CN102062779A - Composition for in-vitro detection of hepatitis B virus C antibody - Google Patents
Composition for in-vitro detection of hepatitis B virus C antibody Download PDFInfo
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- CN102062779A CN102062779A CN2009101988370A CN200910198837A CN102062779A CN 102062779 A CN102062779 A CN 102062779A CN 2009101988370 A CN2009101988370 A CN 2009101988370A CN 200910198837 A CN200910198837 A CN 200910198837A CN 102062779 A CN102062779 A CN 102062779A
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Abstract
The invention discloses a composition for in-vitro detection of a hepatitis B virus C antibody. The composition comprises a hepatitis B virus C antigen with more than two antibody identification epitopes, antibody binding proteins and a labeled antibody labeled by a marker. By coating a specific amount of antibody binding proteins on a detection medium, an antibody to be detected and the composition can be combined more effectively so that a detection effect is improved.
Description
Technical field
The present invention relates to a kind of composition of vitro detection hepatitis B antibody, relate in particular to a kind of composition of vitro detection hepatitis B C antibody.Detect hepatitis B C antibody capable with said composition and make detection more accurate, and the result is more directly perceived.
Background technology
B-mode (viral) hepatitis is by hepatitis type B virus (hepatitis B virus, a kind of worldwide disease that HBV) causes.Developing country's incidence of disease height, according to statistics, the asymptomatic hepatitis carrier in the whole world surpasses 2.8 hundred million.China accounts for 9,300 ten thousand, and most of people are asymptomatic, but 1/3 clinical manifestation that hepatic lesion can occur wherein.
By three kinds of approach hepatitis B is diagnosed at present, as: the antigen of virus, the DNA that detects internal antibody and detect virus detected.Clinically, usually use five kinds of markers to judge, as: hepatitis B surface antigen (hepatitis B surface antigen, HBsAg), hepatitis B surface antibody (antibodies to hepatitis B surface antigen, HBsAb), hepatitis B virus e antigen (hepatitisB core antigen, HBeAg), hepatitis B e antibody (antibodies to the ' e ' antigen, anti-Hbe/HBeAb) and hepatitis B core antibody (antibodies to the core antigen, anti-Hbc/HBcAb), claim again " two double ".Hepatitis B virus DNA then is used for diagnosing and the observation chronic hepatitis B.Viral DNA quantity is stablized or is increased more after a little while in blood, illustrates that the state of an illness is comparatively stable; Be on the increase when viral DNA quantity, illustrate that then the state of an illness worsens.
HBsAg comes across in the hepatitis B patient serum before glutamic-pyruvic transaminase raises, and when convalescence, the titre of HBsAg just can progressively reduce and even disappear, but the sustainable existence of part patient HBsAg is arranged.In most hepatitis B virus infection persons' peripheral blood, contain the HBsAg of 5ng-600 μ g/ml.HBsAg in the serum only is that individuality is subjected to the sign that HBV infects, and whether does not reflect virus replication, the infectiousness power of virus replication degree and virus etc.The acute hepatitis b patient is after convalescence, along with HBsAg progressively reduces and even disappears, can occur in the serum HBV is infected the HBsAb with protective immunity effect.Generally speaking, HBsAb in the serum and HBsAg can not detected simultaneously.
The preceding core (pre-core) of HBV/core (core) gene direct coding two peptide species, it is 21kD subunit p21c that forms the virus nucleocapsid body and the 17kD subunit peptides pl7e (cutting 29 amino acid whose pre-core signal peptides that are positioned endoplasmic reticulum) that produces through secretion process.P21c is the HBcAg that defines on the serology, and pl7e is the HBeAg that defines on the serology.Also have bibliographical information to isolate to have-HBeAg of 10--1 pronucleus section at serum (Virology 1988,163,268-275) and contain whole 29 amino acid whose HBeAg (J.Immunol., 1991,147,3156-3160).HBeAg and HBcAg have 75% homology approximately, and its time of occurrence in serum is slightly later to HBsAg.Generally speaking, during as if the HBeAg test positive, HBsAg detects also positive, and the HBeAg positive findings shows that also the infectiousness of virus is strong.If this situation continued more than 3 months, the chronicity tendency is arranged then.After the HBeAg marker testing result in the serum is negative, in the body HBeAb can appear.Serum detects the HBeAb positive findings and shows that virus replication reduces, and infectiousness weakens.Therefore, the accurate mensuration of HBeAg among the hepatitis B patients serum and HBeAb marker had important directive function to diagnosis, prevention and the treatment of hepatitis B.
HBcAb is an important indicator of judging the hepatitis B state of an illness.Along with the recovery of acute hepatitis b, the HBcAb titre can reduce and even disappear.If this marker continues high titre in serum, then show the tendency of the oriented chronicity development of the hepatitis B state of an illness.In the chronic hepatitis B patient, the higher explanation hepatitis B virus duplication of the recall rate of HBcAb and titre is active, has shown that also viral infectiousness is strong.
In the clinical diagnosis and therapeutic process of hepatitis B, adopt usually several modes that combine in five kinds of markers such as HBsAg, HBsAb, HBeAg, HBeAb and HBcAb are judged the hepatitis B state of an illness.If three kinds of markers of HBsAg, HBeAg and HBcAb detect when all positive among the patients serum, just can determine that this patient just is being subjected to hepatitis B virus infection, and virus enlivening and duplicating, viral load is more, and infectiousness is also stronger.If when three kinds of markers detections of HBsAg, HBeAb and HBcAb were all positive among the patients serum, sb.'s illness took a favorable turn to show hepatitis B, virus replication quantity reduces.
