preferably, it includes intact HIV-1 gpl60 envelope protein (i.e., which contains the principal neutralizing determinant), e.g., gplβOMN, or a fragment thereof or a synthetic peptide having an amino acid sequence corresponding to that of gpl60, any of which may be the immunogen. Preferably, the second antigen is a protein fragment or a synthetic peptide having an amino acid sequence corresponding to that of the PND of gpl60, e.g., the PND of gplβOMN (i.e., gpl60 from the MN isolate) or a neutralizing portion thereof; most preferably, the second antigen is a combination of gplβOMN and the PND of the immunogen. (As used herein, an 'immunogen' is an antigen which causes the production of neutralizing antibodies by the immune system of a mammal, e.g., a rabbit on a human.) Thus, the detection method in which the antigen is gpl60Δ135 results in detection of HIV-1-specific antibodies which recognize regions of gplβo lying outside the PND, whereas the detection method in which the antigen is gpl60 (and may include PND peptides) results in detection of antibodies specific for any portion of the gpiβO molecule, and will include the detection of PND-specific antibodies. A biological sample from a person infected with HIV-1 would include antibodies detectable by both methods, whereas a sample from a person who had been immunized against HIV-1 with PND peptides but who is not infected with the virus, would include antibodies detectable only in the latter method, which employs either the complete gpl60 molecule, i.e., that contains the PND, or PND peptides, or a combination of the two. As used herein, an amino acid sequence 'corresponding to' or 'derived from' means the amino acid sequence is identical to all or a portion of another amino acid sequence. As used herein, 'MN' refers to the MN prototype of HIV or a viral variant of the MN prototype; the MN prototype virus is defined by the amino acid sub-sequence within the PND region of the gplβO envelope protein R-I-H-I-G-P-G-R-A-F-Y; MN viral variants are herein defined as variants which exhibit complete amino acid sequence homology at residues I-G-P-G-R, and at least 36% homology with the remaining 6 amino acids of the MN sequence given above.

In other preferred embodiments, the method further includes contacting the sample with an agent (e.g., an enzymatically or radioactively labeled component) capable of binding to the first and second adsorbed antibodies, and detecting the bound agent as an indication of the presence in the sample of first and second antibodies; for example, the agent may be protein A or a labeled antibody capable of binding to the antibodies. The invention also features an HIV-1 envelope in which the principal neutralizing determinant, i.e., the region lying between amino acid residues 297-331, inclusive, or a region encompassing all or part of the PND, or a neutralizing portion of the PND has been deleted.

In preferred embodiments, this protein is gpl60Δ135, and may be immobilized on a solid support. The invention also features a. it for detecting the presence of HIV-1 antibodies in a human biological sample, which includes a first antigen capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in a human of HIV-neutralizing antibodies, and an agent capable of reacting with first and second antibodies.

In preferred embodiments, the first antigen is immobilized on a solid support; preferably, it is HIV-I gpl60 which contains a deletion of the PND region lying between residues 297 and 331, or a portion of the PND to which neutralizing antibodies are raised; most preferably, the first antigen is gpl60Δ135. The detection agent may be labeled and may be, for example, protein A or a labeled anti-human immunoglobulin, e.g., a labeled Fc-region- specific antibody. The kit may further include the second antigen immobilized on a support; preferably, the second antigen is a peptide having an amino acid sequence corresponding to the amino acid sequence of an immunogen which causes production of HIV neutralizing antibodies, i.e., the HIV-1 PND sequence from the MN variant; more preferably, gpl60 is immobilized on the second support, and the peptides have sequences corresponding to the sequence of the PND of the MN PND. In another aspect, the invention features a method of detecting HIV-1 infection in a person, which includes contacting an antibody-containing biological sample from the person with HIV-1 gplβOMN, formation of immune complexes between the sample antibodies and gpl60MN being diagnostic of HIV infection. Because the MN isolate is a commonly occurring HIV isolate, the use of gpi60 containing the PND sequence of the MN isolate provides for a more sensitive assay and allows for the earlier detection of seroconversion than can be obtained with other gpl60 PND sequences, e.g., IIIB.

In preferred embodiments, the first antigen is immobilized on a solid support. The antigen may further include a peptide having an amino acid sequence corresponding to an HIV-1 gpl60 PND or a neutralizing portion of the PND. When a synthetic peptide corresponding to all or a portion of the PND are added to assays based on a gpl60 adsorbant, the amount of gpl60 used in the assay can be reduced without a loss in assay sensitivity.

