FI99115C - Peptides reactive with antibodies to antigen binding to T-4 receptors and their diagnostic use - Google Patents

Description

99115 Peptides reactive with antibodies to antigen binding to T-4 receptors and their diagnostic use 5 Divided by application FI 885630.

Brief Description of the Invention This invention relates to synthetically produced short peptide sequences that induce the production of antibodies to HTLV-III / 10 LAV, or HIV envelope protein. Consequently, these peptides and monoclonal antibodies raised against them could be used as diagnostic agents for the identification of the HIV virus. Therefore, peptides and antibodies directed against the peptides could have use in the preparation of test kits for the identification of HIV carriers or individuals with AIDS.

Background of the invention

Several researchers have reported the complete nucleic acid sequence of AIDS (HIV) virus-20. (See Lee Ratner et al., Nature 313, p. 277, January 1985; Muesing et al., Nature 313, p. 450, February 1985; and Wain-Habson et al., Cell 40, pp. 9-17, January 1985.) In particular, the envelope bed has been linked to antigenic cause and infectivity. The region of the sheath is also known to include regions that are very variable. The glycoprotein of the HIV viral envelope has been shown to be covalently linked to human, rat, and monkey brain membranes and to cells of the immune system.

30 The understanding that viruses can show selectivity for cells and tissues by attaching to highly specific regions of cell membrane surface receptors has encouraged researchers to look for agents that • ·: could bind to cell membrane viral receptor sites and thus prevent the binding of a particular virus to these. solui- | · 2 99115 hin. It has previously been shown (Epstein et al., Nature 318, pp. 663-667) that infectivity mediated by specific receptors of the vaccinia virus can be shown to be ineffective with synthetic peptides.

The HIV virus has been shown to bind to a surface molecule known as the CD4 or T4 region, which is present in a variety of HIV-susceptible cells, including T lymphocytes and macrophages. (See Shaw et al. [Science 226, pp. 1165-1171] for a review of HTLV-10 III selectivity).

In addition to the symptoms of immunodeficiency, AIDS patients show neuropsychological damage. The central nervous system and immune system share a large number of specific cell surface recognition molecules that act as receptors for intercellular communication mediated by neuropeptides. Neuropeptides and their receptors have been shown to be remarkably stable during development and have remained in almost unchanged form in unicellular organisms as well as in higher animals. In addition to Li-20, common DC4 (T4) cell surface recognition molecules that act as receptors for the HIV envelope glycoprotein (gp120) are detected in the central nervous system and immune system. Because the same largely preserved neuropeptides; · · Substances that carry information that combine immune and ·. * * 25 brain function receptors that are significantly similar to HIV receptors • • ·: \ · have been claimed to be very similar the amino acid sequence of which • t * HIV glycoprotein 120 is a short peptide • · · · · * · *; previously identified in other contexts from the envelope region of Ep-30 Stein-Barr virus may indicate that; the existence of a nuclear peptide essential for binding to the viral receptor. It was suggested that such receptor site-binding peptides would result in the production of antibodies directed against peptide sequences, and that these peptides could be used to provide an immunological basis for the diagnosis of AIDS.

3 99115

Object of the present invention was to provide diagnostic means for detecting antibodies to HIV or HIV envelope protein.

5 Detailed description of the invention

The octapeptide in the HIV envelope glycoprotein (gp120) was identified using computer-assisted analysis. This peptide, named "Peptide T" due to its high threonine content, has been shown to inhibit the binding of gp10120 to brain membranes. The amino acid sequence of the peptide is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Subsequent analysis revealed similar types of pentapeptides with similar binding properties.

The peptide of the present invention is characterized in that it has the formula I: X-Ra-Ser-Thr-Thr-Thr-Asn-Tyr-Rb-X (I) wherein 20 Ra is an amino-terminal end group A- or D- Area-,

Rb is the carboxy terminus -Thr or -Thr-amide, and. . X is Cys or one or both Xs are absent, or have the formula II: * 25 • ·: * X-R1-R2-R3-R4-R5-X (II) • «• * * • · • · · 'In which formula

• · · · «J

R1 is an amino terminus of Thr, Ser, Asn, Leu, Ile, Arg or Glu, R2 is Thr, Ser or Asp, R3 is Thr, Ser, Asn, Arg, Gln, Lys or Trp , R 4 is Tyr, and: R 5 is a carboxy-terminal terminal amino acid group, the corresponding 35 D-amino acid, the corresponding amide derivative, or R 5 is absent, and. 99115 4 X is Cys or one or both Xs are absent, provided, however, that the peptide is not Ser-Thr-Asn-Tyr-Thr, or physiologically acceptable salts thereof.