(Enzyme-linked immunosorbent assay ELISA) is the common method that is used for HBsAg, HBsAb, HBeAg, HBeAb and five kinds of marker vitro detection of HBcAb at present in enzyme linked immunological absorption.Wherein, HBsAg, HBsAb and HBeAg adopt ELISA double antibody (two antigen) sandwich method, as: Chinese invention patent application 200610030783.3 disclosed vitro detection to hepatitis B virus core antigen, HBeAb and HBcAb all adopt the ELISA competition law, promptly in the process of antibodies antigen, the antibody in the testing sample is competed mutually with labelled antibody.When using " sandwich method " to detect, the solution colour developing shows that then this marker testing result is positive (+), and solution is colourless to show testing result be negative (-).This judgment mode is consistent with people's custom.And in " competition law ", it is the competition to conjugated antigen of antibody in the testing sample and labelled antibody, when the concentration of labelled antibody during greater than the antibody in the testing sample, solution color depth, shows marker be negative (-); When the concentration greater than flag antibody of the antibody in the testing sample, solution is colourless or look shallow, shows testing result positive (+).Be applied to enzyme labeling bond that ELISA detects because temperature, humidity or chemical oxidation etc. are former thereby when making proteinase generation sex change, inactivation or degraded, just can't realize that enzymatic reaction adds lustre to substrate, this is for the HBeAb and HBcAb that adopt the ELISA competition law to detect, and it will directly cause the testing result of false positive (+).This has hindered the doctor to adopt correct method or medicine to treat to the patient, makes the medical resource can't high-efficient disposition.In addition, also can produce considerable influence, also can produce all influences its daily life to experimenter's psychological condition.In addition, ELISA competition law detection sensitivity in detecting HBeAb and HBcAb marker is lower, therefore, can not accurately detect the content of HBeAb and HBcAb marker in the sample to be measured.
On the one hand, the ELISA competition law exists problems and defective in detecting HBeAb and HBcAb marker.On the other hand, also because the molecular weight ratio less (less than 20kDa) of HBeAg and these two kinds of antigens of HBcAg, when being applied to ELISA, be difficult to effectively wrap by in the ELISA Plate surface, and the accurate detection of corresponding antibodies is exerted an influence by the mode of physisorption.These problems mainly can solve by two kinds of methods at present, as: company of Abbott Laboratories (Abbott) has released a series of products that chemiluminescence are applied to immune detection---ARCHITECT.In the testing process, sample and the antigen coated paramagnetic particle of HBc/HBe with dilution mixes earlier, and the anti-HBe of anti-HBc/ in the sample is combined in the HBc/HBe of microballoon antigen with bag.After the cleaning, add anti-human IgG antibody's bond to the more stable acridine of hydrolysis (acridinium) (US5,468,646) mark.Afterwards, clean once more, in reaction vessel, add pre-(Pre-Trigger) and triggering (Trigger) solution of triggering.Detect at last the relative light unit that chemiluminescence reaction produces (relative light units, RLUs).The selectivity of this detection can reach 99.37%-99.73%.Chinese invention patent application 200510033411.1 and 200610107394.6 also discloses the method for several chemoluminescence method detection by quantitative hepatitis B antibody, and relatively the back is found, its principle separately is corresponding to.Because these class methods need reconfigure instrumentation, not only will cause vacantly to existing instrument, this instrument can not be applicable to other detection.Therefore, all there are some defectives in all many-sides such as its substitutability, versatility and popularization.
CORNING company provide at present a kind of detection process for treating surface (
AssaySurface), common polystyrene ELISA Plate surface is handled, makes its surface produce functional group, as carboxyl, sulfydryl or amino etc., and then with specific molecule by mode envelope antigens such as covalent bond, hydrophobic interaction or hydrogen bonds.In addition, the ImmobilizerTM ELISA Plate that the Denmark ExiqonA/S company that also has NUNC company to sell permits, on it Covalent Immobilization have glutathione (glutathione, GST), Streptavidin (Streptavidin) or huge legendary turtle close nickel (Nickel-Chelate) etc.These technology only can be satisfied with the needs of scientific research at present, also can't be satisfied with the needs of vitro detection in enormous quantities in medical treatment and the clinical observation.
Summary of the invention
One object of the present invention is to provide a kind of composition of vitro detection antibody, and said composition need not detecting medium in advance, as: 96 hole polystyrene check-out consoles, pan coating antigen or antibody.Avoided producing the back kit has been detected the influence that produces, more helped large-scale application in daily detection.
Another object of the present invention is to provide a kind of composition of vitro detection hepatitis B C antibody, according to ELISA " sandwich method " principle, when HBcAb detects the solution colour developing, then show HBcAb be positive (+), then negative otherwise (-), make testing result consistent, be more convenient for operation and judgement with people's understanding custom.
Another purpose of the present invention is to provide a kind of vitro detection antibody, as: the quantitative detecting method of hepatitis B C antibody, by bioassay standard solution gained curve, to specific antibodies in the serum, as: HBcAb, realize detection by quantitative.
The composition of vitro detection antibody of the present invention comprises the labelled antibody of antigen, antibody binding proteins and label mark with two above antibody identification meter positions.
