CN101523216B - Method of detecting antibodies against a series of human immunodeficiency virus proteins - Google Patents

Method of detecting antibodies against a series of human immunodeficiency virus proteins Download PDF

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CN101523216B
CN101523216B CN200680055877.4A CN200680055877A CN101523216B CN 101523216 B CN101523216 B CN 101523216B CN 200680055877 A CN200680055877 A CN 200680055877A CN 101523216 B CN101523216 B CN 101523216B
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hiv
antigen
structural proteins
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antibody
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CN101523216A (en
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孙旭东
杨晓林
邵一鸣
邢辉
刘勇
秦莉
吴晓东
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Yuande Biological Medicine Engineering Co Ltd Beijing
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
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    • 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
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    • 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

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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 antibodies assay method
Technical field
The present invention relates to the method that there is situation in human immunodeficiency virus in the detection of biological sample (HIV).More specifically, the present invention relates to by having situation, determine that HIV infects the method that whether exists by this human immunodeficiency virus protein's antibody in the combined detection of biological sample of multiple human immunodeficiency virus proteantigen.The invention still further relates to the kit for the method.
Background technology
Acquired immune deficiency syndrome (AIDS) (Human immunodeficiency virus, HIV) is one of the most fiendish enemy of facing of human health, since 1981 are found first, has continued to spread in the whole world with the speed that is difficult to contain.Estimate that according to the World Health Organization (WHO) (WHO) end calendar year 2001 by the end of December, the whole world is HIV the infected of survival/about 4,000 ten thousand people of AIDS case load still, existing about 2,200 ten thousand people die from acquired immune deficiency syndrome (AIDS) during 20 years.Become the life killer who takes the first four place the position, infect in addition and died between twenty and fifty ratio very large, so the disaster that acquired immune deficiency syndrome (AIDS) causes to the mankind is not second to world war.
Since the last century the nineties, the HIV-positive transfers to the Asia from Africa in fastest-rising area.China 1985 finds to import into case first, first HIV occurs in Yunnan in 1991 to infect Endemic Area, and HIV infects popular in all provinces, cities and autonomous regions so far.Since phase, the HIV number of the infected of estimation added up to surpass 640,000 and also will continue to rise from the popular rapid growth that enters of China HIV in 1994.Controlled as not adopting an effective measure, might be broken through 1,000 ten thousand people to China HIV the infected in 2010, become one of the highest country of global HIV the infected's absolute number, thereby had a strong impact on China people's health, the stable and national reform and opening-up of society.
HIV virus mainly contains I type and II type, and I type HIV virus (HIV-1) is the main popular type of infection.HIV the infected overwhelming majority that China finds at present is HIV-1 virus carrier, and HIV-2 virus carrier is less.Other HIV Virus Types also have continuous discovery.HIV can propagate through blood, property contact and mother and baby's approach.At present HIV infection and AIDS be there is no effective medicine and vaccine, the method that control infects mainly is that prevention is infected, and the cut-out route of transmission is the best preventive control strategy, and therefore, reliable cost-efficiently HIV detection method just seems particularly important.
HIV-1 each structural proteins antigen for HIV-1 all can occur after infecting in all the infected's blood, gp41, the gp120, the gp160 that are namely encoded by env gene (ENV); P17, p24, p55 by nucleocapsid gene (GAG) coding reach by whole or most specificity continuation IgG antibody such as P34, the P51 of pol gene (POL) coding, P66.
HIV antibody is the indirect indexes of judging that HIV infects in the serum.After HIV infects body, body will produce antibody to HIV, and antibody is sustainable to be present in the whole course of disease.Therefore detecting of internal antibody means viral existence usually.
The detection of antagonist is the most frequently used, the most simple and effective at present method.In order to make testing result reach high as far as possible accuracy, HIV detects and uses special strategy, namely carries out primary dcreening operation with the high method of susceptibility first, and the sample of the primary dcreening operation positive is confirmed with the method for high specificity again.So antibody detection method divides two kinds usually by its purpose: primary dcreening operation detects and the affirmation experiment.
1. Antibody Screening detects
The purpose that primary dcreening operation detects is the sample of rejecting negative antibody, and dwindles affirming score.The primary dcreening operation feminine gender can be thought negative antibody, and namely nonreactive HIV antibody exists, and provablely gives birth in three noninductive hair dyeings before thoughtful three months.The primary dcreening operation positive does not prove and must infect, and needs can be confirmed whether to infect with Confirmation reagent.At present main screening methods has in the world: 1. enzyme-linked immunosorbent assay (ELISA); 2. gelatin particle aggegation experiment (PA); 3. latex agglutination experiment (LA); 4. various fast detection methods (as: HIV gold mark quick test strips) etc.But the most commonly used with ELISA, ELISA has very high sensitivity and specificity, and easy and simple to handle, is suitable for extensive screening.Since first generation ELISA product in 1985 comes out, have in the world more well-known nearly 100 kinds of company of tens of families in use, domestic also have ten many enterprises to produce HIV ELISA detection kit, but kind is more single, basically be the detection kit of HIV-1/2 type mixed antibody, can only detect the serum/plasma sample.The ELISA developer reagent, so far type different according to the source of antigen or antibody and that detect reagent basically can be divided into four generation product.
