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WO1996012023A1 - Proteine recombinee appelee dev-1 utilisee pour detecter le vih, sequence d'adn codant cette proteine, et dosages immunologiques realises au moyen de cette proteine - Google Patents

Proteine recombinee appelee dev-1 utilisee pour detecter le vih, sequence d'adn codant cette proteine, et dosages immunologiques realises au moyen de cette proteine Download PDF

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Publication number
WO1996012023A1
WO1996012023A1 PCT/US1995/013335 US9513335W WO9612023A1 WO 1996012023 A1 WO1996012023 A1 WO 1996012023A1 US 9513335 W US9513335 W US 9513335W WO 9612023 A1 WO9612023 A1 WO 9612023A1
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Prior art keywords
protein
hiv
dev
seq
dna sequence
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PCT/US1995/013335
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English (en)
Inventor
Yair Devash
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Devaron, Inc.
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Publication date
Application filed by Devaron, Inc. filed Critical Devaron, Inc.
Priority to AU40024/95A priority Critical patent/AU4002495A/en
Publication of WO1996012023A1 publication Critical patent/WO1996012023A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention concerns antigens useful in the diagnosis of the Human Immunodeficiency Virus (HIV). More particularly, the invention relates to a recombinant protein which maximizes sensitive detection of antibodies to HIV and minimizes the danger of false-positive results in immunoassays for HIV, to DNA sequences encoding the protein, and to their use in immunoassays.
  • HIV Human Immunodeficiency Virus
  • HIV Human Immunodeficiency Virus
  • LAV Long Virus
  • ARV a cytopathic lymphotropic retrovirus
  • HIV Acquired Immunodeficiency Syndrome
  • the underlying disease state involves a tropism of HIV for the T4+ lymphocyte subset resulting in a selective depletion of the helper/inducer cells of the immune system, leaving the individual defenseless against a number of opportunistic infections.
  • the env open-reading frame of HIV which consists of 863 amino acids, has been reported to encode a 160 kd precursor glycoprotein, designated gpl60.
  • This precursor glycoprotein is thought to be processed into a 120 kd exterior glycoprotein, designated gpl20, and a 41kd transmembrane protein, designated gp41. All three proteins have been found to react with AIDS patient sera. [Barin et al., Science, 228:1094 (1985); Sarngadharan et al., Science, 224:506 (1984). Recombinant proteins derived from the env reading frame and other regions of the HIV genome had been studied as diagnostic and vaccine candidates.
  • Allan et al., Science, 230:810 (1985) discloses a HIV/LAV 27,000 MW protein having a coding origin 3' to the env gene.
  • U.S. Patent 4,520,113 issued to Gallo et al., discloses serological detection of antibodies to HIV in sera of patients with aids and pre-aids conditions. HIV isolated from AIDS patients and transmitted by cocultivation with an HT cell line is detected by antibodies from human sera taken from AIDS patients. The most prominent reactions were reported to be directed to gp41, a 41,000 MW protein constituting the envelope antigen of the HIV virus.
  • U.S. Patent No. 4,725,669 teaches an assay for detecting infection by human T-cell lymphotropic virus-Ill, by assaying a biological specimen for the presence of either of two specific proteins.
  • the first generation HIV antibody tests used HIV viral lysate, containing multiple antigens (envelop, gag, pol, etc.). In spite of these antigens, the tests lacked sensitivity and picked up false positive samples. As viral lysates were substituted with recombinants, tests were improved both in sensitivity and specificity. Furthermore, some specific recombinants show superiority as antigens although they are not as long as other recombinants and they contain fewer immunological domains. Their superior performance may be due to folding on the matrix in a way that reflects better their in vivo presentation to the immune system.
  • WO 86/02383 deals with purified expression products of DNA sequences derived from the genome of the LAV virus, and particularly to a glycoprotein having a molecular weight of about 110,000 or antigen of lower molecular weight derived from the preceding one, which purified product possesses the capacity of being recognized by sera of human origin and containing antibodies against the LAV virus.
  • the invention accordingly, provides a recombinant protein displaying the antigenicity of Human Immunodeficiency Virus (HIV) viral antigens.
  • the protein designated hereinafter as "DEV-1"
  • DEV-1 comprises the C- terminus of gpl20 and most of gp41.
  • Two E. coli cytotoxic sections are deleted from this gpl20-gp41 fusion protein, DEV-1, in order to facilitate its expression in E. coli cells.
  • DEV-1 starts from HIV-IBRU env amino acid 474, and ends in amino acid 752.