Antigen, or claim target antigen, have antibody identification meter position identical or different more than two (Epitope).This identification epi-position can be albumen or the polypeptide that is made of antibody identification meter position inequality; Also can be two albumen or polypeptide that above same antibody identification epi-position constitutes; Can also add more than one another kind of or several antibody identification meter positions in the antigen of the above same antibody identification epi-position by two forms.For the third mode, those skilled in the art can select the array mode of antigen, as: but be not limited only to the terminal increase of the N end of the antigen of forming two above same antibody Identification Lists positions or C another kind of antibody identification meter position more than; Or between described two above same antibody identification epi-positions, increase another kind of antibody identification meter position more than; Or a kind of antibody identification meter position and another kind of antibody identification meter position apart from one another by.
These antigens all can be applicable to the composition of vitro detection antibody of the present invention, promptly have two above antigen recognizing epi-positions, can discern simultaneously or in conjunction with the antigen of two above antibody.The antibody that combines with these antigens can be identical also can be inequality, as: but be not limited only to, in testing process, antibody is respectively the antibody to be measured that comes from the serum usually and is combined with the labelled antibody of label.
Albumen or the polypeptide of forming antigen can be that self exists in the biosome, also can be synthetic by manual type, and can also be the polypeptide that the antigen in the biosome is obtained through hydrolysis.Genetic engineering is recombinant expressed to be a kind of selectable, obtains the mode of described target antigen by synthetic.The dna sequence dna that is about to similar and different antibody identification meter position is connected in (as: expression plasmid) on the expression vector, after transduceing or being transformed into genetic engineering bacterium, is expressed by modes such as fermentation or cellular incubation, at last through separating and purification step obtains again.Form the gene dosage difference of target antigen, the expression vector that is suitable for also has difference.Different expression vectors also is applicable to different bacterial classifications or bacterial strain.Commonly used gene engineering expression system is as Escherichia coli (Escherichia coli), saccharomycete (Saccharomycescerevisiae) and zooblast etc.Those of ordinary skills just can realize preparation to the target recombinant antigen according to textbook, laboratory manual, Hardware Description Manual (as: GE Healthcare is the purifying Guide Book or the CD of AKTA series purification system configuration), reagent handbook and corresponding carrier design software (as: DNAMAN and Vector NTISuite) and operation instruction thereof.Conventionally known to one of skill in the art, the whole bag of tricks that the technician who possesses general biology general knowledge puts down in writing according to " " molecular cloning experiment guide " (third edition); Science Press; 2002 " book just can be realized the molecular biology operations and the corresponding separation and purification in genetic engineering field.
Antigen of the present invention can also adopt the synthetic or connection of chemical mode (as: organic synthesis).The chemosynthesis of antigen can realize with reference to the synthetic conventional method of polypeptide.It is similar and different antibody identification meter position is linked together and to obtain that the chemistry of antigen connects.Because the molecular weight of antibody identification meter position is less, two identical or different antibody identification meter positions are connected in the resulting antigen of high molecular polymer by chemical mode, the interference that it can avoid the conception of intermolecular space more helps combining of specific antibody and antibody identification meter position.
Antibody binding proteins is the several amino acids that can covalently or non-covalently combine with antibody, as: but be not limited only to, tryptophane, methionine, threonine, valine, lysine, histidine, leucine, isoleucine, alanine, phenylalanine, halfcystine, arginine, glycocoll, serine, tyrosine, glutamic acid, sky (door) winter propylhomoserin, proline, glutamine and asparagine, condensate.These in conjunction with albumen can with some zone of antibody, as Fc fragment and light chain of antibody, non-covalent combination.These non-covalent bonds, as: hydrogen bond, ionic link, hydrophobic interaction and Van der Waals force.
According to the type of binding label on the antibody, need add substrate once more to finish detection.Detection should be understood to separately or combination utilization visual inspection and instrument identification colors, the absorbance value under specific wavelength or chemiluminescent method.As: when the label that is incorporated into antibody is horseradish peroxidase or alkaline phosphatase, make the solution colour developing earlier after need adding the chromogenic reaction thing, distinguish between colors by naked eyes again or the mode that absorbs light value by instrument detecting under specific wavelength determines whether there is antibody to be measured in the sample; When the label that is incorporated into antibody was beta galactosidase, the reaction substrate of adding sent fluorescence earlier, determined whether there is antibody to be measured in the sample by the mode of Instrument measuring again; When the label that is incorporated into antibody is chemiluminescent substance, directly the label of combination on the labelled antibody is measured by instrument, just can determine whether there is antibody to be measured in the sample.
The composition that is used for vitro detection antibody, antibody binding proteins bag be by in the check-out console surface, antibody binding proteins can with antibodies to be measured, antigen combines with antibody to be measured and the labelled antibody that is combined with label simultaneously.According to the kind of label, finish vitro detection behind the substrate of adding necessity to antibody to be measured.
Antibody binding proteins can covalently or non-covalently combine with antibody, as: but be not limited only to anti-human IgG antibody; Staphylococcal protein A (Protein A), it comes from staphylococcic cell membrane, and molecular weight 42kD can combine with the Fc of antibody is non-covalent; Peptostreptococcus magnus albumen L (Protein L), it can combine with light chain of antibody is non-covalent.In addition, can also use streptococcus protein G (Protein G), as: the G148 Protein G of 65kD and the C40 Protein G of 58kD, or the fusion A/G of staphylococcal protein A and streptococcus protein G (Protein A/G).Antibody binding proteins is selected from one or more of these albumen.
