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WO2008034297A1 - Procédé de détection d'anticorps dirigés contre une série de protéines virales d'immunodéficience humaine - Google Patents

Procédé de détection d'anticorps dirigés contre une série de protéines virales d'immunodéficience humaine Download PDF

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Publication number
WO2008034297A1
WO2008034297A1 PCT/CN2006/002461 CN2006002461W WO2008034297A1 WO 2008034297 A1 WO2008034297 A1 WO 2008034297A1 CN 2006002461 W CN2006002461 W CN 2006002461W WO 2008034297 A1 WO2008034297 A1 WO 2008034297A1
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WIPO (PCT)
Prior art keywords
hiv
fragment
analog
antigen
antibody
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PCT/CN2006/002461
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English (en)
Chinese (zh)
Inventor
Xudong Sun
Xiaolin Yang
Yiming Shao
Hui Xing
Yong Liu
Li Qin
Xiaodong Wu
Original Assignee
Beijing Yuande Bio-Medical Engineering Co., Ltd.
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Application filed by Beijing Yuande Bio-Medical Engineering Co., Ltd. filed Critical Beijing Yuande Bio-Medical Engineering Co., Ltd.
Priority to PCT/CN2006/002461 priority Critical patent/WO2008034297A1/fr
Priority to CN200680055877.4A priority patent/CN101523216B/zh
Publication of WO2008034297A1 publication Critical patent/WO2008034297A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates to a method of detecting the presence of human immunodeficiency virus (HIV) in a biological sample. More specifically, the present invention relates to a method for determining the presence or absence of HIV infection by detecting the presence of human immunodeficiency virus protein antibodies in a biological sample by combining a plurality of human immunodeficiency virus protein antigens. The invention also relates to kits for use in such methods. Background technique
  • HIV Human immunodeficiency virus
  • WHO World Health Organization
  • HIV virus mainly has type I and sputum type, and type I HIV virus (HIV-1) is the main infection type.
  • type I HIV virus HIV-1
  • HIV-2 HIV-2
  • Other HIV types are also constantly being discovered. HIV can be transmitted through blood, sexual contact and mother-to-child transmission.
  • the main method of controlling infection is to prevent infection, and cutting off the transmission route is the best prevention and control strategy. Therefore, reliable low-cost and high-efficiency HIV detection methods are particularly important.
  • HIV-1 After HIV-1 infection, all the structural protein antigens of HIV-1 appear in the blood of all infected persons, namely gp41, gpl20 and gpl60 encoded by the envelope gene (ENV); pl7 encoded by the nucleocapsid gene (GAG), P24, p55 and all or most of the specific sustained IgG antibodies of P34, P51, P66 encoded by the polymerase gene (POL).
  • HIV antibodies in serum are an indirect indicator of HIV infection. After HIV infects the body, the body will produce antibodies to HIV, and antibodies can persist throughout the disease. Therefore, the detection of antibodies in vivo usually means the presence of a virus.
  • Detection of antibodies is currently the most common and most effective method.
  • HIV testing uses a special strategy of first screening with a highly sensitive method, and screening for positive screening is confirmed by a specific method. Therefore, the antibody detection method is generally divided into two types according to its purpose: initial screening detection and confirmation of the real face.
  • the purpose of the primary screening test is to eliminate antibody negative samples and narrow the confirmation range. Negative screening may be considered negative for antibodies, ie, no anti-HIV antibodies are present, and no infection may occur between three weeks and three months. Positive screening does not prove inevitable infection, and confirmation reagents are required to confirm infection.
  • the main screening methods in the world are: 1 enzyme-linked immunosorbent assay (ELISA); 2 gelatin particle agglutination test (PA); 3 latex agglutination test (LA); 4 various rapid detection methods (such as: HIV gold standard fast Test strips) and so on.