  • the purified protein was tested for immunoreactivity in ELISA and Western Blots. It is superior to other commercially available antigens in early detection of HIV seroconvertors, and has minimal specificity problems. Furthermore, purified DEV-1 tolerates a wide range of pH and buffer conditions, and can be easily lyophilized and then reconstituted into solution.
  • the invention thus provides a purified protein, which is the protein of SEQ ID NO. 1, biologically active analogs, fragments and derivatives thereof.
  • the invention is directed to a purified protein, which is the protein of SEQ ID NO. 1.
  • the amino acid residues designated "Xaa” are meant to indicate that these residues are not present in the protein DEV-1, rather, they serve to show those residues which were deleted from the new gpl20- gp41 fusion protein. These deleted residues constitute the above-noted E. coli cytotoxic sections.
  • the invention is directed to the use of the protein of SEQ ID NO. 1, as an antigen for the detection of HIV antibodies in human biological samples.
  • the method of detecting antibodies to HIV in a human biological sample comprises providing a support to which the protein of SEQ ID NO. 1 is bound, contacting the solid support with the human biological sample, and examining the solid support to determine whether antibodies have been bound to the protein.
  • the skilled person will easily recognize suitable methods to determine whether antibodies have become bound to the antigen, and any suitable method can be used.
  • the determination of whether antibodies have been bound to the protein is carried out by an ELISA test.
  • the determination of whether antibodies have been found is carried out by the use of gold particles conjugated to an antibody-binding protein.
  • the invention is also directed to a method of producing the protein of SEQ ID NO. 1, comprising providing a vector comprising a DNA sequence essentially the same as SEQ ID NO. 2, and expressing the same in a suitable host cell.
  • the vector is plasmid pDEV-1, which has been seen to express the protein in high yield and stability.
  • the protein of the invention can be expressed in a variety of host cells, which will be recognized by the skilled person.
  • the host cell is an E. coli, e.g., E. coli W31 10.
  • Plasmid pDEV-1 is a novel plasmid and as such also forms a part of the present invention. Also encompassed by the invention is E. coli W3110, containing plasmid pDEV-1, having inserted into it the recombinant DNA sequence, which is SEQ ID NO. 2, that encodes the protein of the invention.
  • the present invention also provides a new DNA sequence which encodes the new protein of the invention.
  • the new DNA sequence of the invention is a recombinant DNA sequence encoding the protein DEV-1, biologically active analogs or fragments thereof, said DNA sequence selected from the group consisting of:
  • SEQ ID NO. 2 DNA sequences capable of hybridization to the DNA sequence of (a) under moderately stringent conditions and which encode biologically active protein DEV-1, analogs or fragments thereof;
  • a preferred embodiment of the above DNA sequence of the invention is a recombinant DNA sequence which is SEQ ID NO. 2.
  • Fig. 1 is a flow diagram illustrating construction of plasmid pDEV-1 described in Example 1.
  • - Fig. 2 is a restriction map of plasmid pDEV-1.
  • a recombinant protein having residues from both the gpl20 and gp41 domains of HIV has been created which is highly immunoreactive with the sera of HIV infected individuals.
  • the protein comprises the C-terminus of gp 120 and most of gp41.
  • Two E. coli cytotoxic sections are deleted from this gpl20-gp41 fusion protein to enable expression thereof in E. coli cells (nuc.acid 7349-7425, and nuc.acid 7839-7920; or AA 517-541 and AA 680-704 of the HIV genome sequence).
  • the protein starts from HIV-1 env. amino acid 474, and ends at amino acid 752.
  • the specified domains of HIV are known in the art.
  • the expression "corresponding to” includes modifications of the specified amino acid sequences which do not adversely affect the antigenic characteristics of the protein of the invention.
  • the protein can contain additional amino acids corresponding to other domains of HIV or other sources, such as a plasmid.
  • One skilled in the art could align the amino acid sequences of peptides from different sources to the scheme in SEQ ID no. 1 to identify the segments therein which correspond to the protein defined herein.
  • biologically active analogs are those analogs which have at least a one amino acid change with respect to the amino acid sequence of SEQ ID NO. 1, but which retain essentially all of the antigenic properties, especially the sensitivity to anti-HIV antibodies, of the DEV-1 protein.
  • the at least one amino acid change present in these analogs may be the result of a deletion of one or more amino acid residues, an addition of one or more amino acid residues or a substitution of one or more amino acid residues with different amino acid residues, all with respect to the sequence of SEQ ID NO. 1.
