WO2008034297A1

WO2008034297A1 - Method of detecting antibodies against a series of human immunodeficiency virus proteins

Info

Publication number: WO2008034297A1
Application number: PCT/CN2006/002461
Authority: WO
Inventors: Xudong Sun; Xiaolin Yang; Yiming Shao; Hui Xing; Yong Liu; Li Qin; Xiaodong Wu
Original assignee: Beijing Yuande Bio-Medical Engineering Co., Ltd.
Priority date: 2006-09-20
Filing date: 2006-09-20
Publication date: 2008-03-27
Also published as: CN101523216B; CN101523216A

Abstract

A method of detecting the situation of existence of human immunodeficiency virus in biological samples is provided. Particularly, by detecting the situation of existence of antibodies against human immunodeficiency virus proteins in biological samples, it is judged whether HIV infection exists. Reagent kits used in the method are also provided.

Description

Human immunodeficiency virus series protein antibody assay method

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

Human immunodeficiency virus (HIV) is one of the most ferocious enemies facing human health. Since it was first discovered in 1981, it has continued to spread around the world at an unstoppable rate. According to estimates by the World Health Organization (WHO), as of the end of December 2001, there were approximately 40 million HIV-infected/AIDS cases in the world, and about 22 million people died of AIDS during the 20-year period. Became the top four life-killers, and the proportion of young and middle-aged adults with infections and deaths is so great that AIDS has caused disasters to humanity as much as the world war.

Since the 1990s, the fastest growing areas of people living with HIV have moved from Africa to Asia. In China, the first case of afferent cases was discovered in 1985. In 1991, the first HIV-infected epidemic area appeared in Yunnan. HIV infection has been prevalent in all provinces, municipalities and autonomous regions. Since the HIV epidemic in China entered a period of rapid growth in 1994, the estimated number of HIV infections has accumulated to more than 640,000 and will continue to rise. If effective measures are not taken to control, by 2010, China's HIV-infected people may exceed 10 million, making it one of the countries with the highest absolute number of HIV-infected people in the world, thus seriously affecting the health of our people, social stability and national reform. open.

The HIV virus mainly has type I and sputum type, and type I HIV virus (HIV-1) is the main infection type. The vast majority of HIV-infected people currently found in China are HIV-1. There are fewer carriers of the virus, and fewer carriers of the HIV-2 virus. Other HIV types are also constantly being discovered. HIV can be transmitted through blood, sexual contact and mother-to-child transmission. At present, there are no effective therapeutic drugs and vaccines for HIV infection and AIDS. 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.

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. In order to achieve the highest possible accuracy of the test results, 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.

1. Antibody screening test

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. At present, 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. However, ELISA is most commonly used, and ELISA has high sensitivity and specificity, and is easy to operate, and is suitable for large-scale screening. Since the first generation of ELISA products came out in 1985 In the world, there are dozens of well-known companies in the world, nearly 100 varieties are in use, and there are more than 10 companies in China that produce HIV ELISA test kits, but the varieties are relatively simple, basically HIV-1/2 mixed antibodies. The test kit can only detect serum/plasma specimens. The development of ELISA reagents has been basically classified into four generations depending on the source of the antigen or antibody and the type of the detection reagent.

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.

To further shorten the 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 'Ό' 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.

2. Antibody confirmation experiment

It needs to be confirmed on the basis of primary screening. The methods currently available for confirmation are: 1 Western blot (WB - Western Blot); 2 immunoassay (Line Immunoassay LIA); 3 immunofluorescence assay (IFA); 4 Radioimmunoprecipitation Test (RIPA); 5 virus isolation method, etc., the first two clinical applications, namely WB method and LIA method.

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.

Their common disadvantage is that the sensitivity is much lower than the ELISA method for primary screening. 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

In one embodiment, 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.

In the present invention, 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.

In the context of the present invention, 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. Thus, the immunogenic portion of the protein described herein can be identified using an antibody binding assay. These 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. Preferably, the substitution is a conservative substitution. Preferably, 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.

As used herein, "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. . Generally speaking, the following 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. For example, 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. For example, the polypeptide with an immunoglobulin F _c region conjugation.

As used herein, "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). Once a DNA sequence encoding an HIV protein antigen is obtained, it 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. The 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. For example, 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. Preferably, 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.

In the present invention, at least five 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 are well known in the art and 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).

Preferably, 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. In one embodiment, 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. A variety of 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. In a preferred embodiment, 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. For example, the solid support can be a sputum or nitrocellulose membrane or other suitable membrane on a microtiter plate. Alternatively, 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. In the context of the present invention, the term "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. In such cases, 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. In general, 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.

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. For example, 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.

[See Pierce Immunotechndogy Catalog and Handbook, 1991, at A12-A13

In certain embodiments, 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.