The first generation and second generation kit all adopt the ELISA indirect method, namely first with hybrid antigen (gp41, gp36, gp120 and p24) coated elisa plate, add serum to be checked again, add at last the anti-human IgG antibody of enzyme labeling.Therefore can only detect IgG antibody.Because the restriction of the aspects such as methodology and raw material is difficult to guarantee that its detection sensitivity and specificity remain on the better level simultaneously.Third generation reagent then adopts dual-antigen sandwich method, namely first with antigen coated ELISA Plate, adds serum to be checked again, adds at last the antigen of enzyme labeling, can detect simultaneously IgG and IgM antibody.On immunology principle, after the immune response of human body was pathogenic infection, IgM antibody appearred first, and IgG antibody appears again.Therefore compare with indirect method, dual-antigen sandwich method can shorten the blind spot in the detection---window phase.
For further shortening window phase, released the 4th generation ELISA product that can detect simultaneously HIV antigen (P24) and antibody (comprising " O " hypotype antibody) in 1998.This product is that antigen and antibody are coated with the polystyrene ELISA Plate simultaneously, adds serum to be checked again, adds at last antigen and the monoclonal antibody of enzyme labeling, thereby can detect simultaneously " O " hypotype antibody of P24 antigen, HIV-1+2 type antibody and HIV-1 in the serum.
The primary dcreening operation experiment is the synthesis result that detects HIV antibody in the sample, and its shortcoming is that specificity is relatively poor, so a lot of positive sample are false positive in the primary dcreening operation.
2. antibody recognize experiment
Need on the basis of primary dcreening operation to confirm that the method that can be used at present confirming has: 1. Western blot (WB-Western Blot); 2. band immunization experiment method (LineImmunoassay LIA); 3. immunofluorescence experiment method (IFA); 4. radioimmunoprecipitation experiment (RIPA); 5. isolation of virus etc., using clinically more is front two kinds, i.e. WB method and LIA method.
HIV antibody recognize reagent commonly used has the HIV-1 type in the international market, HIV-2 type and HIV-1/2 mixed type, detection method has WB's, LIA is also arranged, using clinically more also is WB method and LIA method, all these methods are HIV each structural proteins antigen of virus (natural or artificial expression) preparation on the film bar at present, during use the bar film is immersed reaction overnight in the human serum sample, finally visible bars whether occurs with the relevant albumen of development process and bring detection antibody, and situation about finally occurring by each antigen-antibody comprehensively judges whether infected by HIV, and criterion is referring to for example described in the embodiments of the invention 3.
Their common shortcoming is the ELISA method that sensitivity detects far below primary dcreening operation.The present invention has realized quick, Sensitive Detection that HIV infects, thereby has overcome defective of the prior art by with at least five kinds of HIV-1 structural proteins antigens and the combined detection for the biological sample corresponding antibodies of a kind of HIV-2 structural proteins antigen.
Summary of the invention
The invention provides the method that there is situation in anti-HIV antibody in a kind of detection of biological sample, comprise will at least five kinds HIV-1 structural proteins antigens combined with a kind of HIV-2 structural proteins and contact with described sample respectively, then detect the situation that exists in conjunction with compound.
The present invention also provides and has been used for biological sample is carried out the kit that HIV detects.
Embodiment
In one embodiment, the invention provides the method that there is situation in anti-HIV antibody in a kind of detection of biological sample, comprising will at least five kinds HIV-1 structural proteins antigens combined with a kind of HIV-2 structural proteins and contact with described sample respectively, then detect the situation that exists in conjunction with compound, described at least five kinds of HIV-1 structural proteins antigens comprise (i) P24 or its fragment or analog; (ii) p17 or p55 or its fragment or analog; (iii) at least a or its fragment or the analog among five kinds of P31, P34, P39, P51, the P66; (iv) GP41 or its fragment or analog; (v) GP120 or its fragment or analog.
In the present invention, term " antigen " comprises the derivant of total length HIV albumen, total length HIV albumen, for example but be not limited only to contain protein fragments or synthetic peptide corresponding to the amino acid sequence of one or more part of total length HIV albumen, comprise any modified fragment or the synthetic peptide that have connected aglucon.Term " antigen " also comprises the analog of total length HIV albumen, and the perhaps analog of fragment or peptide comprises but is not limited only to non-peptide molecule with corresponding parent's protein combination same antibody, and the variant of described antigen or fused polypeptide or immunogenic fragments.