  • the above analogs and fragments of the invention may be readily prepared by any skilled artisan using standard recombinant DNA technology.
  • the plasmid of the invention pDEV-1
  • pDEV-1 may be manipulated so as to generate one or more deletions, additions or substitutions of codons in the DEV-1 coding sequence to provide new sequences encoding analogs; or so as to generate substantial deletions in the DEV-1 coding sequence to provide new sequences encoding fragments.
  • Each such analog or fragment may then be readily analyzed for its suitability, i.e., biological activity, by applying the quality tests set forth herein in Examples 2 and 4-7.
  • biologically active derivatives of the invention are those derivatives of the DEV-1 protein, its biologically active analogs and its biologically active fragments, that have been produced by modifying DEV-1, its analogs or fragments, chemically, for example, by acylation, amidation, etc.; or by conjugation with other proteins, such as, for example, enzymes, antibodies, etc., or other molecules such as, for example, lectins, etc.; or by labeling with, for example, radiolabels and fluorescent labels.
  • These derivatives may be prepared by any of the well known methods of the art.
  • the antigenic sequences of the protein DEV-1 of the invention is highly sensitive to the presence of antibodies against HIV and can serve in their early detection.
  • the protein of the invention provides a superior sensitive diagnostic reagent for detecting HIV infection.
  • the sensitivity of the protein permits the early detection of HIV, because it recognizes lower serum titers of antibodies against HIV infection as compared to other known recombinant proteins and peptides.
  • a second advantage is related to the specificity of the protein.
  • the protein is smaller in size compared to prior art recombinant proteins, and thus contains fewer non-critical antigenic domains, thus minimizing the possibility of "false positive" results when employed as a diagnostic reagent to detect HIV infections, as described in more detail below (Table III).
  • the protein shows excellent stability over a large range of temperatures, for long periods of time (see Table IV).
  • the protein of the invention is used in a diagnostic kit used for detecting antibodies to HIV in a biological sample.
  • the protein can be employed in a method for detecting antibodies to HIV comprising contacting a biological sample with the protein and detecting, by means known in the art, whether specific HIV antibodies are present in the sample.
  • the methods employed in assays of this type are well known to the skilled person and comprise, for instance, assays of the type described in US 5,006,464, the specification of which is incorporated herein by reference.
  • the protein comprises an antigenic segment having an amino acid sequence which corresponds to that encoded by the nucleotide sequence of the Bglll to BamHl restriction fragment of HIV env, and most preferably, an amino acid sequence which corresponds to that encoded by the nucleotide sequence of the Bgl II to Hind III restriction fragment of HIV env, which amino acid sequence is shown in SEQ ID NO. 1.
  • the amino acid residues designated "Xaa" in SEQ ID NO. 1 depict the above-noted residues deleted from the gpl20-gp41 fusion protein DEV-1 of the invention, i.e., in the DEV-1 protein, these "Xaa" residues are not present.
  • the bacterial expression host strain was constructed at Peprotech Inc. by inserting the
  • Plasmid lambdaNot/pUT/Hg was transfected into E. coli host strain W3110 using increased resistance to 10 ug/ml HgCl2 as the initial screen for stable insertion of the transposon into the host strain.
  • the use of mercury resistance screen was somewhat tedious and inaccurate due to some natural resistance by the W3110 strain to HgCl2 in the 5-10 ug/ml concentration range.
  • the stable insertion of the lambda CI857/Cro regulatory elements was confirmed by resistance to lambda infection at 30°C and the ability of the resulting strain to regulate, by temperature shift induction, the expression of genes residing on plasmids and transcribed by the lambda PL promotor.
  • the resulting strain was designated ENV1.
  • E. coli strain DH5n, lambda CI857 DNA were purchased from Life Technologies,
  • Plasmids pUC18Not, pUT/Hg and E. coh host CO 18 were obtained from Dr. Burt Ensley, Envirogen Inc., Princeton, NJ. who obtained these materials from the laboratory authoring the aforementioned publication ( Herrero et. al., Journal of Bacteriology, Nov. 1990, p.6557-6567 and 6568-6572).
  • E. coli strain W31 10 was obtained from Envirogen Inc., Princeton, NJ.
  • E. coli W31 10 was used as the host strains.
  • the plasmid employed was derived from the pEGF/PV, which contains the P promotor and the Shine-Dalgarno sequences inserted into the EcoRI-Hindlll sites of pUC18.
  • the plasmid, pEGF/PV contains a cDNA segment encoding for the poliovirus 3C protease placed under trp control.