The invention also features a kit for detecting HIV- 1 antibodies in a human biological sample, which contains immobilized HIV-1 gplβOMN, and an agent capable of reacting with an antibody. In preferred embodiments, the sample is serum and the agent is a labeled molecule.

The invention also features a kit for diagnosing the presence of HIV-1 antibodies in a human biological sample which includes combined immobilized gpl60 and a peptide having an amino acid sequence corresponding to that of the MN PND or a portion thereof, and an agent capable of reacting with the antibodies.

Methods of the invention are useful for distinguishing between HIV-1 infection and immunization, and are inexpensive and safe; for example, recombinant protein components, such as full-length gplβO, which are expensive to produce, may be supplemented with synthetic peptides having the same antigenic sites. Methods of the invention thus reduce expense without sacrificing sensitivity, and enable early detection of seroconversion.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. pesσription pf Preferred Embodiments Before describing preferred embodiments of the invention, the drawings will be described. Drawings Fig. 1 is an ELISA in which gpl60Δ135 and gplβOIIIB

(i.e., gplβO from the IIIB isolate) are compared as antigens for a panel of HIV positive human sera.

Fig. 2 is an ELISA in which the response of the gplβOΔ135-based assay was compared to the response of the gplβOIIIB-based assay to various dilutions of a single serum.

Fig. 3 is an ELISA in which PND-based peptides were used.

Fig. 4 is an ELISA in which the antigen consisted of synthetic peptides in combination with gplβOIIIB.

Fig. 5 is a Western blot in which gpl60Δ135 and gplβOIIIB were probed with normal human serum (NHS) , human HIV positive (+) serum, or anti-RP174C serum.

The invention provides a method of detecting and distinguishing between antibodies which recognize different regions of the HIV-1 envelope protein gplβO, e.g., the principal neutralizing determinant vs. non-PND regions, without a significant loss of sensitivity for the antibodies. This is accomplished by using, as an adsorbant, a recombinant envelope protein from which the amino acids corresponding to only one determinant are deleted; as a result, antibodies directed against the non-deleted determinant(s) are detected, whereas those directed against the deleted determinant are not detected. Preparation of recombinant σplβOIIIB- gp!60Δ135. and σplβOMN/IIIB gpl60Δ135 is a gplβO polypeptide which contains a deletion of the PND region. gpl60Δ135 is used herein as an example of a gpl60 polypeptide having a deletion which renders the protein incapable of reacting with an antibody to the PND; the deletion may encompass one or more amino acids between residues 297 and 331, inclusive. The preparation of gpl60Δ135 was described by Javaherian et al., 1989, Proc. Nat. Aca. Sci. 86:6768. gpl60Δ135 was derived from the gplβOIIIB envelope gene cloned into the baculovirus transfer vector pAcβlO (Rusche et al, 1987, supra) . The Bglll fragment of this envelope gene, containing the PND, was subcloned into an M13 vector for site-directed mutagenesis. A synthetic oligonucleotide

(5'<^ACTAATGTTAC_AATGTGCGGGTC_TTGTAα\ATTAATTT3') was used to direct the deletion of nucleotides encompassing the PND resulting in the deletion of amino acids 300-328 of the envelope gene. This mutagenized fragment was then cloned back into the baculovirus transfer vector and recombinant baculovirus obtained as described in Rusche et al., 1987, supra. In the assays described below, a gplβOMN/IIIB hybrid envelope gene, in which sequence from the IIIB isolate surrounds PND region consisting of MN determinants, was used as an antigen. Although the gpl60MN/IIIB hybrid was used, any MN polypeptide or fragment, or any MN hybrid which retains the PND of the MN isolate may be used, e.g., gpl^βOMN. The gp 60MN/IIIB hybrid was constructed by direct replacement of the Bglll fragment of the MN envelope. This resulted in a hybrid IIIB/MN envelope gene having an MN PND and contained in a IIIB gplβO framework. Antibodies generated to the resulting PND region of the hybrid molecule are specific for MN. This hybrid envelope gene contained in the baculovirus transfer vector pAcβlO, was then used to generate recombinant baculovirus, as described in Rusche et al., 1987, supra.