The antibody binding proteins bag is 0.1ng/cm by the amount in the check-out console surface
2-9,000ng/cm
2, be generally 1ng/cm
2-5,000ng/cm
2, further select 1ng/cm
2-1,000ng/cm
2, preferentially select 100ng/cm
2-500ng/cm
2Antibody binding proteins can use the quantitative detecting method of albumen to determine in the check-out console package amount, as: direct ultraviolet absorption process, Bradford method, Lorry method and BCA method etc. are determined.The preferential BCA method of using is determined the package amount of antibody binding proteins on the check-out console surface among the present invention.
Check-out console is a solid-phase media, is selected from plastics or glass.
Plastics should be understood to all or partly by carbon and oxygen, hydrogen, organic and the inorganic elements chemical combination of nitrogen and other forms, become solid in the final stage of making, some stage is that (plastic material is becoming before the final products liquid in the mill, must want and to flow in some stage), thereby can heat or plus-pressure, or the mode of the two and usefulness, make it form different shape, any in this huge and protean material same clan, as resin, thermoset resin, cellulose derivatives etc. exist numerous repetition atom or molecule in the molecular structure of a long-chain.Plastics comprise synthetic or nature organic material.Make the used plastics of mounting medium or use a kind of plastic material, or use several plastic materials physically stack or addition after compound, its concrete form is a plastic plate.
Glass is appreciated that frangible amorphous material, these materials can be transparent also can be translucent, merge by molten silicon and silicon-carbon hydrochlorate usually and form.Glass can also think that a class does not have crystallization process, but is solidified and next material by molten state, substantially by Na
2O, CaO and 6SiO
2Chemical oxide is formed, and has optical properties and various mechanical attributes.
Specifically, described check-out console is made by polystyrene, tygon, Polyvinylchloride, polycarbonate or glass etc., has vesicular structure, as: 96 holes or 48 hole ELISA Plate.
Antigen should have antibody identification meter position identical or different more than two, and can select the genetic recombination mode to realize.For the antigen after albumen with identical or different antibody identification meter position or the polypeptide fusion, it makes up principle and method is that those of ordinary skills institute is known.In order not influence the function separately for the treatment of the fusogenic peptide section, generally need between different peptide sections, add a joint.Document Protein Eng., 2001,14, provide and describe in detail the multiple joint that is used for polypeptide or albumen fusion among the 529-532, these contents can be used as those of ordinary skills and select suitable joint that some references are provided.
Generally speaking, the selected polypeptide of recombinant antigen or the protein that are used for antigen recognizing epi-position identical or different more than two is connected and form, its sequence length is generally 1-100 amino acid, usually selecting length is 4-30 amino acid whose polypeptide, and preferentially selecting length is 4-15 amino acid whose polypeptide.The amino acid of forming these polypeptide and protein refers to not only have the organic molecule of the asymmetric center carbon atom of covalently bound amino but also covalently bound carboxyl.
Chemical mode (as: organic synthesis) synthesizes or connects is the mode that another kind obtains antigen of the present invention.The chemosynthesis of antigen can be with reference to the synthetic conventional method of polypeptide, as, but be not limited only to Fmoc polypeptide synthesis method (J.Org.Chem., 1972,37,3404-3409; Eur.J.Immunol., 1994,24,3188-3193; Huang Weide, old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985).It is similar and different antibody identification meter position is linked together and to obtain that the chemistry of antigen connects.These connected modes, as: but be not limited only to, two identical or different epitopes are directly connected through chemical process; Or be that 4-15 amino acid whose peptide linker links to each other all with two identical or different epitopes with a length by chemical mode; Or two identical or different epitopes are all linked to each other with an organic molecule or organic macromolecule by chemical mode.
In addition, because the antibody identification meter position molecular weight is less usually, two identical or different antibody identification meter positions can also be obtained target antigen by the mode that chemical mode is connected in high molecular polymer.
High molecular polymer (polymer) is meant molecular weight greater than 2, and the compound of 000Da is intermolecularly interconnected via covalent bond by structural units (structural unit) or monomer.Its structure can be linear pattern (linear), ramiform (branch/multi-arm) or branch type (dendrimer).Described polymkeric substance is selected from, but be not limited only to, polyglycol, polyaminoacid, polynucleotide, PLA, polylysine, poly-imines and 10 polysaccharides (polysaccharide) that above monose forms by glycosidic bond, as: but be not limited only to starch and hydrolysate thereof, cellulose, chitin or glucosan.
Branch type polymkeric substance (dendrimer) is generally poly-amino amine (poly (amidoamine), PAMAM), can be starting material preparation (Clin.Chem., 1994,40 by ammonia or ethylenediamine respectively, 1845-1849), also have and use amino acid be polymerized (J.Immuno.Method., 1996,196,17-32), as lysine and arginine.Resulting polymers has reactive group, and as amino, hydroxyl or carboxyl, aggregation number is many more, and its molecular weight is big more, and functional group is also many more, energy in conjunction with the also corresponding increase of molecule.
Polyglycol is selected from the polyglycol that two ends are not all sealed or an end seals, and blocking groups is selected from the C1-C30 alkyl, C1-C30 fatty acid or C3-C30 glycosyl.Polyglycol is selected from molecular weight 2,000-100, and 000Da can select 2,000-50,000Da generally selects 5,000-50,000Da.Further, described blocking groups is selected from the C1-C20 alkyl, selects the C1-C10 alkyl usually, preferentially selects methyl, ethyl or propyl group.