R5 is preferably a carboxy-terminal end group -Thr, Arg or Gly or a derivative thereof. A peptide terminating in R4 (tyrosine) retains the binding properties of the group discussed herein. The active compounds of this invention may exist as physiologically acceptable salts of the peptides.

Peptides in this group have been found to bind to T4 viral receptors.

The most preferred peptides, as well as the peptide T described above, are the following octa-15 peptides of formula (I): D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr and D-Ala-Ser-Thr-Thr-Thr -Asn-Tyr-Thr-amide and the following pentapeptides of formula (II): Thr-Asp-Asn-Tyr-Thr 20 Thr-Thr-Ser-Tyr-Thr

Thr-Thr-Asn-Tyr-Thr and analogs thereof having D-Thr at the amino terminus and / or an amide derivative at the carboxy terminus.

The peptides disclosed herein may also be modified to have a limited opening structure to improve stability.

• · '·' The following abbreviations are used below: • · ·:. * Amino acid Three-letter One-letter ··· code code • · · · ^ ____________ • · · • · ·

* · * 30 arginine arg R

asparagine asn N

• ·

i ’* · aspartic acid asp D

cysteine cys C

: glycine Gly G

«· *

35 serine ser S

threonine thr T

· ... · tyrosine tyr Y

5 99115

Unless otherwise indicated, the amino acids are, of course, in their natural L-stereoisomeric forms.

A comparison of the amino acid sequences of the twelve pentapeptides is shown in Table 1. Although historically our initial computer search revealed that peptide T (a peptide contained in ARV isolation) is a relevant sequence, it became clear that new viral amino acid sequences could be shorter pentapept. 10 consists of peptide T (4-8), or the sequence TTNYT. In the isolations we compared (Table 1), significant homology was observed only in this shorter range. Most of the changes are the exchange of serine (S) and threonine (T), two closely resembling amino acids, in one to the other. Tyrosine at position 7 of peptide T is an unchanged feature of all these forms, suggesting that it may be necessary for bioactivity. The substitution events occurring at position 5 include T, G, R, and S. Positions 4 and 6 were first delineated to include (with one exception) S, T, and N, all of which are amino acids with uncharged polarities that are closely similar in space structure. . element. The determination of the uniformity of the sequences between the five different AIDS virus isolation preparations reveals that the region adjacent to the peptide T sequence and to which it itself belongs is highly variable. Such variability may indicate the development of sub-strains with a strong activity (s).

«M

ί through an increase in diversity, which is vulnerable to selection events and which is located in this area.

· * · .. Like opiate peptides, these peptide T analogs appear to exist in many forms, resembling met and Leu encephalins. These pentapeptide sequences, which are present in these various AIDS virus isolation preparations, are biologically active and capable of interacting with the CD4 receptor, previously more widely known as a "marker" surface of helper T helper cells.

Table 1. Comparison of the ENV sequence from several AIDS virus isolation-5 preparations

Isolation Preparation Section Reference Peptide T ASTTNYT Pert, C.B., and Others PNAS (w / w) 10 XARV (195-199) TTNYT Willey, R.L. and others LAV TTSYT PNAS 83, p.5083, 1986

Z3 SSTYR

NY5 NTSYT

15 B10 (HTLV-III) TTSYT Starcich, B.R., and others WMJ-1 SSTYR Cell, 45, p. 637, 1986

HAT-3 NTSYG

Successive insulation products STNYR

20 WMJ-1 SSTYR Hahn, B.L. and others WMJ-2 SSRYR Science 232 p. 1548, WMJ-3 SSTYR 1986

: TTSYS

· / · '· 25 1 Numbers refer to relative positions of amino acids: **. ARV in the env period (9).

The seven amino acid peptide CYS-THR- • ·.:. THR-ASN-TYR-THR-CYS is also active. The addition of cysteines to the ···· core period does not significantly impair activity. Pep- • · · 30 tids were synthesized as a service provided by Peninsula Laboratories under a confidentiality agreement between the inventors and the manufacturer. was used

Merrifield solid phase peptide synthesis. (See U.S. Patent No. 3,531,1258, which is incorporated herein by reference.) The synthesized peptides are in the primary position 799115. Although peptide T and the pentapeptide included in it could be isolated from a virus, the peptides synthesized according to Merrifield do not contain residues from cells or viruses. As a result, reactions to site 5 contamination do not occur when synthesized peptides are used.