More specifically, once the antigen is immobilized on the support as described above, the residual protein binding site on the support is typically blocked. Many suitable blocking agent ordinary skill in the art is known in the negative, such as bovine serum albumin or Tween 20 ^TM (Si _g ma Chemical Co., St. Louis, MO). 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). In general, the appropriate contact time (i.e., incubation time) is sufficient to determine the presence of antibodies to the HIV protein antigen in the sample. Optimum level of at least 95% of the time. One of ordinary skill in the art will recognize that 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.

Then, use a suitable buffer (such as PBS containing 0.1% Tween 20TM) The unbound sample is removed by washing the solid support and the binding between the coated antigen and the antibody is detected by a suitable method. Preferably, the binding between the antigen and the antibody in the biological sample is detected using a secondary antibody or a related antigen. More preferably, the second antibody or related antigen is in a labeled form comprising a reporter group. Preferred 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. The judgment of the result can be observed by the naked eye. In order to eliminate the influence of the subjective experience of the experimenter, exposure film can also be used to observe. Of course, 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.

To determine the presence or amount of antibodies to the HIV protein antigen in the sample, the measured signal of the reporter group still bound to the solid support is typically compared to a standard curve. In a preferred embodiment, 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. In a related example, 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. Then, when the solution containing the second antibody or the related antigen contacts the microbeads, 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.

In one embodiment, the HIV-1 structural protein antigen, gp41, gpl20, gpl60 encoded by the 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. If 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. Next, 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.

In the present invention, 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).

In the present invention, the HIV protein antigen is immobilized on a solid phase support in an appropriate amount (concentration). (Concentration) This amount is preferably 0.05-2.0μ _§ / πι1, in particular 0.5 g / ml. Typically, 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 ΙΟΟ μ 。. Of course, the ordinarily skilled artisan is aware of the buffers that can be employed, the concentration of protein antigens, and the volume of protein antigen solution applied, and can be determined by routine experimentation.

In one embodiment of the invention, five HIV-1 structural protein antigens and one HIV-2 structural protein antigen are used for detection. Preferably, the HIV-1 structural protein antigens are p24, p55, P66, gp41 and gpl20, and the HIV-2 structural protein antigen is GP36. In another embodiment, 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. Preferably, the at least six HIV-1 structural protein antigens are p24, p55, p31, P66, gp41, gpl20. The HIV-2 structural protein is GP36. In the present invention, these structural protein antigens can be immobilized on a solid phase support by the method described above and detected as described above.

In a further embodiment, other HIV structural protein antigens may also be included in the methods of the invention.

As used herein, "patient," means any warm-blooded animal, preferably a human, a patient may It is sick or does not have a detectable disease.

In the context of the present invention, a "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.

The invention also provides kits for HIV detection of biological samples. The kit of the present invention 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. In one embodiment, the kit optionally includes an agent suitable for detecting the label. For example, if 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). In addition, 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. For example, the HIV antigen can be prepared by recombinant methods, such as those described herein. Alternatively, HIV protein antigens are commercially available from commercial suppliers, for example, from Biodesign International (Maine, USA). In the present invention, 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

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.

Example 2 Preparation of HIV Protein Antigen Coating Plate

The HIV protein antigen prepared according to Example 1 was prepared into a coating solution of 0.1 μg/mL with a pH 9.6, 0.02 M carbonate buffer solution, and coated with a microplate of 100 μl per well, 2 ~ After 8 ° C overnight, the coating was discarded. Add 1% BSA in PBS blocking solution at 200 μl 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.

To the wells of the microplate of HIV protein antigen prepared according to Example 2, 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. ΙΟΟμΙ 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. L-ring diamine tetraacetic acid (CDTA) and a mixture of 4 mmol/L H ₂ 0 ₂ and 4 mmol/L NaB0 ₃ according to the formulation disclosed in Chinese Patent No. 91120621.9, the relative luminescence intensity was measured and compared with the critical value. To determine whether an HIV antigen is positive or negative, and finally the comprehensive specimen is responsive to various HIV antigens, as follows The well-established discriminant criteria in the art lead to the conclusion that HIV antibody-positive, HIV-negative or HIV antibodies are uncertain:

Analysis results Protein antibody positive (S/CO greater than 1.0)

Negative No specific protein antibody positive

At least 2 ENV proteins (gp41, gpl20) antibodies were detected positive, or

HIV-1 positive detected 1 ENV protein (gp41, gpl20) antibody positive and 1

GAG ( p55, p24 ) or POL ( p66, p31 ) protein antibody positive

HIV-1 P day sex, and tips

HIV-1 antibody positive results, and gp36 protein antibody positive HIV-2 positive infection

Uncertain, specific protein antibody is positive, but not enough to be positive

Uncertain, suggesting that HIV-2 is positive for specific protein antibodies, but not enough to be positive, and positive for positive gp36 protein antibody was detected in the experiment, including a mouse anti-human I _g G monoclonal antibody coated well System quality control comparison. Whether or not specific for HIV I _{g G,} IgG in the sample will be combined with the package murine monoclonal anti-human IgG, and subsequent addition of the conjugated anti-human horseradish peroxidase biological sample I _g G binds, showing a positive reaction, indicating whether the biological sample and reagent have been added and whether the reagent is effective. At the same time, BSA blocking protein control wells were used as a background control.