Polyaminoacid comprises the oligothiophene molecule that is polymerized by same amino acid, also comprises the poly molecule that is polymerized by different molecular, and protein or polypeptide are its natural existence forms.These protein comprise natural products and the hydrolysate thereof that separation obtains from biosome, also comprise the product and the hydrolysate thereof of genetic engineering reorganization, also comprise by chemical mode product directly synthetic or that different polypeptide fragments are formed by connecting.The length of polypeptide should 15 more than the amino acid, comprise separation obtains from biosome natural products and hydrolysate thereof, the product and the hydrolysate thereof that also comprise genetic engineering reorganization also comprise by chemical mode product directly synthetic or that different polypeptide fragments are formed by connecting.
Various identical or different antibody identification meter positions and high molecular polymer covalent bond (covalentconjugate), the covalent bond of formation as: but be not limited only to amido link (amide bond), urine key (urine bond), ester bond or disulfide bond.Antibody identification meter position combines with high molecular polymer by its amino acid side chain, N terminal amino group or C terminal carboxyl group.This combination can by as: but be not limited only to, reagent such as N-hydroxy-succinamide, maleic anhydride or third/aldehyde-base activate the back with amino acid side chain, N terminal amino group or C terminal carboxyl group and combine with high molecular polymer, also can be suitable for aforementioned identical or different mode, behind the group of activation high molecular polymer, combine with target antigen (as: hepatitis B C antigen) more earlier.Other also have by hydrazone (hydrazone), oxime (oxime), sulfydryl or thiazolidine modes such as (thiazolidine) in conjunction with (J.Immuno.Method, 1996,196,17-32).
Various identical or different antibody identification meter positions and the combination between the carbohydrate molecule be referring to J.Pharma.Sci.87,326-332; Adv.Drug deliv.Rev., 6,103-131; Adv.Drugdeliv.Rev., 13,251-267.The combination of polyglycol and various epitopes is referring to Adv.Drug deliv.Rev., 28,275-299; Adv.Drug deliv.Rev., 54,453-609; Adv.Drug deliv.Rev., 60,1-88.Because epitope combines related chemical reaction with high molecular polymer same or similar, those of ordinary skills can be applied to it in the middle of various antibody identification meter positions and the reaction that other polymkeric substance combines equally to obtain target antigen under the situation with reference to these combinations.
Various identical or different antibody identification meter positions can also be connected with high molecular polymer with chemical ways of connecting indirectly by connexon (space linker).This connexon is selected from the polypeptide of amino acid length 1-500 or protein, molecular weight at 100-2, the polymkeric substance of 000Da or organic molecule.As: but be not limited only to, molecular weight is 200-2, the two ends activated polyglycol (NOFCorporation) of 000Da, NH
2(CH
2)
nCOOH, SH (CH
2)
nThe fatty acid of the C1-C30 of COOH or two ends activation (J.Gene.Med., 2005,7,604-612).The chemistry connection can be that antibody identification meter position N end or C activated group terminal and on the above-mentioned connexon is connected, and also can be connected with activated group on the connexon by the some amino acid side chains of antibody identification meter position.Form covalent bond between connexon and the antibody identification meter position, as: but be not limited only to, amido link, urine key, ester bond,, (CH=N-NH-), the oxime key (CH=N-O-) and disulfide bond for thioether bond, hydrazone key.Formed covalent bond is selected from one or more of these chemical bonds.
The purpose of usage flag thing of the present invention is, when the antibody that is combined with label with in conjunction with serum in after the antigen of antibody combines, this mark directly can be detected by instrument, or detect solution absorbance, thereby whether exist antibody to be measured to judge serum by chromogenic reaction.These labels are selected from, but are not limited only to, fluorescent marker (as: fluorescein isothiocyanate (fitc)), isotope labeling, enzyme labeling thing (as: alkaline phosphatase or horseradish peroxidase) and chemiluminescent labels (United States Patent (USP) 5,468,646) etc.With enzyme labeling for being example, when use horseradish peroxidase (horse radishperoxidase, HRP is EC.1.11.1.7) during labelled antibody, used chromogenic reaction thing can be for 3,3 ', 5,5 '-tetramethyl benzidine (3,3 ', 5,5 '-Tetramethylbenzidine, TMB).
In testing process, the difference of label content just makes detected value produce height difference, cooperates a typical curve again, just can realize that antibody to be measured carries out detection by quantitative in the blood serum sample to unknown concentration.
In order to improve the detection effect, can add the IgG albumen of serum or purifying simultaneously at adding antigen and the antibody that is combined with label.At cumulative volume is in the reaction system of 100 μ L, the addition of serum 〉=1 μ L; Or the IgG albumen addition of purifying 〉=50 μ g.
In the vitro detection for people's specific antibody (as: Anti-HBc), preferred, the human IgG albumen that uses healthy human serum (containing human IgG) or purifying to obtain comes blocking antibody in conjunction with the free site of albumen, occurs to prevent false positive results.
Those skilled in the art can directly buy or entrust and customize the human IgG protein reagent of purifying, also can be by the mode purifying human IgG albumen from healthy human serum in textbook or the laboratory manual, as: adopt commercial affinity chromatography reagent, commercial IgG in conjunction with protein reagent (
Thermo Scientific) human IgG is carried out purifying; Can also use ammonium sulfate precipitation and DEAE-cellulose chromatography method to the human IgG purifying, or human IgG directly be extracted by DEAE-cellulose or DEAE-SephadexA50 resin.In addition, can also pass through the genetic engineering mode, obtain containing the fermentation liquor of the human IgG albumen (as: monoclonal IgG albumen) of reorganization, obtain human IgG albumen by subsequent purification again by zooblast and expression vector.The detection method of purifying afterproduct purity, as: but be not limited only to agar two-phase double-diffusion process, immunoelectrophoresis or disc electrophoresis.When testing result is Yi Tiao district band, think to obtain the pure product of human IgG albumen.