In the context of the present invention, can cause immune response and the part that can be combined with the specific antibody that produces for corresponding protein antigen equally after " the immunogenicity part " of HIV proteantigen refers in being inoculated into individual body.Therefore, protein immunogenicity part available antibodies described herein is in conjunction with test for identification.Any carrying out in the common available several different methods known to persons of ordinary skill in the art of these tests, for example, at Harlow and Lane, the method described in " antibody: laboratory manual " (cold spring harbor laboratory, cold spring port, New York, 1988).
Polypeptide used herein " variant " is to distinguish the immunogenic polypeptide that only is one or more amino acid whose replacement, disappearance and/or insertion and has still kept polypeptide with described polypeptide.Preferably, described replacement is that conservative property substitutes.Polypeptide variants and the polypeptide of having identified preferably have at least about 80%, more preferably at least about 90%, most preferably at least about 95% homogeneity.There is immunocompetent HIV proteantigen variant to be identified by the amino acid sequence of for example modifying one of aforementioned polypeptides and the immunocompetence of assessing this modified polypeptide.
Used herein " conservative property substitutes " refers to that certain monoamino-acid is substituted by the amino acid of another tool similar characteristic, and the chemistry of peptides those of skill in the art can predict not material alterations of the secondary structure of this polypeptide and water-wet behavior.Usually in fact, following amino acid group has represented the conservative property change: (1) alanine, proline, glycocoll, glutamic acid, aspartic acid, glutamine, asparagine, serine, threonine; (2) halfcystine, serine, tyrosine, threonine; (3) valine, isoleucine, leucine, methionine, alanine, phenylalanine; (4) lysine, arginine, histidine; (5) phenylalanine, tyrosine, tryptophane, histidine.
Variant can be simultaneously or is only comprised other modification, comprises deletion or adds this polypeptide antigen, secondary structure and the small amino acid of water-wet behavior impact.For example, can be with amino terminal and the conjugation of polypeptides of signal (or leading) sequence at protein, this sequence can be translated with protein, and instructs the transfer of protein after translation.Polypeptide also can be puted together with joint or other sequence, and polypeptide is synthetic to be easy to, purifying or evaluation (such as the poly histidine) or strengthen the combination of polypeptide and solid support.For example, can be with the F of polypeptide and immunoglobulin (Ig) CPut together in the district.
" antigen protein " used herein also comprises combination or fused polypeptide." combination polypeptide " is to contain at least one above-mentioned immunogenicity part and one or more extra immunogenic HIV antigen proteins, and they connect into an amino acid strand by peptide bond.These sequences can directly link together in (namely without the amino acid that inserts), maybe can link together by can obviously not weakening the immunogenic catenation sequence of polypeptide fractions (such as Gly-Cys-Gly).
In the available several different methods well known in the art any obtains HIV antigen protein of the present invention and this type of protein DNA molecule of coding.Be equivalent to one of code book invention HIV proteantigen gene (or its part) dna sequence dna can by to wild type strain check order and compare with known HIV nucleic acid sequence after obtain.The partial dna sequence that obtains like this can be used for the design oligonucleotides primer, with amplification full length DNA sequence in RT-PCR, used technology be well known in the art (visible such as people such as Mullis, Cold Spring Harber Symp.Quant.Biol., 51:263,1987; Erlich compiles, round pcr, Stockton Press, New York, 1989).In case obtained the dna sequence dna of coding HIV proteantigen, namely the induced-mutation technique of available standard such as the oligonucleotide mediated direct mutagenesis of instructing in (DNA, 2:183,1983) such as Adelman, is introduced above-mentioned any modification easily.Sequence of the present invention also can directly be synthesized.
Also available synthetic or recombination form produces HIV albumen disclosed herein or its immunogenicity part.Be less than about 100 amino acid and usually be less than about 50 amino acid whose synthetic polypeptide and can prepare with the technology that those of ordinary skills know.For example, this class polypeptide can be synthetic with any solid phase technique for utilizing, such as the Merrifield solid phase synthesis process, wherein amino acid be added to successively on the amino acid chain that is prolonging (be found in as, Merrifield, U.S. chemical institute magazine (J.Am.Chem.Soc.85:2149-2146,1963).The polypeptide automatic synthesizer can be available from such as branch of Perkin Elmer/ applying biological system (Foster city, the producer such as CA), and can operating by the indication of manufacturer.
The generation of all can recombinating of any aforementioned polypeptides, the dna sequence dna that is about to coded polypeptide inserts in the expression vector, and in suitable host marking protein.In all expression vectors known to those of ordinary skills any all can be used to express recombinant polypeptide of the present invention.Transform or transfection contain in any suitable host cell of expression vector of recombinant polypeptide coding DNA molecule and all can express.Suitable host cell comprises prokaryotic, yeast and higher eucaryotic cells.Preferred used host cell is Escherichia coli, yeast or cells of mamma animals system, such as Chinese hamster ovary celI.The naturally occurring polypeptide of dna sequence dna codified, the natural part that has polypeptide expressed in this way, or their other variant.