  • ELISA enzyme-linked immunosorbent assay
  • PA gelatin particle agglutination test
  • LA latex agglutination test
  • 4 various rapid detection methods such as: HIV gold standard fast Test strips
  • Both the first and second generation kits use the ELISA indirect method, which first coats the ELISA plate with mixed antigens (gp41, gp36, gpl20, and p24), adds the serum to be tested, and finally adds the enzyme-labeled anti-human IgG antibody. . Therefore, only IgG antibodies can be detected. Due to limitations in methodology and raw materials, it is difficult to ensure that the sensitivity and specificity of detection are maintained at a good level.
  • the third-generation reagent adopts the double antigen sandwich method, that is, the antigen-coated ELISA plate is first added, the serum to be tested is added, and finally the enzyme-labeled antigen is added, and the I g G and IgM antibodies can be simultaneously detected. In the immunological principle, after the human immune response is infected with the pathogen, IgM antibodies first appear, followed by IgG antibodies. Therefore, compared with the indirect method, the double antigen sandwich method can shorten the blind spot on the detection-window period
  • a fourth-generation ELISA product was launched in 1998 to simultaneously detect HIV antigen (P24) and antibodies (including "O" subtype antibodies).
  • the product is to simultaneously coat the antigen and the antibody on the polystyrene plate, add the serum to be tested, and finally add the enzyme-labeled antigen and the monoclonal antibody, thereby simultaneously detecting the P24 antigen in the serum, HIV-1+2.
  • Type antibodies and HIV-1's ' ⁇ ' subtype antibodies were launched in 1998 to simultaneously detect HIV antigen (P24) and antibodies (including "O" subtype antibodies).
  • the primary screening test is a comprehensive result of detecting HIV antibodies in the sample.
  • the disadvantage is that the specificity is poor, so many positive samples in the primary screening are false positives.
  • the HIV antibody confirmation reagents commonly used in the international market include HIV-1 type, HIV-2 type and HIV-1 1/2 hybrid type.
  • the detection method is WB and LIA.
  • the clinical application is also WB method and LIA. At present, all of these methods are to prepare various structural protein antigens (natural or artificial expression) of HIV virus on the membrane strip. When used, the membrane is immersed in a human serum sample for overnight reaction, and finally the color-related method is used to see whether the relevant protein is related. A visible strip is present to bring the detection antibody, and finally, whether or not the HIV infection is caused by the occurrence of each antigen antibody, the judgment criteria are as described in, for example, Example 3 of the present invention.
  • the invention overcomes the prior art by realizing the rapid and sensitive detection of HIV infection by combining at least five HIV-1 structural protein antigens and one HIV-2 structural protein antigen for the detection of corresponding antibodies in biological samples. Defect in the middle. Summary of the invention
  • the present invention provides a method for detecting the presence of an anti-HIV antibody in a biological sample, comprising combining at least five HIV-1 structural protein antigens and an HIV-2 structural protein, respectively, in contact with the sample, and then detecting binding. The presence of the complex.
  • the invention also provides kits for performing HI V detection on biological samples. detailed description
  • the invention provides a method of detecting the presence of an anti-HIV antibody in a biological sample, comprising combining at least five HIV-1 structural protein antigens and one HIV-2 structural protein, respectively The sample is contacted, and then the presence of the binding complex is detected, the at least five HIV-1 structural protein antigens comprising (i) P24 or a fragment or analog thereof; (ii) pl7 or p55 or a fragment or analog thereof; (iii) at least one of P31, P34, P39, P51, P66 or a fragment or analog thereof; (iv) GP41 or a fragment or analog thereof; and (V) GP120 or a fragment or analog thereof.
  • the term "antigen" includes a full-length HIV protein, a derivative of a full-length HIV protein, such as, but not limited to, a protein fragment or a synthetic peptide containing an amino acid sequence corresponding to one or more portions of the full-length HIV protein, Any modified fragment or synthetic peptide to which a ligand is attached is included.
  • the term "antigen” also includes analogs of full-length HIV proteins, or analogs of fragments or peptides, including, but not limited to, non-peptide molecules that bind to the same antibody as the corresponding parent protein, as well as variants or fusion polypeptides of the antigen. Or immunogenic fragments.