A kind of composition of vitro detection hepatitis B C antibody of the present invention, mainly comprise antibody binding proteins bag quilt check-out console, have the hepatitis B C antigen of two above antibody identification meter positions, healthy human serum or purifying human IgG albumen, be combined with the hepatitis B C antibody and the chromogenic substrate of label.
The composition of another kind of vitro detection hepatitis B C antibody of the present invention mainly comprises the antibody and the tmb substrate of the hepatitis B C antigen of the check-out console of antibody binding proteins bag quilt, the human IgG albumen with the hepatitis B C antigen of two above antibody identification meter positions, healthy human serum or purifying, horseradish peroxidase-labeled.
Antibody binding proteins is selected from one or more of anti-human IgG antibody, Protein A, Protein L, Protein G and Protein A/G, preferentially selects Protein A.The antibody binding proteins bag is 1ng/cm by the amount in check-out console
2-5,000ng/cm
2, be generally 1ng/cm
2-1,000ng/cm
2, further select 1ng/cm
2-500ng/cm
2, preferentially select 100ng/cm
2-200ng/cm
2
In the system of antibody-antigen to be measured-labelled antibody of cumulative volume 100 μ L (being reaction system), healthy human serum addition 〉=1 μ L is preferentially selected 5 μ L.
In the system of antibody-antigen to be measured-labelled antibody of cumulative volume 100 μ L (being reaction system), the human IgG albumen addition of purifying 〉=50 μ g is preferentially selected 100 μ g.
In the detection, need elder generation with antibody binding proteins, as: staphylococcal protein A, the bag quilt adds dilution or undiluted test serum sample in the ELISA Plate surface, and the antibody binding proteins on the ELISA Plate is combined with antibody in the sample; After the cleaning, add HBcAg, the anti-HBcAg antibody (preferentially selecting monoclonal antibody) of HRP mark and the human IgG albumen of healthy human serum or purifying, the hepatitis B C antibody in the sample and the anti-HBcAg antibody of HRP mark are all combined with HBcAg.After cleaning once more, add chromogenic substrate TMB and reaction terminating liquid, under the 450nm wavelength, detect at last.Along with the amount difference of contained HBcAb in the sample, its colour developing back solution absorbance also changes, and just can obtain a typical curve by several check points like this, quantitative in order to unknown content HBcAb in the serum.
The external detection method of another kind of hepatitis B C antibody of the present invention comprises the steps:
With the staphylococcal protein A bag by in the porous ELISA Plate, 4 ℃ are spent the night;
2. remove coating buffer, add 200 μ l/ hole confining liquids (as: pH7.4, the PBS solution of 1% (w/v) BSA), 4 ℃ spend the night or 37 ℃ hatched 1-2 hour;
3. remove confining liquid, the cleaning of enzyme target adds the HBcAb standard items of known variable concentrations and dilution or undiluted test serum sample respectively in each hole of ELISA Plate, and hatched 1 hour for 37 ℃ in 100 μ l/ holes;
4. the cleaning of enzyme target adds HBcAg, the anti-HBcAg monoclonal antibody of HRP mark and the human IgG albumen of healthy human serum or purifying, mixing, hatched 30 minutes for 37 ℃, the cleaning of enzyme target adds chromogenic substrate TMB once more, hatched 15-30 minute for 37 ℃, add stop buffer;
5. under the 450nm wavelength, detect solution absorbency value in each hole.
By setting up the coordinate relation of variable concentrations HBcAb and absorbance, obtain a typical curve and formula thereof, just can obtain corresponding HBcAb content in the blood serum sample absorbance substitution formula with unknown concentration then.Draw actual quantitative result in conjunction with the diluted sample degree at last.
Hepatitis B C antigen of the present invention comprises the sequence shown in the SEQ ID No:1, also comprises the recombinant C antigen that obtains after the polypeptide of antibodies epi-position is recombinated more than 2 kinds with 70% above homology that obtains from its antigen sequence.
Hepatitis B C antigen of the present invention can also use the protein epitope forecasting software, as: BIOSUN (calculation biology center, Institute of Basic Medical Sciences, Beijing), choose have one or more epi-positions in the antigen, the corresponding peptides section of advantage epi-position (can excite stronger immunoreactive epi-position) especially, the molecular biology method by routine angles to be got its coded sequence and gives expression to this antigen polypeptide; Or the C epitope is screened (J.Immunol., 1988,141,4376-4380 according to the function section; Immuno.Lett., 1991,30,59-68; J.Gen.Virol., 1993,74,1335-1340).From the specific antigen epi-position, select suitable antibody, can adopt the display technique of bacteriophage in immune antiboidy storehouse to screen (J.Immunol Method, 2008,329,176-183).
Hepatitis B C antigen of the present invention can be for as sequence shown in the SEQ ID No:1.Also can select plural antibody identification meter position, use gene engineering method to recombinate external from SEQ ID No:1, as: with Escherichia coli (Virus Res, 1989,14,27-48), yeast (J Biotechnol., 1993,29,243-255; United States Patent (USP) 6,277,631), red complete yeast (J Biotechnol., 2008,138,1-8), mammalian cell (Antiviral Res, 2006,72,116-124) or insect cell (Biotechnology, 1995,13,261-264) be recombinant expression system, make after recombinating the recombinant hepatitis B virus C antigen (rHBcAg) of expressing have antibodies (identification) epi-position more than 2.