In the present invention, can adopt various ways use in conjunction at least five kinds of HIV-1 structural proteins antigen and a kind of HIV-2 structural proteins that biological sample is carried out HIV detects.Such technology is well known in the art, including, but not limited to enzyme immunoassay (EIA), for example enzyme-linked immunosorbent assay (ELISA), Western blotting and other Western blot, radioimmunoassay (RIA), radioimmuno-precipitation assay (RIPA), particle agglutination determination method and immunofluorescence assay (IFA).
Preferably, described at least five kinds of HIV-1 structural proteins antigens and a kind of HIV-2 structural proteins carry out the HIV detection by enzyme immunoassay or immunofluorescence assay to biological sample.In one embodiment, described HIV structural proteins antigen is individually fixed on the solid support.Described solid support can be as known in the art, such as polystyrene bead, plastic microporous plate etc.The mensuration mode of the corresponding antibodies in the known multiple use antigen test sample of this area routine techniques personnel.For example see Harlow and Lane, " antibody: laboratory manual ", cold spring harbor laboratory, 1988.In a preferred embodiment, measuring the antigen that comprises with being fixed on the solid support separates in conjunction with the corresponding antibodies in the biological sample and with its remainder with sample.In conjunction with HIV proteantigen-antibody complex can detect by conventional method subsequently, for example comprise and to adopt the second antibody that contains reporter group, perhaps contain the related antigen (being dual-antigen sandwich method) of reporter group.
Solid support can be any material that those of ordinary skills are known, protein can be attached to it.For example, solid support can be inspection hole or nitrocellulose filter or other the suitable film on the microtiter plate.Perhaps, holder can be pearl or dish, such as glass, fibrous glass, latex, collaurum or plastic material, such as polystyrene or Polyvinylchloride.Holder also can be magnetic-particle or fiber optic sensor, for example is disclosed in U.S. Patent number 5,359, those materials in 681.The HIV antigen protein can be fixed on the solid support with multiple technologies well known by persons skilled in the art, and these technology have sufficient description in patent and scientific literature.In the context of the present invention, " immobilization " word refers to non-covalent combination (as absorption), and covalent bond (can be direct connection the between functional group on antigen and the holder, or the connection by cross-linking reagent).By on the hole that is adsorbed onto microtiter plate or on the film and immobilized method is preferred.In this class situation, can be by in the damping fluid that is fit to, make the HIV proteantigen contact one suitable period with solid support and finish absorption.Change with temperature duration of contact, but normally between about 1 hour to 1 day.In general, with plastic microtiter (such as polystyrene or Polyvinylchloride) aperture with about 10ng-10 μ g, preferably the antigen of about 100ng-1 μ g contacts, and is enough to the fixedly bond of appropriate amount.
Generally can be by bifunctional reagent and holder at first being reacted to finish the covalent attachment of proteantigen and solid support, described difunctional dose can with holder and bond on the two reaction of functional group's (such as hydroxyl or amino).For example, utilize benzoquinones, or by between the aldehyde radical on the solid support and the amido on the antigen and reactive hydrogen, condensation occuring, can antigen is covalently bound with the holder with suitable polymer coating.[consult Pierce Immunotechndogy Catalog and Handbook, 1991, in A12-A13].
In certain embodiments, can measure the HIV infection conditions in the sample by enzyme-linked immunosorbent assay.The HIV proteantigen that at first will be fixed on the solid support (often being the hole of microtiter plate) contacts with sample, so that the antibody in the sample is attached on the immobilized antigen specifically, thereby finishes this analysis.Subsequently unconjugated sample is removed from immobilization proteantigen-antibody complex, and add anti-human IgG antibody (be called as two anti-) or related antigen (puted together suitable reporter group on the two anti-or related antigens, comprised such as horseradish peroxidase, Streptavidin etc.).Then measure the second antibody that still is incorporated on the solid support or the amount of related antigen with the method that is suitable for detecting the particular report group.
More particularly, in case antigen is fixed on the holder as mentioned above, usually seal protein binding site remaining on the holder.Many suitable sealers are known to those of ordinary skills, such as bSA or polysorbas20 TM(Sigma chemical company, the St. Louis, MO).Then immobilized antigen and sample are incubated jointly, antigen is combined with antibody.Available suitable diluted sample before the incubation is such as phosphate buffer (PBS).In general, be the time that is enough to determine to exist for the antibody of HIV proteantigen in the sample situation suitable duration of contact (being temperature retention time).Preferred duration of contact be enough to make in conjunction with level reach combination and not during the Binding peptide balance in conjunction with time of at least 95% of level.Those of ordinary skills will appreciate that, by in a period of time in conjunction with level, can measure easily the necessary time of balance that reaches.During room temperature, about 30 minutes temperature retention time is usually just enough.