  • the "immunogenic portion" of an HIV protein antigen refers to a moiety which, upon inoculation into an individual, is capable of eliciting an immune response and is also capable of binding to a specific antibody produced against the corresponding protein antigen.
  • the immunogenic portion of the protein described herein can be identified using an antibody binding assay.
  • assays can generally be performed by any of a variety of methods known to those of ordinary skill in the art, for example, in Harlow and Lane, Antibody: Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988). Said method.
  • a polypeptide "variant" as used herein is a polypeptide which differs from the polypeptide only by the substitution, deletion and/or insertion of one or more thio acids while still retaining the immunogenicity of the polypeptide.
  • the substitution is a conservative substitution.
  • the polypeptide variant and the identified polypeptide have at least about 80%, more preferably at least about 90%, and most preferably at least about 95% identity.
  • An immunologically active HIV protein antigen variant can be identified by, for example, modifying the amino acid sequence of one of the above polypeptides and assessing the immunological activity of the modified polypeptide.
  • conservative substitution means that an amino acid is replaced by another amino acid of similar character, and those skilled in the art of peptide chemistry predict that the secondary structure and hydrophilic properties of the polypeptide are not substantially altered.
  • amino acid groups represent conservative changes: (1) alanine, proline, glycine, glutamic acid, aspartame Acid, glutamine, asparagine, serine, threonine; (2) cysteine, serine, tyrosine, threonine; (3) valine, isoleucine, leucine, Methionine, alanine, phenylalanine; (4) lysine, arginine, histidine; and (5) phenylalanine, tyrosine, tryptophan, histidine.
  • Variants may contain other modifications at the same time or only, including deletion or addition of amino acids that have minimal effect on the antigenicity, secondary structure and hydrophilic properties of the polypeptide.
  • a signal (or leader) sequence can be conjugated to a polypeptide at the amino terminus of the protein, which can be translated with the protein and direct the transfer of the protein after translation.
  • the polypeptide may also be conjugated to a linker or other sequence to facilitate polypeptide synthesis, purification or identification (e.g., polyhistidine), or to enhance binding of the polypeptide to a solid support.
  • the polypeptide with an immunoglobulin F c region conjugation can be conjugated to a polypeptide at the amino terminus of the protein, which can be translated with the protein and direct the transfer of the protein after translation.
  • the polypeptide may also be conjugated to a linker or other sequence to facilitate polypeptide synthesis, purification or identification (e.g., polyhistidine), or to enhance binding of the polypeptide to a
  • antigen protein also includes a combination or fusion polypeptide.
  • a “combined polypeptide” is an HIV antigenic protein comprising at least one of the above immunogenic portions and one or more additional immunogenicities which are joined by peptide bonds into an amino acid sheep chain. These sequences can be joined directly together (i.e., without an inserted amino acid) or can be joined together by a linker sequence (e.g., Gly-Cys-Gly) that does not significantly impair the immunogenicity of the polypeptide component.
  • the HIV antigenic proteins of the invention and the DN A molecules encoding such proteins can be obtained by any of a variety of methods well known in the art.
  • a DNA sequence equivalent to a gene (or a portion thereof) encoding one of the HI V protein antigens of the present invention can be obtained by sequencing a wild type strain and aligning it with a known HIV viral nucleic acid sequence.
  • the partial DNA sequence thus obtained can be used to design oligonucleotide primers to amplify full-length DNA sequences in RT-PCR, and the techniques used are well known in the art (see, for example, Mullis et al., Cold Spring Harber Symp. Quant. Biol., 51:263, 1987; Erlich, PCR, Stockton Press, New York, 1989).
  • DNA sequence encoding an HIV protein antigen can be mediated by oligonucleotides as taught in Adelman et al. (DNA, 2: 183, 1983) using standard mutagenesis techniques. Site-directed mutagenesis, easily introducing any of the above modifications.