Hepatitis B C antigen of the present invention is as sequence shown in the SEQ ID No:1, or from the plural antibodies epi-position of SEQID No:1 selection, by the antigen that obtains after the chemical mode connection, or select plural antibodies epi-position from SEQ ID No:1, by chemical mode and the antigen that obtains after high molecular polymer is connected.
The beneficial effect that technical solution of the present invention realizes
The composition of vitro detection antibody of the present invention uses the ELISA Plate of the antibody binding proteins of specific package amount, makes the Fc fragment of antibody combine with antibody binding proteins, effectively improves and detects effect.
When said composition is used for hepatitis B C antibody vitro detection, use healthy human serum or purifying obtains from serum human IgG albumen simultaneously at the antibody that adds antigen and be combined with label, strengthened the detection effect.
Term involved in the present invention is identical with its general notion.
Described " antibody identification meter position is connected in high molecular polymer by chemical mode " refers to that antigen is direct or passes through the indirect and high molecular polymer covalent bond of connexon.
Described " hepatitis B C antigen and high molecular polymer covalent bond " refers to that hepatitis B C antigen is direct or passes through the indirect and high molecular polymer covalent bond of connexon.
Described " C1-C10 alkyl ", " C1-C20 alkyl " and " C1-C30 alkyl " refer to the straight or branched alkyl, as: methyl, ethyl, propyl group, isopropyl, butyl or isobutyl etc.Wherein, letter C is represented carbon atom, and numeral is a positive integer thereafter, as: 1,2,3,4 or 5 etc., the contained carbon atom number of expression group.
Described " fatty acid of C1-C30 " refers to saturated or undersaturated straight or branched carbochain, has a carboxyl on it at least, as: formic acid, acetate, propionic acid, oleic acid, linoleic acid, palmitic acid, parinaric acid, succinic acid and malic acid etc.Wherein, letter C is represented carbon atom, and numeral is a positive integer thereafter, as: 1,2,3,4 or 5 etc., the contained carbon atom number of expression group.
Described " glycosyl of C3-C30 ", as: triose base, tetrose base, pentose base and hexose-based etc.Wherein, letter C is represented carbon atom, and numeral is a positive integer thereafter, as: 3,4 or 5 etc., the contained carbon atom number of expression group.
Described " serum " refers to that it is the blood of the individuality of health after diagnosing that serum derives from, and the antibody that does not contain antibody to be measured and detection is produced cross reaction (false positive).
Healthy individuality is that clinical diagnosis is that healthy people, animal doctor is diagnosed as healthy wild animal and domestic animal (Livestock).Wild animal is without domestication's animal under the state of nature.Domestic animal is the animal of the artificial feeding for food source is provided, as: dog, mouse, hamster, pig, rabbit, milk cow, buffalo, bull, sheep, goat, goose and chicken etc.
Described " healthy human serum " refers to that serum derives from the blood that clinical diagnosis is a healthy human body, and the antibody that does not contain antibody to be measured and detection is produced cross reaction (false positive).Hbv antibody with the vitro detection patient is an example, and healthy human serum should be understood to hepatitis B surface antigen (HBsAg), HCV antigen/antibody combination and anti-HIV antibody test and is sera negative.Detect hepatitis B C antibody, should not contain the serum of antibody to be measured simultaneously.As: when composition is that healthy human serum is the blood that comes from non-hepatitis and AIDS patient when detecting hepatitis B C antibody.
Embodiment
Below describe technical scheme of the present invention in detail.The embodiment of the invention is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
The used reagent of the present invention is not if clearly indicate, then all available from Sigma-aldrich (Sigma-Aldrich).
Embodiment 1Protein A bag quilt is in ELISA Plate
Select undressed polystyrene ELISA Plate, add 1.2 μ g/ml Protein A and be dissolved in carbonate buffer solution, pH9.2-9.8,100 μ l/ holes, 4 ℃ are spent the night.Remove coating buffer, every hole add 1% (w/v) BSA solution (PBS, pH7.4) 200 μ l seal, 4 ℃ spend the night (12-18 hour) or 37 ℃ 1-2 hour, gained ELISA Plate pan coating has 100-200ng/cm
2Protein A.
Embodiment 2 blood serum sample detection methods
With PBST (the PBS damping fluid that contains 0.05%Tween-20) cleaning of enzyme target 1-2 time, pat dry at every turn.
After every hole added 90 μ l PBS, every hole added test serum 10 μ l, hatches 60 minutes in 37 ℃ behind the mixing.
After removing reactant liquor and patting dry, wash plate 4 times, all pat dry at every turn with PBST.
Every hole adds 40 μ l reorganization HBcAg (SEQ ID NO:1, provide by ShangHai RongSheng Biology Pharmacy Co., Ltd) (with PBS dilution 1: 2000), 40 μ l through the anti-HBcAg antibody (with PBS dilution 1: 20000) of HRP mark and 20 μ l contain the unsoundness human serum (25%, v/v).
Every hole adds substrate A and each 50 μ l of B liquid respectively, and mixing was hatched 15 minutes for 37 ℃, added stop buffer then, and the 450nm microplate reader is measured.
The preparation of substrate A liquid:
Take by weighing citric acid 17.9g and Na
2HPO
412
2O 4.67g is dissolved in the 400ml deionized water, slowly adds 30% (w/w) H then
2O
2330 μ L after stirring, add water to 500ml.Be distributed into the 10ml/ bottle, preserve down for 2～8 ℃.