The peptides of this invention can be produced by conventional peptide synthesis methods. Both solid and liquid phase methods can be used. We have ha-10 assumed the solid phase of Merrifield to be particularly useful. In this method, the peptide is synthesized stepwise with the carboxy terminus of the chain covalently attached to an insoluble support. During the intermediate steps of the synthesis, the peptide remains in the solid phase and is thus easily handled. The solid support phase is a chloromethylated styrene-divinylbenzene copolymer.

The N-protected form of the terminal amino acid of the carboxy terminus, e.g., the t-butoxycarbonyl (Boc-) -protected amino acid, is reacted with the chloromethyl group of the chloromethylated styrene-divinylbenzene-20-copolymer resin as a bimethyl ester to give the protected aminoacyl. This. . the deprotection is carried out and reacted with the next protected form of the amino acid to give the protected dipeptide attached to the resin. The amino acid is usually used in its activated form, e.g., using a carbodiimide or active ester. These steps are repeated and the peptide chain grows group by group by condensing Amici: with the desired N-protected amino acids 30 until the desired peptide is assembled on a support. The peptide-resin is then treated with anhydrous hydrofluoric acid, which cleaves the ester bond connecting the assembled peptide to the support to release the desired peptide. At the same time, amino acid side chain functional groups which have had to be protected during the synthesis steps can be liberated by conventional methods. The synthesis of a peptide containing an amide group at its carboxy terminus can be carried out conventionally using 4-methylbenzhydrylamine resin.

Another object of the present invention relates to test kits for detecting AIDS viruses, the antigen source of which comprises a peptide of the present invention. The assay kit of the invention for determining antibodies to an antigen that binds to the T4 receptor is characterized in that it comprises a peptide of the invention bound to a porous surface or solid support. For example, the peptide of this invention can be used in a test kit to detect AIDS infection and the diagnosis of AIDS and pre-AIDS conditions by using it as a test reagent in an enzyme-linked immunosorbent assay (ELISA) or an enzyme-linked immunostaining assay. Such assay kits may include an insoluble porous surface or solid support to which the antigenic peptide has been pre-adsorbed or covalently attached, such surface or support being primarily in the form of a microtiter plate or wells; test sera; secondary antisera that specifically bind to and saturate the surface or carrier adsorbed • antigen or antibody; I 1 several different diluents and buffers; labeled conjugates to detect specifically associated antibodies and other signaling reagents, such as enzyme substrates, cofactors and chromogens.

The present invention further relates to the use of a peptide of the invention in a diagnostic method for detecting human immunodeficiency virus (HIV) infection in body fluid or tissue, which method is useful for the treatment of the acquired immunodeficiency symptom group ( AIDS) or its: ··: for the diagnosis of a precursor, wherein • · iu 't 1999115 a) reacting a sample with said antigenic peptide, b) forming an antigen-antibody complex, and c) detecting the formation of an antigen-antibody complex. 5 punches.

The peptide of this invention can also be used as an immunogen by conventional methods to induce the production of monoclonal antibodies that specifically bind to appropriate regions in the envelope region of the AIDS virus.

Experimental methods and test results

Radiolabeling of gp120, preparation of membranes from the brain, binding and cross-linking of gp120 to the receptor, and immunoprecipitation of T4 antigen 15 strains of HIV HTLV-IIIb were added in H9 cells and gp120 was isolated by immunoaffinity chromatography and pre-preparative NaD Using PAGE. Purified gp120 was labeled with 125 I by the chloramine-T method.

Fresh human, monkey, and rat cerebellum was rapidly homogenized (Polytron, Brinkmann Instruments) in 100 volumes of ice-cold Hepes (pH 7.4) buffer. The membranes collected by centrifugation (15,000 x g) were washed with the original buffer. in the volume of the control solution and used fresh or stored at -70 ° C. Brain membranes and highly purified T cells (Ref. 16; donation from Larry Wahl) were preincubated for 15-30 min in phosphate buffered saline before use. in saline (PBS). A 20 mg aliquot (wet weight at the beginning) of brain-derived membranes (approximately 100 pg protein) was incubated with a 28,000 cpm aliquot of 125 I-gp 120 for 1 hour at 37 ° C in 200 μl ( final volume) 50 · * · .. mM Hepes buffer containing 0.1% bovine serum albumin and the peptidase inhibitors bacitracin (0.005%), aprotinin (0.005%), leupeptin (0.001%) and: chymostatin ( 0.001%). The incubation mixtures were rapidly filtered under vacuum and the radioactivity was counted to determine the amount of material attached to the receptor.