Production of recombinant envelope proteins by infection of insect cells (S_ fruσiperda) with recombinant baculovirus was performed as described by Rusche et al., 1987, supra. preparation of Synthetic Peptides

Synthetic peptides corresponding to the principal neutralizing determinant were prepared by the Merrifield solid-phase synthesis procedure (Merrifield, 1963, J. Amer. Chem. Soc. 85:2149). Peptides were purified by reverse- phase HPLC and characterized by amino acid composition analysis, according to procedures well-known in the art. The designation of the peptides, the isolate to which they correspond, and their sequences are as follows: RP135 (IIIB) NNTRKSIRIQRGPGRAFVTIGKIGC RP174C (IIIB) CNNTRKSIRIQRGPGRAFVTIGKIGC RP1 2 (MN) YNKRKRIHIGPGRAFYTTKNIIGC

RP70C (MN) INCTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAHCNIS Peptides RP174c and RP70c each contain an intramolecular disulfide bond between the two cysteine residues. A method for creating such a bond is described in Zhang et al., 1988, Biochemistry 27:3785. The existence of these bonds was confirmed by mass spectrometric analysis. Detection of Antibodies The adsorbed antibodies are detected by any of several well-known methods, one example of which is an enzyme-linked i munosorbant assay (ELISA) . The antigen (gpl60Δ135, gplβOIIIB, gpl60MN/IIIB, synthetic peptides, or a combination of these reagents) is diluted in suitable buffer such as a sodium carbonate buffer (pH 9.6) to a concentration of >0.125 ug/ml (preferably 1 ug/ml) and 50 ul is added to each well of a polystyrene 96-well microtiter plate (Immunodynamics, Chantilly, VA) . After incubation for 16-20 hours at room temperature (RT) , the wells are emptied, refilled with a blocking solution such as 10 mg/ml bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.02% sodium azide, and allowed to incubate :> 60 min at RT. The wells are then emptied and washed three times with PBS containing 0.1% Tween-20 (PBS-T) . Serum samples to be tested for the presence of anti-gplβO antibodies are diluted in PBS-T and added to the plate (50 ul per well) . Several serum dilutions may be selected. After incubation for 90 min at room temperature (RT) , the wells are emptied and again washed three times with PBS-T. In order to detect human antibodies adsorbed to the gplβO antigen, a labeled antibody-detecting agent solution is added to the wells and allowed to incubate for 30-90 min at RT. Examples of agents which may be used are goat anti- human IgG:horseradish peroxidase conjugate, goat anti-human IgG:alkaline phosphatase conjugate, Staphylococcal protein A:horseradish peroxidase conjugate, or any conventional conjugate. Following incubation with the probe, the wells are washed three times with PBS-T and treated with a suitable substrate solution such as the horseradish peroxidase substrate 3,3*-azino-bis(3-ethyl benzthiazoline- 6-sulfonic acid) (ABTS) and hydrogen peroxide. The solutions are allowed to incubate 15-20 min and the absorbance of the solution in each well (at 410 nm for ABTS) is determined. The absorbance of each solution is proportional to the amount of antibody adsorbed by the antigen. Testing of Serum Samples Using σpiβo Antigen

The procedure described above was used to test a panel of fourteen HIV-1-positive human sera for binding to gpl60Δl35 or gpl60HIB (each coated at lug/ml in carbonate buffer) . Serum samples, which were obtained from the Interstate Bloodbank, Memphis, TN, and shown to test positive in the HTLVIII EIA (Abbott Laboratories, Chicago, IL) , were tested at a 1:200 dilution. A goat anti-human IgG:horseradish peroxidase conjugate was used as the detection agent. Signal-to-background ratios for assay of each serum are shown in Fig. 1. The data demonstrates that immunoassays which employ gpl60Δ135 and gplβOIIIB are equally effective for detecting anti-HIV antibodies in all of the human serum samples tested. A single HIV-positive serum was tested at varying dilutions using the above procedure. Dilutions ranged from 1:1 to 1:8192. As shown in Fig. 2, the response of the gpl60Δ135-based assay was identical to the response of the gpl60IIIB-based assay at each dilution, within experimental error.

Testing of Serum Samples Using crplβO and PND Peptides as Antigens

In order to show that antisera elicited by PND- based synthetic peptides do not cause a response in the immunoassay based on gpl60Δ135, the following experiments were performed. Animals were immunized with the following reagents, prepared as described above:

RP174c (guinea pig 596, guinea pig 598) gpl60Δ135 (goat 1116) gplβOIIIB (rabbit #1)

Antisera were prepared according to conventional methods by immunizing the designated animal with the corresponding peptide or protein. Each serum was tested in immunoassays using each of four different antigens: RP135, RP142, gpl60Δ135 and gplβOIIIB. Antigens were coated at 1 ug/ml in carbonate buffer, and bound antibodies were detected using goat anti-human IgG:horseradish peroxidase conjugate. The resulting absorbance values are shown in Fig. 3. Antisera from the guinea pigs immunized with RP174c (corresponding to the IIIB PND) produced significant absorbance when RP135 and gplβOIIIB were used as antigens, but produced no response when gpl60Δ135 or RP142 (a peptide from the MN PND) were used. Antiserum elicited by gpl60Δ135 or gpl60IIIB produced a similar response when gpl60Δ135 of gplβOIIIB were used as antigens.