Then, available suitable damping fluid (as contains 0.1% polysorbas20 TMPBS) clean solid support and remove not in conjunction with sample, and adopt suitable method that the combination between envelope antigen and the antibody is detected.Preferably, adopt two anti-or related antigens to detect the combination between the antibody in envelope antigen and the biological sample.More preferably, described second antibody or related antigen are the forms that is labeled, and wherein contain reporter group.Preferred reporter group comprises enzyme (such as horseradish peroxidase), substrate, accessory factor, inhibitor, dyestuff, radioactive nuclide, luminophore, fluorophor and biotin.The standard method of adhering to or puting together known to available those of ordinary skills of antibody or related antigen and reporter group is finished.
Then with second antibody or related antigen and immobilized antibody-polypeptide complex incubation long enough time, to detect related polypeptide.Usually determine suitable incubative time in conjunction with level according to test after a period of time.Then remove unconjugated second antibody or related antigen, and detect second antibody or the related antigen of combination by reporter group.The method of examining report group depends on the character of reporter group.For the radioactivity group, scintillation counting technique or radioautograph method are normally feasible.Spectroscopic methodology can be used to detect dyestuff, luminophore and fluorophor.Biotin can be with coupling the avidin of other reporter group (normally radioactivity or fluorophor or enzyme) detect.Usually by adding substrate (generally lasting special time), with spectroscopic methodology or other methods analyst reaction product the enzyme reporter group being detected again.Result's judgement can be observed by naked eyes.For the impact that the subjective experience of getting rid of the experimenter brings, can also adopt exposed plate to observe.Certainly, this mode also by experiment personnel's naked eyes judge.Therefore more preferably, in the situation of chemoluminescence method, can adopt the single photon analytic approach to detect, so can greatly improve the sensitivity of detection.
For determine in the sample for the antibody of HIV proteantigen have situation or content, signal and the typical curve of the reporter group that still is incorporated into solid support that usually will record compare.In a preferred example, typical curve is by with the HIV positive incubation of immobilized HIV proteantigen with serial dilution, and the signal that detects and Sample Dilution multiple mapped obtains.
In a related example, can carry out this analysis by circulating form.Wherein antigen is fixed on the microballon, is the polystyrene microbeads of 4.5mm such as diameter.In process of the test, when sample contact microballon, the antibody in the sample namely combines with immobilized antigen.Then, when the solution contact microballon that contains second antibody or related antigen constantly, the second antibody of mark or related antigen namely combine with antigen-antibody complexes.In conjunction with second antibody or the detection of related antigen can carry out according to said method.
In one embodiment, with each structural proteins antigen of HIV-1, gp41, the gp120, the gp160 that are namely encoded by env gene (ENV); By p17, the p24 of nucleocapsid gene (GAG) coding, p55 and be coated on respectively in the microwell plate by whole specific antigens such as P34, the P51 of pol gene (POL) coding, P66, each sample carries out compound detection, and situation about finally occurring by each antigen-antibody comprehensively judges whether infected by HIV.
Enzyme immunoassay comprises the steps.At first, by virolysis thing purifying HIV antigen, by recombinant DNA technology or the preparation of peptide synthetic method, and coated in the hole of microwell plate, form " solid phase " of determination method.With biological sample to be checked for example serum join in the hole.If there is the HIV specific antibody in the sample, then it will react with fixing antigen on the solid phase, then other content in the hole be removed by washing.The Xiang Kongzhong interpolation contains anti-human antibody or the related antigen of being combined with enzyme or other detection system.If contain the HIV specific antibody in the sample, they will remain adhered on the solid phase antigen, puted together the anti-human antibody of enzyme or related antigen in connection with to these antibody, thereby indirect joint be on solid phase.Then carry out another washing step.If individual serum contains anti-HIV antibody, enzyme will keep being attached on the solid phase by antibody, thereby add suitable substrate to Kong Zhonghou with the catalysis color producing reaction.Can measure the variation of color by spectrometer.Absorbance is higher than the critical value of being calculated by control sample and means that this sample is responding property.
In the present invention, anti-human antibody (being referred to as in the art " two is anti-") or related antigen are the forms of having puted together mark.Described mark can be such as horseradish peroxidase, Streptavidin etc.Can be used for that two among the present invention is anti-to comprise complete molecule and fragment thereof, for example comprise Fab, F (ab ') 2 and albumin A and Protein G etc., they can be in conjunction with the epi-position determinant.The method for preparing these fragments is as known in the art, consults for example Harlow and Lane, " antibody: laboratory manual ", publishing house of cold spring harbor laboratory, New York (1988).This document is hereby incorporated by.Can be used for related antigen of the present invention and refer to antigen or its fragment or the analog consistent with coated (being fixed on the solid support) antigen.