10 99115

Immune precipitation

Immunoprecipitates were prepared by incubating (overnight at 4 ° C) with 0.5% Triton-X-100 / PBS solubilized lactoperoxidase / glucose oxidase / 125 I iodinated membranes or intact T cells against the indicated monoclonal antibodies. with the substances used at 10 pg per reaction. Solid phase immunoadsorbent 10 (Immunobeads, Bio-Rad) was used to precipitate immune complexes before separation by NaDodSO 4 / PAGE. Control incubations did not contain primary monoclonal antibodies or contained a subset of control monoclonal antibody (0KT8).

15 Chemical Nerve Anatomy and Computer-Aided Density Measurement 25 μm cryostat sections from fresh frozen human, monkey, and rat brains were thawed and dried on gelatin-coated slides, and receptors were detected as described in reference (18). Incubations without antibodies against T4, T4A, T8 and T11 (10 pg / ml) or. . these were performed overnight at 0 ° C in RPMI solution, cross-linked to their antigens, and challenged with 125 I-labeled goat mice. agent. Incubations of slide sections attached to slides to label antigen receptor 125I-gp120 were performed in 5 ml slides: with or without unlabeled gp120 (1 pM) or with 30 0KT4A monoclonal antibody (10 pg / ml). ml) (Ort-ho Diagnostics) or without this.

Separation of T lymphocyte subsets • T cell subsets were obtained by treating peripheral circulating T cells purified in a Percoll density with specific monoclonal antibodies (T4 or 1 «•»

I · I

• <11 99115 T8) at a concentration of 10 pg / ml. The treated cells were allowed to stand for 30 min at 4 ° C in a plastic petri dish fixed with immunoglobulin against goat [F (ab ') 2] mouse (Sero Lab, Eastbury, MA).

The remaining cells were then removed and washed and examined for reactivity with a flow cytometer. Separated T4 and T8 cell populations have other T cell subsets at <5% contamination level. The cells were then cultured in the presence of phytohemagglutinin (1 pg / ml) and exposed to HIV as described below. The nature of the appearance of HIV-bound cells was elucidated when cell toxicity assays were performed.

virus Commitment

The HTLV virus used for binding was isolated from a cultured T cell line requiring in-15 terleukin 2 (IL-2) isolated from fresh AIDS patient material and inoculated into HuT 78, a permissive cell line that is independent of IL-2. 2.

Description of the Drawings Figure IA shows the cross-linking of 125 I-gp120 to brain membranes and T cells. (a) 125 I-gp 120 alone; (b) monkeys; (c) rat; (d) the human brain; and (e) human. . T cells.

* ·

Figures IB and 1C show the immune-> «! Of 125 I-labeled monkey <<· '· *' 25 brain membranes and human T cells, respectively. * precipitation; (f, i) a control without a primary antibody; (g, j) monoclonal antibody OKT4; (h, k) 0KT8 mo-% noclonal antibody.

Figure 2A shows the displacement of specific 125 I-gp120 binding to fresh rat cerebellar membranes. Each assay was performed in triplicate; the results f of one experiment giving three times similar results are shown. The degree of specific binding that was displaceable by 10 pg / ml 0KT4 and 4A ranged from 27 to 85% of the total binding, which in the experiment shown was 2201 ± 74 cpm.

. «

I I I

Ψ · 12 99115

Figure 2B shows that peptide T and its synthetic analogs render viral binding ineffective. Each assay was performed in two replicates. The results represent one experiment repeated three times with similar results.

Example 1 Specific binding of 125 I-gp120 to membranes of skull, rat or human brain yields one radiolabeled, approximately 180 Kd cross-linked product that is indistinguishable from the corresponding product bound to human T cells ( Figure IA). This result indicates that gp120 can be coupled to a protein of about 60 Kd; the unreacted 125I-gp 120 travels next to the membrane-free control (channel a).

Immunoprecipitation of radiolabeled human brain membranes with 0KT4 and OKT8 (10 pg / ml) (Figure IB) indicates that the brain membranes contain T4 antigen of approximately 60 Kd in size and cannot be distinguished from human T-antigen. the corresponding 20 antigens identified from lymphocytes (Figure 1C); in contrast, immunoprecipitation with OKT8 precipitates a low molecular weight (approximately 30 Kd) protein from T lymphocytes (Figure 1C) that is not present in the meninges (Figure IB), suggesting that brain T4 is not derived from these localized V 25 lymphocytes. . Similar results (data not shown) have been obtained using monkey and rat cerebellar membranes. These results indicate that the T4 antigen acts as a viral receptor and is a well-conserved 60 Kd molecule common to the immune system and central nervous system.