The results of these ELISAs show that an enzyme immunoassay based on gpl60Δ135 is as effective as an assay based on gplβOIIIB in responding to HIV-positive human serum, but that the former assay, in contrast to the latter, does not respond to antisera raised against peptides corresponding to the PND. Recombinant gplβOMN/IIIB as Antigen Recombinant gpl60MN/IIIB was compared with gplβOIIIB for use as an antigen in the enzyme immunoassay described above. Wells of the icrotiter plate were coated with solutions of gplβO (in carbonate buffer) at concentrations of 0.125, 0.25, 0.50, 1.0, and 2.0 ug/ml. A pool of four HIV-positive human sera was assayed on each antigen at dilutions of 1:100, 1:1000, and 1:10,000. In addition, a pool of normal human sera was assayed at a dilution of 1:100. Goat anti-human IgG:horseradish peroxidase conjugate was used as probe and ABTS was used as the substrate. Measured absorbance values (at 410 nm) are shown in Table 1. The results presented in Table 1 show that the response of the assay when gpl60MN/IIIB was used as the antigen was greater. Table 1 also shows that the response of the gpl60MN/IIIB-based assay to the normal human serum pool was also greater than the. response of the gpl60IIIB-based assay, but if these background values are subtracted, the response to the HIV-positive serum pool remains greater when gpl60MN/IIIB is used as the antigen, particularly at lower concentrations of gplβO. PND Peptides in Combination with gplβO as Antigen

Synthetic peptide RP142, corresponding to the principal neutralizing determinant of the MN isolate, was used in combination with gplβOIIIB as a antigen. Wells of a microtiter plate were coated with varying concentrations of gplβOIIIB (in carbonate buffer) , either with or without peptide RP142. The response of each assay to an HIV- positive human serum (at 1:200 dilution) is shown in Fig. 4. The data in Fig. 4 shows that a combination of the synthetic peptide and gpl60 results in higher sensitivity in the ELISA and allows for use of lower concentrations of gplβO than when gplβO is used alone as an antigen. Western Blot analysis using gpl60-Δ135

Gpl60Δ135 and gplβOIIIB were individually subjected to SDS polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters (Towbin et al., 1979, Proc. Nat. Aca. Sci. 76:4350). Blots were treated with an HIV-positive human serum sample, antisera raised against RP174c in guinea pigs, and normal human serum. Adsorbed antibodies were visualized by treatment of the blots with goat anti-human IgG:horseradish peroxidase conjugate or goat anti-mouse IgG:horseradish peroxidase conjugate, followed by a diaminobenzidine solution and hydrogen peroxide. Fig. 5 shows that the HIV positive human serum recognized both of the blotted gplβOs, whereas the anti-RPl74c serum recognized gpl60IIIB, but not gpl60Δ135. Normal human serum did not recognize either protein.

Other embodiments are within the following claims.

What is claimed is:

Claims

1. A method of diagnosing an HIV-1 infection in a human comprising contacting a biological sample from said human with a irst antigen capable of forming immune complexes with antibodies specific for native HIV-1 but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes the production in humans of HIV-neutralizing antibodies, formation of immune complexes with said first antigen being diagnostic of an HIV infection in said human.

2. The method of claim 1 wherein said first antigen is immobilized on a solid support.

3. The method of claim 2, further comprising the steps of contacting said sample with said second antigen immobilized on a solid support and then detecting immune complexes with said second antigen as an indication of thm presence of antibodies capable of binding to said second antigen.

4. The method of claim 1, said first antigen comprising an HIV-1 gpl60 envelope protein which lacks the principal neutralizing determinant or a neutralizing portion thereof.

5. The method of claim 4, said first antigen comprising gplβO having a deletion of one or more amino acids between residues 297 and 331, inclusive.

6. The method of claim 5, said first antigen comprising gpl60Δ135. ^~

7. The method of claim 3 wherein said second antigen comprises the principal neutralizing determinant of the HIV gpl60 envelope protein.