  • sequences of the invention can also be synthesized directly.
  • the HIV proteins disclosed herein, or immunogenic portions thereof, can also be produced synthetically or recombinantly.
  • Synthetic polypeptides of less than about 100 amino acids and typically less than about 50 amino acids can be prepared by techniques well known to those of ordinary skill in the art.
  • such polypeptides can be synthesized using any solid phase technique available, such as Merrifield solid phase synthesis, in which amino acids are sequentially added to the extended amino acid chain (see, for example, Merrifield, J. Am. J. Am. Chem. Soc. 85: 2149-2146, 1963)
  • the automated peptide synthesizer is available from Perkin Elmer/Applied Biosystems (Foster City, CA) and can be operated according to the manufacturer's instructions.
  • any of the above polypeptides can be produced recombinantly by inserting a DNA sequence encoding the polypeptide into an expression vector and expressing the protein in a suitable host.
  • Any of a variety of expression vectors known to those of ordinary skill in the art can be used to express the recombinant polypeptides of the invention.
  • Expression can be carried out in any suitable host cell transformed or transfected with an expression vector containing the recombinant polypeptide-encoding DNA molecule.
  • Suitable host cells include prokaryotic cells, yeast, and higher eukaryotic cells.
  • the host cell used is an E. coli, yeast or mammalian cell line, such as a CHO cell.
  • a DNA sequence expressed in this manner can encode a naturally occurring polypeptide, a portion of a naturally occurring polypeptide, or other variants thereof.
  • HIV-1 structural protein antigens and one HIV-2 structural protein can be used in combination in various ways for HIV detection of biological samples.
  • Such techniques include, but are not limited to, enzyme immunoassays (EIAs), such as enzyme-linked immunosorbent assays (ELISA), Western blotting and other immunoblotting, radioimmunoassay (RIA), Radioimmunoprecipitation assay (RIPA), particle agglutination assay, and immunofluorescence assay (IFA).
  • EIAs enzyme immunoassays
  • ELISA enzyme-linked immunosorbent assays
  • RIA radioimmunoassay
  • RIPA Radioimmunoprecipitation assay
  • particle agglutination assay and immunofluorescence assay (IFA).
  • the at least five HIV-1 structural protein antigens and one HIV-2 structural protein are subjected to biological samples by enzyme immunoassay or immunofluorescence assay. HIV testing.
  • the HIV structural protein antigen is immobilized separately on a solid support.
  • the solid support can be known in the art, such as polystyrene beads, plastic microplates, and the like.
  • assays for determining the corresponding antibodies in a sample using an antigen are known to those of ordinary skill in the art. See, for example, Harlow and Lane, Antibody: Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • the assay comprises using an antigen immobilized on a solid support to bind the corresponding antibody in the biological sample and separate it from the rest of the sample.
  • the bound HIV protein antigen-antibody complex can then be detected by conventional methods, including, for example, the use of a second antibody containing a reporter group, or a related antigen containing a reporter group (i.e., a double antigen sandwich method).
  • the solid support can be any material known to those of ordinary skill in the art to which proteins can be attached.
  • the solid support can be a sputum or nitrocellulose membrane or other suitable membrane on a microtiter plate.
  • the support may be a bead or disc, such as glass, fiberglass, latex, colloidal gold or a plastic material such as polystyrene or polyvinyl chloride.
  • the support may also be a magnetic particle or fiber optic sensor such as those disclosed in U.S. Patent No. 5,359,681. HIV antigenic proteins can be immobilized on solid supports using a variety of techniques known to those skilled in the art, which are well described in the patent and scientific literature.
  • immobilized refers to non-covalent binding (e.g., adsorption), and covalent attachment (which may be a direct linkage between an antigen and a functional group on a support, or by attachment of a crosslinking reagent).
  • a method of immobilization by adsorption onto a well or a membrane of a microtiter plate is preferred.