In the present invention, the HIV proteantigen is to be fixed on the solid support with suitable consumption (concentration).This consumption (concentration) is 0.05-2.0 μ g/ml preferably, especially 0.5 μ g/ml.Usually, proteantigen is to be in 0.02mol/l, and the solution form in the carbonate buffer solution of pH9.6 is applied on the solid support, generally is fixed with 100 μ l volumes.Certainly, those of ordinary skill is known the damping fluid that can adopt, the volume of the concentration of proteantigen and applied proteantigen solution, and can determine by normal experiment.
In one embodiment of the invention, adopt five kinds of HIV-1 structural proteins antigens and a kind of HIV-2 structural proteins antigen to detect.Preferably, described HIV-1 structural proteins antigen is p24, p55, and P66, gp41 and gp120, described HIV-2 structural proteins antigen is GP36.In another embodiment, adopt at least six kinds of HIV-1 structural proteins and a kind of HIV-2 structural proteins antigen to detect, wherein said HIV-1 structural proteins antigen comprises at least two kinds among five kinds of P31, P34, P39, P51, the P66.Preferably, described at least six kinds of HIV-1 structural proteins antigens are p24, p55, p31, P66, gp41, gp120.Described HIV-2 structural proteins are GP36.In the present invention, these structural proteins antigens can be fixed on the solid support by method mentioned above, and detect according to preceding method.
In further embodiment, can also comprise in the method for the invention other HIV structural proteins antigen.
" patient " described herein refers to any warm-blooded animal, people preferably, and the patient can be ill, or does not suffer from perceptible disease.
In the context of the present invention, " sample " can be any sample that may contain for the antibody of HIV proteantigen, comprises for example serum, whole blood, urine, pleural effusions and ascites, cerebrospinal fluid and tissue specimen.
The present invention also provides and has been used for biological sample is carried out the kit that HIV detects.Kit of the present invention can comprise the wrapped reagent that exists with scheduled volume, comprises the anti-human IgG antibody of HIV proteantigen and mark pattern or related antigen etc., and the instructions that is used for implementing the inventive method.Preferred labelling groups comprises enzyme (such as horseradish peroxidase), substrate, accessory factor, inhibitor, dyestuff, radioactive nuclide, luminophore, fluorophor and biotin.The standard method that is connected known to available those of ordinary skills of antibody or related antigen and labelling groups (also can be called reporter group) is finished.In one embodiment, comprise alternatively the reagent that is suitable for detecting described mark in this kit.For example, if mark is enzyme, then can comprise the required co-factor of substrate and this enzyme (the substrate precursor of chromophore or fluorophore for example can be provided) in the kit.In addition, can also comprise other adjuvant, such as stabilizing agent, damping fluid etc.The relative quantity of various reagent can extensively change, in order to can make the sensitivity optimization of this determination method.Reagent can provide by dry powder form, and normally freeze-dried powder will make reagent solution have the excipient of debita spissitudo when being included in dissolving.Can obtain to be used for from multiple source as known in the art the HIV antigen of kit of the present invention.For example, described HIV antigen can pass through recombination method, for example described those method preparations herein.Perhaps, the HIV proteantigen can be buied from the commodity suppliers, for example can obtain from BiodesignInternational (Maine, USA).In the present invention, the HIV proteantigen can be fixed on microwell plate or microballon or microballoon or other Special plastic and glass material, the form in the holder of the pattern of measuring in parallel or shape simultaneously.These microwell plates can be to assemble form, thereby as required with the combined mensuration for the HIV infection conditions in different HIV proteantigens hole.
The present invention combines the advantage of original primary dcreening operation and confirmation method, with its remarkable sensitivity and perfect specificity accuracy and time of having improved AIDS diagnosis.
Below be an embodiment of this invention, this embodiment only is used for explanation the present invention, but content of the present invention is not limited to this.
Embodiment
The preparation of embodiment 1HIV-1 proteantigen and HIV-2 proteantigen
About HIV-1 proteantigen gp41, gp120, p66, the sequence information of p31, p55, p24 and HIV-2 proteantigen gp36 is produced 6 kinds of HIV-1 antigens (ENV district: gp41, gp120 by gene engineering method according to well known in the prior art; POL district: p66, p31; GAG district: p55, p24; ) and a kind of HIV-2 antigen (ENV district: gp36).Antigen protein sequence information and preparation method's list of references see Table 1.