  • the pEXC derivative employed in the Examples maintains the original PvuII site within the pBR322 sequence and thus contains a single Bglll site.
  • the plasmid pDEV-1 was constructed by combining the Ndel/BamH 1 env fragment obtained by recombinant PCR with the vector pJGl digested with Ndel.
  • the env fragment was obtained in two steps. First, three different env fragments were obtained by PCR, and second, they were linked in a second PCR to produce an env fragment encoding for the DEVI HIV env antigen (See Fig. 1). Restriction endonucleases, T4 DNA ligase, calf intestinal phosphatase, and the Klenow Fragment of DNA polymerase I were obtained from New England Biolabs or Life Technologies, Inc. and used as recommended.
  • Pfu DNA polymerase was obtained from Stratagene Cloning Systems and used as recommended.
  • the plasmid pLAI-3 which contains the HIV-1 /B RU ) provirus was used as template to produce three fragments of the env coding sequence by the polymerase chain reaction.
  • Each reaction mix contained 100 pg of pLAI-3, 200mM of dNTP's, 2.5 units of Pfu DNA polymerase, 20 mM Tris-HCl (pH 8.2), 10 mM KC1, 60 mM (NH ) 2 SO 4 , 2 mM MgCl2, 0.1% Triton X-100, 10 mg/ml nuclease-free BSA, and 0.2 mM of each oligonucleotide primer pair (la and lb, Ha and lib, or Ilia and Illb).
  • the resulting fragments (1,170 bp; 11,460 bp; and 111,178 bp) were loaded on an agarose gel and purified using the gene-clean procedure according to the manufacturer's instructions (Bio-101, Inc.).
  • the three fragments were linked in a second PCR in which about 20 ng, 60 ng, and 20 ng of the fragments I, II, and III, respectively were used as templates in a single amplification reaction with the oligonucleotide primers la and Illb.
  • the resulting fragment (808 bp) was digested with the restriction endonucleases Ndel and BamHl for three hours at 37 C in a buffer containing 1 mM Tris-HCl (pH 7.4), 30 mM KC1, 0.1 mM EDTA, 0.1 DTT, 0.0015% Triton X-100 and 5% glycerol. 10 mg of the vector pJGl were digested with the enzyme Ndel under identical conditions and incubated for an additional hour after adding 2 units of calf intestinal phosphatase. Both the env fragment and the pJGl vector were loaded onto an agarose gel, electrophoresed, and purified by the gene-clean procedure as described previously.
  • E. coli strain W31 10 containing the plasmid pDEV-1 was grown at 30 C in Luria Broth containing 100 ⁇ g/ml of penicillin until an OD ⁇ of 0.2-0.4 and then transferred to 42 C for 6 hours. Aliquots were taken at 0 time and 6 hours after induction. The cell pellet from 500 ul of each sample was resuspended in 50 ul of SDS loading buffer, heated at 100 C for 3 min followed by short centrifugation. 5 to 20 ul of sample were analyzed by polyacrylamide gel (12.5%) electrophoresis under denaturing conditions. For quantity control, samples containing different amount of bovine serum albumin were loaded in the same gel. Proteins were visualized by Coomassie blue staining.
  • Enzyme Linked Immunosorbant Assay Purified protein was diluted in 60mM carbonate pH 9.6 buffer containing 0.01% azide and 0.00006% SDS to obtain a protein concentration of 100 ng/well. 100 ⁇ l aliquots of the protein solution were placed in each well of Immulon II microtiter plates. The volume used in the binding step set the total volume used for all the rest of the incubations with the exception of the blocking steps. Binding took place at 4°C for about 18 hours. The plates were then washed with PBS + 0.05% Tween 20 (PBS-T).
  • PBS-T PBS + 0.05% Tween 20
  • the plates were blocked with 0.5% casein diluent (6.4 g/1 NaCL, 0.16 g/1 KCL, 0.16 g/1 potassium phosphate, 0.916 g/1 sodium phosphate, 0.5 ml Tween-20, 3.6 ml of 0.1% Rhodamine-B, 100 ml of 5% casein, tota l .O L, pH 7.4) for 1 hour at 37°C and were then washed 3X with PBS-T and stored dry at 4 C until they were used.