  • adsorption can be accomplished by contacting the HIV protein antigen with a solid support in a suitable buffer for a suitable period of time. The contact time varies with temperature, but is usually between about 1 hour and 1 day.
  • contacting a microtiter plate of a plastic microtiter plate (e.g., polystyrene or polyvinyl chloride) with an antigen of about 10 n g - 10 g , preferably about 100 ng-lg, is sufficient to immobilize an appropriate amount of binding agent.
  • a plastic microtiter plate e.g., polystyrene or polyvinyl chloride
  • Protein resistance can generally be achieved by first reacting the functional reagent with the support. Covalent attachment of the original to the solid support, which can react with both the support and a functional group (such as a hydroxyl or amino group) on the binding agent.
  • the antigen can be covalently attached to a support having a suitable polymer coating by the use of phenylhydrazine, or by condensation between the aldehyde group on the solid support and the amine and active hydrogen on the antigen.
  • HIV infection in a sample can be determined by an enzyme-linked immunosorbent assay. This analysis is accomplished by first contacting an HIV protein antigen immobilized on a solid support (often a well of a microtiter plate) with a sample to specifically bind the antibody in the sample to the immobilized antigen. The unbound sample is then removed from the immobilized protein antigen-antibody complex and an anti-human IgG antibody (referred to as a secondary antibody) or a related antigen (the secondary antibody or related antigen is conjugated to a suitable reporter group, Including, for example, horseradish peroxidase, streptavidin, etc.). The amount of secondary antibody or related antigen still bound to the solid support is then determined by a method suitable for detecting a particular reporter group.
  • a suitable reporter group Including, for example, horseradish peroxidase, streptavidin, etc.
  • the immobilized antigen is then incubated with the sample to bind the antigen to the antibody.
  • Samples can be diluted with a suitable dilution before incubation, such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the appropriate contact time i.e., incubation time
  • the time necessary to reach equilibrium can be conveniently determined by the level of binding over a period of time. A holding time of about 30 minutes is usually sufficient at room temperature.
  • a suitable buffer such as PBS containing 0.1% Tween 20TM
  • a suitable buffer such as PBS containing 0.1% Tween 20TM
  • the binding between the antigen and the antibody in the biological sample is detected using a secondary antibody or a related antigen.
  • the second antibody or related antigen is in a labeled form comprising a reporter group.
  • reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorophores, and biotin. Attachment or conjugation of the antibody or related antigen to the reporter group can be accomplished by standard methods known to those of ordinary skill in the art.
  • the second antibody or related antigen is then incubated with the immobilized antibody-polypeptide complex for a sufficient period of time to detect the relevant polypeptide.
  • the appropriate incubation time is usually determined based on the level of binding after a period of testing.
  • the unbound genomic antibody or related antigen is then removed and the bound genomic antibody or related antigen is detected by a reporter group.
  • the method of detecting a reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiography methods are generally feasible. Spectroscopy can be used to detect dyes, luminescent groups, and fluorophores. Biotin can be detected by avidin coupled to other reporter groups (usually radioactive or fluorescent groups or enzymes).
  • the enzyme reporter group is typically detected by the addition of a substrate (typically for a specific period of time), followed by spectroscopy or other methods of analysis of the reaction product.
  • a substrate typically for a specific period of time
  • spectroscopy or other methods of analysis of the reaction product The judgment of the result can be observed by the naked eye.
  • exposure film can also be used to observe.
  • this method should also be judged by the naked eye of the experimenter. Therefore, it is more preferable that in the case of the chemiluminescence method, single photon analysis can be used for detection, which can greatly improve the sensitivity of detection.
  • the measured signal of the reporter group still bound to the solid support is typically compared to a standard curve.
  • the standard curve is obtained by incubating the immobilized HIV protein antigen with serially diluted HIV positive samples and plotting the detected signal to the sample dilution factor.
  • the analysis can be performed in a flow-through format.