Table 1
Figure GPA00000630304000141
The preparation of the coated plate of embodiment 2HIV proteantigen
To use respectively pH9.6 according to the HIV proteantigen of embodiment 1 preparation, the carbonate buffer solution of 0.02M is formulated as the coating buffer of 0.1 μ g/mL, by the coated microwell plate of every hole 100 μ l, 2～8 ℃ spend the night after, abandon coating buffer.Add the PBS confining liquid that contains 1%BSA by every hole 200 μ l, 37 ℃ of sealing 2h; Putting into the sealing bag vacuum after the drying preserves.
Embodiment 3 adopts the coated plate enzyme of HIV proteantigen that biological sample is detected
The sample that this example is measured is from the disease prevention and control center, Sichuan Province, and wherein sample 1, sample 2, sample 3 are the HIV antibody positive sample of confirming; Sample 4 and the HIV negative antibody sample of sample 5 for confirming; Sample 6, sample 7 and sample 8 are HIV antibody indeterminate sample.
Add in the micropore according to the microwell plate of the HIV proteantigen of embodiment 2 preparation by 1:1000 and be diluted in biological sample to be checked 100 μ l among the PBS that contains 1%BSA, incubation is 2 hours under 37 ℃ of temperature.Then, clean each hole with the PBS cleansing solution, to remove unconjugated component.Xiang Kongzhong adds 100 μ l take the PBS of 1%BSA as damping fluid and has contained puting together of about 10ng/ml the anti-human IgG of horseradish peroxidase, anti-human IgG can buy from the commodity suppliers, for example can obtain from Biodesign International (Maine, USA).The anti-human IgG that has puted together horseradish peroxidase adopts conventional periodate oxidation legal system standby, again reacts 1 hour.In the hole, add luminous substrate (1.25mmol/L luminol, the p-iodophenol of 0.136mmol/L, 10mmol/LTrisHCL (pH8.6), 0.2% ethanol, 0.3mmol/L NaCL, the H of 5mmol/L cyclohexanediaminetetraacetic acid (CDTA) and 4mmol/L again 2O 2NaBO with 4mmol/L 3Mixed liquor, according to disclosed prescription preparation in the Chinese patent 91110621.9), measure relative luminous intensity, and with critical value relatively, thereby determine antibody positive or the feminine gender of certain HIV antigen, last comprehensive sample according to art-recognized discrimination standard as described below, draws the conclusion of HIV antibody positive, HIV negative antibody or HIV antibody indeterminate to the reactivity of various HIV antigens:
In experiment, comprised that also the coated hole of a mouse-anti human IgG monoclonal antibody is as system's Quality Control contrast.No matter whether the specific IgG of HIV is arranged in the biological sample, IgG in the sample will be combined with coated mouse-anti human IgG monoclonal antibody, and be combined with the anti-human IgG that has puted together horseradish peroxidase that adds subsequently, biological sample is pointed out in the demonstration positive reaction and whether reagent has added and whether reagent is effective.Simultaneously, contrast as background with BSA closed protein control wells.
Experimental result is as follows:
Table 2
Quality Control Background P24 P31 GP41 P55 P66 GP120 GP36 The result
Sample 1 6785 129 10719 20973 25242 13204 15917 21932 420 +
Sample 2 8472 120 18272 249 25495 17194 9969 25011 292 +
Sample 3 7736 128 16903 8193 24695 8519 16120 5147 430 +
Sample 4 6251 239 134 404 442 292 569 305 197 -
Sample 5 7340 119 126 182 228 348 206 175 216 -
Sample 6 3640 71 324 124 154 1333 197 10062 118 +
Sample 7 4049 67 2035 507 341 1912 841 10309 301 +
Sample 8 5666 191 698 1607 584 10200 900 17786 206 +
Can find out that therefrom sample 1, sample 2, sample 3, sample 6, sample 7 and sample 8 are positive sample, sample 4 and sample 5 negative samples.Method of the present invention is when detecting biological sample, all positive for the testing result of confirming positive biological sample, and all negative for the testing result of confirming negative biological sample, this illustrates that method of the present invention is fully reliable, and its maximum characteristics are fast and convenient.
For infection state unknown biological sample 6-8 sheerly, be the correctness of confirming testing result of the present invention, also adopted PCR method that the HIV nucleic acid of dna form in this sample is detected.This dna form can be to be incorporated into the intrachromosomal provirus of infection cell, or the RNA that synthesizes in the infection cell of active list da virus, perhaps is in the virion in the cell-free plasma.Cell in the biological sample is carried out the detergent cracking, lysate is carried out the pcr amplification of HIV DNA according to the operational manual among the kit Comparison of NucliSens EasyQ HIV-1 of bioM é rieux China Limited (France).
The result shows, PCR method is not to the testing result of sample 6, sample 7 and sample 8 all positive (/ representative detects), illustrate that really to have HIV in the sample viral.This is in full accord with the result who detects according to the inventive method, proved that further the inventive method is fully reliable, but than PCR method, it is very fast and convenient.