The experimental results are as follows:

Table 2

It can be seen that 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. When the biological sample is tested, the detection result of the biological sample which is confirmed positive is positive, and 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.

Biological samples that are purely unknown for the infection status 6 - 8, In order to confirm the correctness of the test results of the present invention, 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).

The results showed that the PCR method was positive for samples 6, 7 and 8 (or no detection), indicating that HIV was indeed present in the sample. This is in complete agreement with the results of the method according to the invention, further proving this The method of the invention is completely reliable, but it is very fast compared to the PCR method. Applicants also tested biological samples 1-8 using Western blotting methods known in the art to compare sensitivity with the present invention. The results of the Western blot are shown in Table 2 below, where ± indicates that the results are uncertain. Sample WB band type and results

Example 4 Detection of a large number of biological samples

Using the same method as in Example 3, 1009 samples confirmed by the Western blotting method were determined, and the detection results were sensitivity (true positive rate) of 99.66%, specificity (true negative rate) of 100.00%, and false positive rate (misdiagnosis rate). ) is 0.00%, and the false negative rate (missing rate) is 0.34%.

The above results indicate that by means of the method of the present invention, by combining at least five HIV-1 structural protein antigens with the HIV-2 structural protein antigen GP36, HIV infection in a biological sample can be easily determined with high accuracy. The sensitivity of the assay is higher than the Western blot confirmation in conventional HIV testing, even comparable to the sensitivity of PCR assays. The invention has been exemplified above by way of illustration, but the invention is not limited thereto. A person skilled in the art will be able to make various modifications to the invention without departing from the spirit and scope of the invention.

Claims

Rights request

A method for detecting the presence of an antibody against an HIV viral protein antigen in a biological sample, comprising combining at least five HIV-1 structural protein antigens and HIV-2 structural protein GP36 together and independently The sample is contacted, and then the presence of the binding complex is detected, wherein the at least five HIV-1 structural protein antigens comprise (1) P24 or a fragment or analog thereof, (2) pl7 or p55 or a fragment or analog thereof, 3) at least one of P31, P34, P39, P51, P66 or a fragment or analog thereof, (4) GP41 or a fragment or analog thereof, and (5) GP120 or a fragment or analog thereof.

2. The method of claim 1 wherein said at least five HIV-1 structural proteins and HIV-2 structural proteins are coated or covalently coupled to a support material of a pattern or shape that can be simultaneously measured in parallel.

3. The method of claim 1, further comprising reacting a mixture of said at least five HIV-1 structural protein antigens and a HI V-2 structural protein antigen with said sample with a second antibody or antibody directed against said antibody The step of contacting the antigen.

The method according to any one of claims 1 to 3, wherein at least two of the five antigens P31, P34, P39, P51 and P66 or a fragment or analog thereof are used.

5. The method of claim 1, wherein the HIV structural protein antigen P24, p55, P66, GP41, GP120, GP36, or a fragment or analog thereof is employed

The method of any one of claims 1 to 5, wherein the biological sample is selected from the group consisting of serum, whole blood, urine, pleural effusion, cerebrospinal fluid, and tissue specimens.

7. A kit comprising at least five HIV-1 structural protein antigens and an HIV-2 structural protein GP36 or a fragment or analog thereof, wherein the at least five HIV-1 structural protein antigens comprise (1) P24 or a fragment or analog thereof, (2) pl7 or p55 or a fragment or analog thereof, (3) at least one of P5, P34, P39>P51, P66 or a fragment or analog thereof, (4) GP41 or Its fragments or analogues and (5) GP120 or a fragment or analog thereof, and optionally a buffer for detection.

8. The kit of claim 7, further comprising an anti-human antibody or a related antigen, preferably in a labeled form, such as conjugated horseradish peroxidase or streptavidin , or in the form of a radioactive label.

9. The kit of claim 8 further comprising an agent, such as an enzyme substrate, for detecting a label conjugated to the anti-human antibody or the associated antigen.

10. The kit of claim 7, wherein said plurality of protein antigens are supported by a pattern or shape that can be simultaneously measured in parallel on said microplate or microbead or microsphere or other shaped plastic and glass material. Things.

11. The kit of claim 7 further comprising instructions.

The kit according to any one of claims 7 to 11, which comprises at least two of the five antigens P31, P34, P39, P51 and P66 or a fragment or analog thereof.

The kit according to claim 7, which comprises HIV structural protein antigens P24, p55, P66, GP41, GP120, GP36, or a fragment or analog thereof.