  • the antigen is immobilized on the microbeads, such as polystyrene beads having a diameter of 4.5 mm. During the test, when the sample contacts the microbeads, the antibody in the sample binds to the immobilized antigen.
  • the labeled secondary antibody or related antigen binds to the antigen-antibody complex. Detection of the bound scorpion antibody or related antigen can be carried out as described above.
  • envelope gene ENV
  • pl7, p24, p55 encoded by the nucleocapsid gene (GAG) and the polymerase gene All specific antigens such as P34, P51, and P66 encoded by POL are coated in a microplate, and each sample is tested in combination, and finally, whether or not HIV is infected is comprehensively determined according to the occurrence of each antigen antibody.
  • Enzyme immunoassays include the following steps. First, the HIV antigen is purified from the virus lysate, prepared by recombinant DNA technology or peptide synthesis method, and coated into the well of the microplate to form a solid phase of the assay. The biological sample to be tested, such as serum, is added. Into the well. If there is an HIV-specific antibody in the sample, it will react with the antigen immobilized on the solid phase, and then remove the other contents of the well by washing. Adding to the well contains the enzyme or other detection system Anti-human antibody or related antigen.
  • the sample contains HIV-specific antibodies, they will remain attached to the solid phase antigen, and the anti-human antibody or related antigen conjugated with the enzyme will bind to these antibodies, thus indirectly attached to the solid.
  • another washing step is carried out. If the individual's serum contains anti-HIV antibodies, the enzyme will remain attached to the solid phase by the antibody, thus catalyzing the chromogenic reaction after the appropriate substrate is added to the wells. The change in color can be determined. The absorbance is higher than the critical value calculated from the control sample, meaning that the sample is reactive.
  • an anti-human antibody (collectively referred to in the art as "secondary antibody") or a related antigen is conjugated to a labeled form.
  • the label can be, for example, horseradish peroxidase, Streptavidin and the like.
  • Secondary antibodies useful in the present invention include intact molecules and fragments thereof, including, for example, Fab, F(ab')2, and Protein A and Protein G, among others, which are capable of binding epitope determinants. Methods for preparing these fragments are known in the art, see, for example, Harlow and Lane, Antibody: Laboratory Manual, Cold Spring Harbor Laboratory Press, Group (1988). This document is incorporated herein by reference.
  • a related antigen which can be used in the present invention means an antigen or a fragment or analog thereof which is consistent with an antigen coated (immobilized on a solid support).
  • the HIV protein antigen is immobilized on a solid phase support in an appropriate amount (concentration). (Concentration) This amount is preferably 0.05-2.0 ⁇ ⁇ / ⁇ 1, in particular 0.5 g / ml.
  • concentration concentration
  • the protein antigen is applied to the solid support in the form of a solution in a carbonate buffer of 0.02 mol/l, pH 9.6, typically in a volume of ⁇ ⁇ ⁇ .
  • concentration of protein antigens typically in a volume of protein antigen solution applied, and can be determined by routine experimentation.
  • HIV-1 structural protein antigens and one HIV-2 structural protein antigen are used for detection.
  • the HIV-1 structural protein antigens are p24, p55, P66, gp41 and gpl20, and the HIV-2 structural protein antigen is GP36.
  • the detection is performed using at least six HIV-1 structural proteins and an HIV-2 structural protein antigen, wherein the HIV-1 structural protein antigen comprises five of P31, P34, P39, P51, and P66. At least two of them.
  • the at least six HIV-1 structural protein antigens are p24, p55, p31, P66, gp41, gpl20.
  • the HIV-2 structural protein is GP36.
  • these structural protein antigens can be immobilized on a solid phase support by the method described above and detected as described above.
  • HIV structural protein antigens may also be included in the methods of the invention.
  • patient means any warm-blooded animal, preferably a human, a patient may It is sick or does not have a detectable disease.
  • sample can be any sample that may contain antibodies against HIV protein antigens, including, for example, serum, whole blood, urine, pleural effusion, cerebrospinal fluid, and tissue specimens.