The applicant also adopts Western blot method well known in the prior art that biological sample 1-8 is detected, in order to compare with regard to sensitivity with the present invention.The result of Western blotting sees table 2, wherein ± and the result is uncertain in expression.
WB banding pattern and the result of table 3 sample
P17 P24 P31 GP41 P55 P66 GP120 GP160 The result The nucleic acid result
Sample 1 + + + + + + + + + /
Sample 2 + + + + + + + + + /
Sample 3 + + + + + + + + + /
Sample 4 - - - - - - - - - Do not detect
Sample 5 - - - - - - - - - Do not detect
Sample 6 - - - - - - - + ± 2.06×10 6
Sample 7 + - - - - - - + ± 2.14×10 4
Sample 8 - - - - - - - + ± 2.15×10 6
The detection of embodiment 4 large number of biological samples
Sample 1009 examples of Western blot method affirmation have been measured in utilization with embodiment 3 identical methods, testing result is that sensitivity (True Positive Rate) is 99.66%, specificity (true negative rate) is 100.00%, false positive rate (misdiagnosis rate) is 0.00%, and false negative rate (rate of missed diagnosis) is 0.34%.
The above results explanation by method of the present invention, by at least five kinds of HIV-1 structural proteins antigens and HIV-2 structural proteins antigen GP36 are combined, can pin-point accuracy be determined HIV infection conditions in the biological sample easily.The highly sensitive Western blot method of ascertainment in conventional H IV detects of its detection, in addition suitable with the sensitivity of PCR detection.
The above illustrates the present invention in illustrative mode, but the present invention never is limited to this.Those of ordinary skills fully clearly can do various changes to the present invention after having read entire description, and still without departing from the spirit and scope of the present invention.

Claims (16)

  1. In the detection of biological sample for the method that has situation of the antibody of HIV virus protein antigen, comprising with at least five kinds of HIV-1 structural proteins antigens and HIV-2 structural proteins GP36 combines and independently contact with described sample respectively, then detect the situation that exists in conjunction with compound, wherein said at least five kinds of HIV-1 structural proteins antigens comprise at least a, (4) GP41 and (5) GP120 among five kinds of (1) P24, (2) p17 or p55, (3) P31, P34, P39, P51, the P66.
  2. 2. the process of claim 1 wherein described at least five kinds of HIV-1 structural proteins and the HIV-2 structural proteins are coated or covalent coupling on the support material of the simultaneously pattern of measuring in parallel or shape.
  3. 3. the process of claim 1 wherein and also comprise described at least five kinds of HIV-1 structural proteins antigens and HIV-2 structural proteins antigen and potpourri after described sample contacts and second antibody or the contacted step of related antigen for described antibody.
  4. 4. each method among the claim 1-3 has wherein adopted at least two kinds in P31, P34, P39, P51, five kinds of antigens of P66.
  5. 5. the process of claim 1 wherein and adopted HIV structural proteins antigen P24, p55, P66, GP41, GP120, GP36.
  6. 6. each method among the claim 1-5, wherein said biological sample is selected from serum, whole blood, urine, pleural effusions and ascites, cerebrospinal fluid and tissue specimen.
  7. 7. kit, wherein comprise respectively independently at least five kinds of HIV-1 structural proteins antigens and HIV-2 structural proteins GP36, wherein said at least five kinds of HIV-1 structural proteins antigens comprise at least a, (4) GP41 and (5) GP120 among five kinds of (1) P24, (2) p17 or p55, (3) P31, P34, P39, P51, the P66, and alternatively for detection of damping fluid.
  8. 8. the kit of claim 7 wherein also comprises anti-human antibody or related antigen.
  9. 9. the kit of claim 8, wherein said anti-human antibody or related antigen are the forms that is labeled.
  10. 10. the kit of claim 8, wherein said anti-human antibody or related antigen have been puted together horseradish peroxidase or Streptavidin, or radiolabeled form.
  11. 11. the kit of claim 8 wherein also comprises the reagent for detection of the mark of puting together on anti-human antibody or the related antigen.
  12. 12. the kit of claim 11, wherein the reagent for detection of the mark of puting together on anti-human antibody or the related antigen is zymolyte.
  13. 13. the kit of claim 7, wherein said multiple protein antigen are to be fixed on pattern described microwell plate or microballon or microballoon or other Special plastic and glass material, that simultaneously parallel connection is measured or the holder of shape.
  14. 14. the kit of claim 7 wherein also comprises instructions.
  15. 15. each kit among the claim 7-14 wherein comprises at least two kinds in P31, P34, P39, P51, five kinds of antigens of P66.
  16. 16. the kit of claim 7 wherein comprises HIV structural proteins antigen P24, p55, P66, GP41, GP120 or GP36.
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