  • casein diluent 6.4 g/1 NaCL, 0.16 g/1 KCL, 0.16 g/1 potassium phosphate, 0.916 g/1 sodium phosphate, 0.5 ml Tween-20, 3.6 ml of 0.1% Rhodamine-B, 100 ml of 5% casein, tota l .O L, pH 7.4
  • Example 1 Construction of Plasmid pDEV-1 The plasmid pDEV-1 was constructed by combining the Ndel/BamH 1 env fragment obtained by Polymerase Chain Reaction (PCR) with the vector pJGl digested with Ndel. The env fragment was obtained in two steps. First, three different env fragments were obtained by PCR, and second, they were linked in a second PCR to produce an env fragment encoding for the DEVI HIV env antigen (See Fig. 1). Restriction endonucleases, T4 DNA ligase, calf intestinal phosphatase, and the Klenow Fragment of DNA polymerase I were obtained from New England Biolabs or Life Technologies, Inc. and used as recommended.
  • PCR Polymerase Chain Reaction
  • Pfu DNA polymerase was obtained from Stratagene Cloning Systems and used as recommended.
  • the plasmid pLAI-3 which contains the HIV-1 (BRU) provirus was used as template to produce three fragments of the env coding sequence by the polymerase chain reaction.
  • Each reaction mix contained 100 pg of pLAI-3, 200 ⁇ M of dNTP's, 2.5 units of Pfu DNA polymerase, 20 mM Tris-HCl (pH 8.2), 10 mM KCI, 60 mM (NH ) 2 SO4, 2 " ⁇ MgCl2, 0.1% Triton X-100, 10 ⁇ g/ml nuclease-free BSA, and 0.2 ⁇ M of each oligonucleotide primer pair (la and lb, Ila and lib, or Ilia and Illb).
  • the primers were designed so that upon amplification 40 bp overlaps would be generated between the 3' end of fragment I and the 5' end of fragment II and between the 3' end of fragment II and the 5' end of fragment III ].
  • the three reaction mixtures were subject to 30 cycles of DNA amplification in a thermal cycler following a single denaturation step of 3 min at 95 C. Each cycle consisted of a denaturation step of 30 sec at 95 C, an annealing step of 30 sec at 30 C, and a polymerization step of 2 min at 72 C.
  • the resulting fragments (1, 170 bp; 11,460 bp; and 111,178 bp) were loaded on an agarose gel and purified using the gene-clean procedure according to the manufacturer's instructions (Bio-101, Inc.).
  • the three fragments were linked in a second PCR in which about 20 ng, 60 ng, and 20 ng of the fragments I, II, and III respectively were used as templates in a single amplification reaction with the oligonucleotid'. primers la and Illb.
  • the resulting fragment (808 bp) was digested with the restriction endonucleases Ndel and BamHl for three hours at 37°C in a buffer containing 1 mM Tris-HCl (pH 7.4), 30 mM KCI, 0.1 mM EDTA, 0.1 DTT, 0.0015% Triton X-100 and 5% glycerol. 10 ⁇ g of the vector pJGl were digested with the enzyme Ndel under identical conditions and incubated for an additional hour after adding 2 units of calf intestinal phosphatase. Both the env fragment and the pJGl vector were loaded onto an agarose gel, electrophoresed, and purified by the gene-clean procedure as described previously.
  • the env fragment inserted into the pJGl vector was sequenced by conventional procedures and the predicted protein product encoded by the env fragment of the resulting plasmid (DEV-1) was shown to contain 233 amino acids which correspond to 38 amino acids of the C-terminal of the gpl20 domain and 241 amino acids of the N- terminal of the gp41 domain, but from which gpl20-gp 1 fusion protein there has been deleted two sections observed to be cytotoxic to E. colt cells (results not shown).
  • the sequence of the env fragment encoding this gpl20-gp-41 fusion protein, herein designated DEV-1 is set forth in SEQ ID NO.
  • the E. coli strain W3110, containing plasmid pDEV-1 was deposited on June 29, 1994 with, and made permanent part of the collection of, the ARS Patent Culture Collection, Peoria Illinois, U.S.A., under Accession Number NRRL 21291.
  • the protein DEV-1 was purified from the E. coli cells containing the vector pDEV-1 described in Example 1 according to the following procedure. The E. coli were grown
  • Q-sepharose (Pharmacia) was pre-equilibrated with extract buffer. The extract was passed through 200 ml of Q-sepharose column, collecting the flow through. PAGE was run to determine DEV-1 concentration. 150-200 mis of S-sepharose(Pharmacia) was pre-equilibrated with S-sepharose binding buffer (8M Urea, 25 mM BICINE pH 8.5, 10 mM DTT,5 mM EDTA, 0.1% Tween 20).