The finding that Epstein-Barr and HTLV-f «· III / LAV have nearly identical octapeptide sequences led to the synthesis and investigation of" peptide T ". Figure 2 shows,; that the binding of 125 I-gp 120 to freshly prepared rat I · '' f 35 brain membranes occurs with high affinity (0.1 »t * Λ * *» - · * · 13 99115 nM) and that this is saturable. Specificity (Figure 2B) is indicated by inhibition by 0KT4 and 0KT4A, but not by 0KT3 (0.1 pg / ml). Peptide T and two of its synthetic analogs significantly inhibited 125 I-gp120 binding in the 0.1 nM range (Figure 2C) (but this was not done by the non-associated octapeptide substance P (1-8)). The D-threoninamide substitution at position 8 caused at least a 100-fold loss of binding activity to the receptor. The classical replacement of (L-domain) with (D-domain) 10 resulted in a permanently more potent, apparently more peptidase-resistant analog than peptide T; The C-terminal terminal threonine produces a permanently somewhat increased potency (Figure 3).

When the ability of synthetic peptides to prevent receptor binding in human T cells was tested, the experimenter was unaware of the results of the binding experiments. At level 10'7, the three peptides active in the binding assay reduce the detectable level of reverse transcriptase by almost 9-fold. The less active binding displacement (D-thre) peptide T also proved to be greatly impaired in its inhibition of binding and was required 100-fold to achieve significant inhibition; higher concentration. Thus, not only the order between the classes of the four peptides ((D- (Ala ^ peptide T-amide> (D- »· ♦ 25 (Ala ^ peptide T> peptide T> (D- (Thr) 8 peptide T- amide), • · *.. but also the inhibition of their binding to the receptor correlates • · · 14 99115 cannot be distinguished from the T4 antigen in terms of their properties.Using the method of detecting antibody-binding receptors in brain sections, the a character that is in the terminal network of neural causes of the ti-5 hay bark and is arranged analogously in all three mammals.

Radiolabeled HIV envelope glycoprotein binding also produced a similar character in successive sections, suggesting that T4 was the 10 HIV receptor.

Example 3

Chemical nerve anatomy, computer-assisted ti-density measurement

25 micron cryostat sections of fresh frozen human, monkey, and rat brains were thawed and dried on gel-coated slides, and the receptors were detected as described in Herkenham and Pert, J. Neurosci., 2, pages 1129-1149 (1982). Incubations with and without antibodies to T4, T4A, T8, and 20 T11 (10 pg / ml) were performed overnight at 0 ° C in RPMI, cross-linked to their antigens, and detected. 125I goat anti-mouse antibody. Tissue-mounted tissue sections for antigen / receptor labeling with 125 I-gp 120. . incubations were performed with a 5 ml slide. · * In broths using or omitting (10’6 unlabeled gp120 or 0KT4A (10 • · · pg / ml) monoclonal antibody as described above for the use of membranes.

30 A computer-aided conversion of autoradiographic film to quantitative color images was performed. Exposure in parallel with sections of monkey brain using standards with known radioactivity enhancement resulted in a linear plot (4 => 0.99) of 35 log 0D and cpm from which the relative concentration of radioactivity can be meaningfully extrapolated. Staining of brain sections with thionine was performed by classical methods and receptors were detected in the stained tissue.

5 Example 4

Experiments have been performed to determine the distribution of T4 antigen in a series extending from the anterior to posterior part of the skull monkey brain or in horizontal sections. These experiments show that the binding of the T4 monoclonal antibody to areas relevant to the cellular structure of the brainstem (e.g., the substantia nigra) occurs at detectably high levels, but a striking cortex-enriching presence is evident at each level of the germ axis. 0KT8, a monoclonal antibody directed against T-lymphocyte from the same subgroup as 0KT4, does not form any recognizable character. The uppermost surface layers of the cerebral cortex generally contain the densest concentration of T4 antigen; the frontal lobe of the cerebral cortex and the portion of the cortex surrounding the limbis-20 system above the tonsil nucleus contain a particularly high number of receptors located throughout the inner layers. Re-. _._ The densest concentration of receptors targets the edema area in the monkey, rat, and human brains. Film development solution,. Dark field microscopy of 25 immersed skull monkey sections revealed that the zone of densest labeling of receptors is located in the molecular layer of the dentition and in the cortex itself (which contains very few neurons). Thus, the receptors appear to be precisely located to cover the terminal network of neural pathways (nerve extensions of nerve terminals and exporting branches) or to be located in a specific subtype of stained cells in the star support tissue.