  • kits for HIV detection of biological samples may comprise pre-packaged reagents present in predetermined amounts, including HIV protein antigens and labeled forms of anti-human IgG antibodies or related antigens, and the like, as well as instructions for practicing the methods of the invention.
  • Preferred labeling groups include enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorophores, and biotin.
  • the attachment of the antibody or related antigen to a labeling group (which may also be referred to as a reporter group) can be accomplished by standard methods known to those of ordinary skill in the art.
  • the kit optionally includes an agent suitable for detecting the label.
  • the label is an enzyme
  • the substrate may comprise a substrate and a cofactor required for the enzyme (e.g., a substrate precursor that provides a chromophore or fluorophore).
  • a cofactor required for the enzyme e.g., a substrate precursor that provides a chromophore or fluorophore
  • other additives such as stabilizers, buffers and the like may also be included.
  • the relative amounts of the various reagents can vary widely so that the sensitivity of the assay can be optimized.
  • the reagent may be provided in the form of a dry powder, usually a lyophilized powder, comprising an excipient which, when dissolved, will provide the reagent solution at an appropriate concentration.
  • the HIV antigen used in the kit of the present invention can be obtained from a variety of sources known in the art.
  • the HIV antigen can be prepared by recombinant methods, such as those described herein.
  • HIV protein antigens are commercially available from commercial suppliers, for example, from Biodesign International (Maine, USA).
  • the HIV protein antigen may be in the form of a support or a shape of a microplate or microbead or microsphere or other shaped plastic and glass material that can be simultaneously measured in parallel. These microplates can be assembled to combine different HIV protein antigen pores for the determination of HIV infection as needed.
  • the invention combines the advantages of the original screening and confirmation methods, and is excellent in sensitivity Degree and perfect specificity improve the accuracy and time of AIDS diagnosis.
  • the following is an embodiment of the invention, which is merely illustrative of the invention, but the content of the invention is not limited thereto.
  • Example 1 Preparation of HIV-1 protein antigen and HIV-2 protein antigen Sequences according to the prior art for HIV-1 protein antigens gp41, gpl20, p66, p31, p55, p24 and HIV-2 protein antigen gp36 Information, genetically engineered to produce six HIV-1 antigens (ENV region: gp41, gpl20; POL region: p66, p31; GAG region: p55, p24;) and one HIV-2 antigen (ENV region: gp36). References to antigenic protein sequence information and preparation methods are shown in Table 1.
  • the HIV protein antigen prepared according to Example 1 was prepared into a coating solution of 0.1 ⁇ g/mL with a pH 9.6, 0.02 M carbonate buffer solution, and coated with a microplate of 100 ⁇ l per well, 2 ⁇ After 8 ° C overnight, the coating was discarded. Add 1% BSA in PBS blocking solution at 200 ⁇ l per well, and block at 37 °C for 2 h; dry and place in a sealed bag and store in vacuum.
  • Example 3 Detection of Biological Samples Using HIV Protein Antigen-coated Plate Enzymes
  • the samples determined in this example were from the Sichuan Provincial Center for Disease Control and Prevention, in which Sample 1, Sample 2, and Sample 3 were confirmed HIV antibody positive samples; Sample 4 and Samples 5 is a confirmed HIV antibody negative sample; Sample 6, Sample 7 and Sample 8 are HIV antibody uncertain samples.
  • a biological sample to be tested diluted 1:1000 in PBS containing 1% BSA, was added, and incubated at 37 ° C for 2 hours. Then, each well was washed with a PBS washing solution to remove unbound components. ⁇ was added to the wells with 1% BSA in PBS as buffer and about 10 ng/ml of anti-human IgG conjugated with horseradish peroxidase.
  • Anti-human IgG can be purchased from commercial suppliers, for example Obtained from Biodesign International (Maine. USA).
  • the anti-human I g G conjugated with horseradish peroxidase was prepared by a conventional periodic acid oxidation method and reacted again for 1 hour.