  • DEV-1 was eluted with HA Elute #2 (400 mM NaH2PO4, pH 6.0, 1 mM DTT, 0.1% SDS). The eluted DEV-1 was concentrated to about 20-30 mis, and 0.9% SDS and 9 mM DTT were added.
  • S-300 was pre-equilibrate with S-300 sizing buffer (8M Urea, 25 mM BICINE pH 8.5, 5 mM b-Me 5 mM EDTA, 0.1% SDS).
  • the seropositive samples used in this study were diagnosed AIDS patients. These were confirmed by other ELISA assays as well as by immunoblot. Samples were run at a dilution of 1:20.
  • Example 5 A kit containing DEV-1 was manufactured, essentially as described in U.S. Patent No. 5,006,464, and employed to determine its ability to detect early seropositive HIV infected individuals. Seroconversion panels were obtained from Boston Biomedica, Inc. The ability to detect HIV antibodies was compared to a state-of-the-art ELISA (Abbott) and to western blot results. Table II shows that the DEV-1 kit performs as well as the ELISA and Western Blot in detection of infection. Note that the time required to obtain a result with the Dev-1 Kit is minutes, compared to hours for the ELISA and even days for Western Blot.
  • PRB 904-4 positive positive positive positive s/co 12.0 18.24.41,55.65.120.160
  • Example 6 The Dev-1 kit was tested in performance tests in many different parts of the world, with samples from widely different populations. In all studies the kit has performed at least as well as the much more complex ELISA-based assays and yet is much easier and faster to perform. In addition, the kit does not produce false positive results known to occur in ELISA assays and is extremely well correlated with the Western Blot.
  • the kit was compared to a leading ELISA test (Abbott) and to the confirmatory Western Blot method (Table III) with the following types of samples:
  • Sensitivity 100% compared to Western Blot Note that the Dev-1 Kit performed better than the ELISA in cases of indeterminate western blot results. Out of 50 such sera that are not considered HIV positive, the ELISA falsely recognized 33 sera as positive, while the Dev-1 Kit showed a negative result for all 50 cases.
  • Example 7 Stability Tests The Dev-1 Kit was stored at three temperatures and then tested according to the following schedule:
  • the devices were tested at each date using a seven member panel derived from the rabbit anti-DEV-1 and the anti-DEV-2 Ab.
  • the results show excellent stability as measured by the sensitivity to the panel .
  • the results indicate that the device is stable for 12 months, at room temperature, and for two months under very harsh conditions.
  • the test carried out according to the invention achieves two important goals: it is as accurate as the western blotting test, but is very quick. Accordingly, each single test becomes very inexpensive, while avoiding false positive results.
  • the invention provides an important solution to a problem with which the art has been struggling daily, viz. it provides an accurate, quick and inexpensive means of testing for HIV.
  • Lys Val Val Lys lie Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys 20 25 30
  • CAGCTCCAGG CAAGAATCCT GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTGGGG 300

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Abstract

L'invention concerne une protéine purifiée (No. d'identification de séquence 1), des analogues, des fragments et des dérivés biologiquement actifs de celle-ci, qui peuvent être utilisés, entre autres, comme antigène pour la détection de la présence d'anticorps dirigés contre le VIH, dans des échantillons biologiques humains.
PCT/US1995/013335 1994-10-17 1995-10-11 Proteine recombinee appelee dev-1 utilisee pour detecter le vih, sequence d'adn codant cette proteine, et dosages immunologiques realises au moyen de cette proteine WO1996012023A1 (fr)

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AU40024/95A AU4002495A (en) 1994-10-17 1995-10-11 A recombinant protein designated dev-1, useful in the detection of hiv, dna sequence encoding the protein, and immunoassays using the protein

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IL11131194A IL111311A0 (en) 1994-10-17 1994-10-17 A recombinant protein designated DEV-1, useful in the detection of HIV, DNA sequence encoding the protein, and immunoassays using the protein
IL111311 1994-10-17

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6605427B2 (en) 2000-02-10 2003-08-12 Panacos Pharmaceuticals, Inc. Assay for detection of viral fusion inhibitors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000594A1 (fr) * 1992-06-23 1994-01-06 Abbott Laboratories Procedes d'utilisation des proteines de fusion cks

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000594A1 (fr) * 1992-06-23 1994-01-06 Abbott Laboratories Procedes d'utilisation des proteines de fusion cks

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6605427B2 (en) 2000-02-10 2003-08-12 Panacos Pharmaceuticals, Inc. Assay for detection of viral fusion inhibitors

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