Assays have been performed that provide evidence for the specificity of the corneal -35 neuronal anatomy, and the results of 16,99115 indicate that T4 and the viral envelope protein recognition molecule do not differ. Horizontal sections of rat brain revealed a very similar pattern of receptor distribution in the cerebral cortex / ai-5 voturs, regardless of whether 0KT4 or 125I-gp 120 was used for detection. In the presence of 0KT4A (10 pg / ml) or 0KT4 (10 pg / ml). No identifiable figure was obtained from rat brain with other murine monoclonal antibodies directed against other human T cell antigens, including 0KT8 and 0KT11, when detected with 125 I-goat anti-mouse IgG secondary antibody, as well as using a 15 secondary antibody alone, no detectable detectable antigen / receptor was found.

m m • · • · • · • · · · · · · · · · mmm mmm m · · • · · m m m m m m mm m

Claims (10)

17 99115 Peptide, characterized in that it has the formula I: X-R * -Ser-Thr-Thr-Thr-Asn-Tyr-Rb-X (I) in which Ra has the amino-terminal end group Ala- or D-Ala-, Rb is a carboxy-terminal end group -Thr or -Thr-amide, and X is Cys or one or both Xs are absent, or the peptide has the formula II: X-R1-R2-R3-R4-R5-X (II) in which R1 is an amino-terminal end group Thr, Ser, Asn, Leu, Ile, Arg or Glu, R2 is Thr, Ser or Asp, R3 is Thr, Ser, Asn, Arg, Gln, Lys or Trp, R4 is Tyr, and R5 is a carboxy-terminal amino acid group, the corresponding D-amino acid, the corresponding amide derivative or R5 is absent, and X is Cys or one or both Xs are absent, provided that the peptide is not Ser-Thr-: Asn-Tyr-Thr, .... or physiologically acceptable salts thereof. v A peptide according to claim 1, characterized in that it is a peptide of formula II, wherein R 1 is Thr, Ser or Asn, R 3 is Thr, Ser, Asn or Arg, and R 5 is -Thr, -Arg or -Gly. 18 99115 Peptide according to Claim 1, characterized in that it is a peptide of the formula II in which R 5 is absent. Peptide according to Claim 1, characterized in that it is Cys-R1-R2-R3-R4-R5-Cys. Peptide according to Claim 1, characterized in that it is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, Thr-Thr-Asn-Tyr-Thr, 10 Ser-Ser-Thr-Tyr-Arg, Asn-Thr-Ser-Tyr-Thr, Thr-Thr-Ser-Tyr-Thr Asn-Thr-Ser-Tyr-Gly, Ser-Thr-Asn-Tyr-Arg, 15 Ser-Ser-Arg-Tyr-Arg, Thr-Thr-Ser-Tyr-Ser, or Cys-Thr-Thr-Asn-Tyr-Thr-Cys. A test kit for the determination of antibodies directed against an antigen which binds to the T4 receptor, characterized in that it comprises a peptide according to claim 1 bound to a porous surface or a solid support. Test kit according to Claim 6, characterized in that the antigenic peptide is bound to the walls of the microtiter plate. Use of a peptide according to any one of claims 1 to 5 in a diagnostic method for detecting human immunodeficiency virus (HIV) infection in body fluid or tissue, which method is useful for the acquisition of an immunodeficiency virus. for the diagnosis of syndrome (AIDS) or a precursor thereof, wherein a) the sample is reacted with said antigenic peptide, b) an antigen-antibody complex is formed, and c) the antigen-antibody complex formed is detected. II • I 19 99115 Use according to claim 8, characterized in that the peptide is used in a diagnostic method in which the antigen-antibody complex is determined by an immunoassay method. Use according to claim 9, characterized in that the peptide is used in a diagnostic method, wherein the antigen-antibody complex is determined by a radioimmunoassay method, an enzyme-linked immunoassay method or an immunoblot assay method. • · • · • «« • • · «· • · t« · «« • · «« I 1 · Φ ♦ '• · 1 I I · • · • · * 20 99115