8. The method of claim 6 wherein said second antigen comprises the HIV-1 gpl60 envelope protein.

9. The method of claim 8, said second antigen comprising the HIV-1 gplβOMN envelope protein.

10. The method of claim 3, said second antigen comprising a peptide having an amino acid sequence corresponding to an amino acid sequence of the principal neutralizing determinant of gplβO or a neutralizing portion thereof.

11. The method of claim 10 wherein said principal neutralizing determinant is derived from the principal neutralizing determinant of the MN variant of HIV.

12. The method of claim 9 wherein said second antigen further comprises a peptide having an amino acid sequence corresponding to the amino acid sequence of an immunogen which causes production of HIV neutralizing antibodies.

13. The method of claim 3, further comprising contacting said sample with an agent capable of binding to said first and second antibodies, and detecting said bound agent as an indication of the presence in said sample of said first and second antibodies.

14. The method of claim 13, said agent being protein A.

15. The method of claim 13, said agent being a labeled antibody capable of binding to both of said antibodies.

16. An HIV envelope protein containing a deletion of the principal neutralizing determinant or a neutralizing portion thereof.

17. The HIV-1 envelope protein of claim 16, containing a deletion of one or more amino acids between residues 297 and 331, inclusive.

18. The protein of claim 17, said protein being gpl60Δ135.

19. The protein of claim 18, said protein being immobilized on a solid support.

20. A kit for detecting the presence of HIV-1 antibodies in a human biological sample, comprising a first antigen capable of forming immune complexes with antibodies specific for native HIV-I but substantially incapable of forming immune complexes with antibodies specific for a second antigen which causes production in an individual of HIV-neutralizing antibodies, and an agent capable of reacting with said first and second antibodies.

21. The kit of claim 20, said agent being protein A.

22. The kit of claim 20 wherein said agent is a labeled antibody.

23. The kit of claim 19, said first antigen being immobilized on a solid support.

24. The kit of claim 20, said first antigen comprising an HIV-1 gplβO envelope protein which lacks the principal neutralizing determinant or a neutralizing portion thereof.

25. The kit of claim 24 wherein said first antigen is HIV-1 gpl60 envelope protein having a deletion of the principal neutralizing determinant, which lies between residues 297 and 331.

26. The kit of claim 25, said antigen being gpl60Δ135.

27. The kit of claim 20, further comprising said antigen immobilized on a support.

28. The kit of claim 27, said second antigen comprising HIV-1 gplβO.

29. The kit of claim 28, said second antigen comprising gplβOMN.

30. The kit of claim 28 wherein said second antigen further comprises a peptide having an amino acid sequence corresponding to that of an immunogen which causes the production mammals of HIV neutralizing antibodies.

31. The kit of claim 20 further comprising said second antigen immobilizeσLjon a support, wherein said second antigen comprises a peptide having an amino acid sequence corresponding to the amino acid sequence of an immunogen which causes production in mammals of HIV neutralizing antibodies

32. The kit of claim 31, said immunogen being the principal neutralizing determinant of gpl60MN, or a portion thereof.

33. A method of diagnosing HIV-1 infection in a human comprising contacting an antibody-containing biological sample from said human with an antigen, said antigen being gplβOMN, formation of immune complexes between said sample antibodies and said gplβOMN being diagnostic of an HIV infection in said human.

34. The method of claim 33 wherein said gplβOMN antigen is immobilized on a solid support.

35. The method of claim 33, said antigen further comprising a peptide having an_amino acid sequence corresponding to an amino acid sequence of a PND of HIV-1 gplβO, or a neutralizing portion thereof.

36. A method for diagnosing HIV infection in a human comprising contacting a biological sample from said human with an antigen comprising a combination of gplβO and a peptide having an amino acid sequence which correspond to the amino acid sequence of the principal neutralizing determinant of HIV-l gplβOMN or a neutralizing portion thereof.

37. A kit for diagnosing the presence of HIV-l antibodies in a human biological sample, comprising immobilized gplβOMN, and an agent capable of reacting with said antibodies.

38. Useful kit of claim 37 wherein said sample is serum and said agent is a labeled molecule.

39. A kit for detecting the presence of HIV-l antibodies in a human biological sample, comprising combined immobilized gplβO and peptides having amino acid sequences corresponding to the amino acid sequence of the principal neutralizing determinant of HIV-l gplβOMN, and an agent capable of reacting with said antibodies.