  • a luminescent substrate (1.25 mmol/L luminol, 0.136 mmol/L p-iodophenol, 10 mmol/L Tris-HCL (pH 8.6), 0.2% ethanol, 0.3 mmol/L NaCL, 5 mmol/ was added to the wells.
  • CDTA L-ring diamine tetraacetic acid
  • the relative luminescence intensity was measured and compared with the critical value.
  • At least 2 ENV proteins (gp41, gpl20) antibodies were detected positive, or
  • GAG p55, p24
  • POL p66, p31
  • HIV-1 antibody positive results results, and gp36 protein antibody positive HIV-2 positive infection
  • Sample 1, Sample 2, Sample 3, Sample 6, Sample 7 and Sample 8 are all positive samples, and Sample 4 and Sample 5 are negative samples.
  • the detection result of the biological sample which is confirmed positive is positive
  • the detection result of the biological sample which has confirmed negative is negative, which indicates that the method of the invention is completely reliable, Its biggest feature is quick and easy.
  • a PCR method is also used to detect the DNA form of HIV nucleic acid in the sample.
  • the DNA form may be a provirus integrated into the chromosome of an infected cell, or an RNA synthesized in an infected cell in which the virus is actively expressed, or a virus particle in a cell-free plasma.
  • the cells in the biological sample were subjected to detergent lysis, and the lysate was subjected to PCR amplification of HIV DNA according to the instructions in the kit of Comparison of NucliSens EasyQ HIV-1 of bioM rieux China Limited (France).

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Abstract

L'invention concerne un procédé de détection de l'existence d'un virus d'immunodéficience humain dans des échantillons biologiques. Particulièrement, la détection de l'existence d'anticorps dirigés contre les protéines virales d'immunodéficience humaine dans des échantillons biologiques permet de déterminer la présence ou non de l'infection par le VIH. L'invention concerne également les kits réactifs utilisés dans ce procédé.
PCT/CN2006/002461 2006-09-20 2006-09-20 Procédé de détection d'anticorps dirigés contre une série de protéines virales d'immunodéficience humaine WO2008034297A1 (fr)

Priority Applications (2)

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PCT/CN2006/002461 WO2008034297A1 (fr) 2006-09-20 2006-09-20 Procédé de détection d'anticorps dirigés contre une série de protéines virales d'immunodéficience humaine
CN200680055877.4A CN101523216B (zh) 2006-09-20 2006-09-20 人类免疫缺陷病毒系列蛋白抗体测定方法

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PCT/CN2006/002461 WO2008034297A1 (fr) 2006-09-20 2006-09-20 Procédé de détection d'anticorps dirigés contre une série de protéines virales d'immunodéficience humaine

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CN104345048B (zh) * 2013-08-07 2016-12-07 杨晓林 一种基于上转换发光物质检测生物分子的方法及实施其的检测系统
CN105738622A (zh) * 2015-10-23 2016-07-06 北京玛斯玛克生物科技有限公司 一种胶体金层析法检测人体尿液hiv1/2抗体试纸及其制备方法
CN105527426A (zh) * 2015-12-31 2016-04-27 天津医科大学总医院 液相芯片法hiv抗原抗体联合检测试剂盒及检测方法
CN114502957A (zh) * 2019-10-14 2022-05-13 深圳迈瑞生物医疗电子股份有限公司 用于检测hiv抗体的试剂盒以及方法
CN110907645B (zh) * 2019-12-17 2022-03-01 郑州安图生物工程股份有限公司 一种人类免疫缺陷病毒抗体的检测试剂盒

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WO2005090983A2 (fr) * 2004-02-27 2005-09-29 Board Of Regents, The University Of Texas System Systeme de dosage a membranes comprenant des particules prechargees
CN1673749A (zh) * 2005-03-23 2005-09-28 北京科卫临床诊断试剂有限公司 Hiv病毒抗体/抗原诊断试剂盒及其制备方法、检测方法

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