The preparation of substrate B liquid:
Take by weighing TMB 0.1g in the 100ml deionized water, add DMSO 2.5ml, when stirring, slowly add 0.5ml 6M HCl,, add water to 500ml at last until dissolving fully.Be distributed into the 10ml brown vial, preserve down for 2～8 ℃.
The preparation of substrate reactions stop buffer:
Measure 98%H respectively
2SO
455ml and deionized water 445ml slowly join the concentrated sulphuric acid in the deionized water then, and mixing is distributed into the 10ml/ bottle, preserve down for 2～8 ℃.
The known HBcAb standard items of 5 kinds of variable concentrations of adding and the test serum sample of dilution add respectively in each hole of ELISA Plate in ELISA Plate, use identical detection method just can realize the detection by quantitative of HBcAb.
Embodiment 3HBcAb detects parallel control
HBcAb competitive ELISA detection kit (ShangHai RongSheng Biology Pharmacy Co., Ltd) with present use contrasts.
Have 350 routine samples and be used for this experiment, wherein,
Great three positive (HBsAg, HBeAg and the HBcAb positive): 110 examples;
"small three positive" (HBsAg, HBeAb and the HBcAb positive): 100 examples;
Healthy human serum (from blood bank): 140 examples.
Testing result sees Table 1.
Table 1HBcAb detects parallel control
Can get from table 1:
Positive concordance rate: 203/203=100%;
Negative concordance rate: 140/147=95.2%;
Total concordance rate: 343/350=98%.
Embodiment 4HBcAb sensitivity detects
Select 6 examples at random from the little positive sample that is used for embodiment 3 detections, (0.1M, pH7.4) serial dilution is respectively: 1/16,1/32,1/64,1/128,1/256,1/512 and 1/1024 with phosphate buffer.Do (feminine gender) contrast simultaneously, promptly do not add dilute serum, replace with PBS, other step is identical, detects the OD value and the results are shown in Table 2.
Table 2HBcAb sensitivity testing result
Annotate: the method detection threshold (Cut-Off value): X+2SD, that is, Cut-Off=0.025OD450 〉=0.025 is positive, and OD450＜0.025 is negative.
From the result as seen, competition inhibition method ELISA sensitivity is no more than dilutability 1/512, detects HBcAb and use detection reaction method of the present invention, and its dilutability can reach 1/1024 at least, and sensitivity is significantly improved.
Embodiment 5HBcAb selectivity detects
Collection clinical samples totally 90 examples (comprises Anti-HAV, Anti-HCV and Anti-HIV (1+2) positive, all detect through ShangHai RongSheng Biology Pharmacy Co., Ltd's corresponding reagent box) and healthy people's sample 100 examples, adopting said method (forward reaction) detects HbeAb, and the result is summarized as follows table 3.
Table 3HBcAb selectivity detects
Detect selectivity=true negative/(true negative+false positive)=188/190=99%
Embodiment 6HBcAb detection by quantitative
1. proofread and correct the preparation of product
This kit adopts the correction product of 5 different relative concentrations, and concentration unit is relative unit (RU/ml), and its establishing method is as follows:
OD
450=1.9～2.0 o'clock, the setting concentration unit was 100RU/ml, as the upper limit;
OD
450=0.9～1.0 o'clock, the setting concentration unit was 50RU/ml;
OD
450=0.4～0.5 o'clock, the setting concentration unit was 25RU/ml;
OD
450=0.2～0.3 o'clock, the setting concentration unit was 12RU/ml;
OD
450=0.01～0.2 o'clock, the setting concentration unit was 0RU/ml, as lower limit.
The drawing standard curve is set 5 measuring points, and (0,12,25,50 and 100RU/ml) are proofreaied and correct product 1～No. 5 in preparation respectively.
Proofread and correct the preparation of product No. 5: get the anti-HBcAb positive and OD
450=1.9～2.0 hepatitis B patient blood serum 1ml is with the dilution of 9ml sample diluting liquid, fully mixing; Get 5ml wherein, in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-5 ", preserves down for 2～8 ℃.
Proofread and correct the preparation of product No. 4: get the 5ml of above-mentioned remainder, add sample diluting liquid 5ml, fully mixing is got 5ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-4 ", preserves down for 2～8 ℃.
Proofread and correct the preparation of product No. 3: get the 5ml of above-mentioned remainder, add sample diluting liquid 5ml, fully mixing is got 5ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-3 ", preserves down for 2～8 ℃.
Proofread and correct the preparation of product No. 2: get the 5ml of above-mentioned remainder, add sample diluting liquid 5ml, fully mixing is got 5ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-2 ", preserves down for 2～8 ℃.
Proofread and correct the preparation of product No. 1: the sample thief dilution, in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-1 ", preserves down for 2～8 ℃.
2. the preparation of weak positive reference substance
Get the anti-HBcAb positive and OD
450=0.2～0.3 hepatitis B patient blood serum 1ml, the centrifuging and taking supernatant, with 0.22 μ m biofilm filtration degerming, with the dilution of 9ml sample diluting liquid, abundant mixing, in the packing standard vial, every bottle of 1ml is labeled as " weak positive reference substance ", preserves down for 2～8 ℃.
3. the preparation of negative control product
Get many parts of normal human serums, equal proportion is mixed, and gets 1ml, the centrifuging and taking supernatant, and with 0.22 μ m biofilm filtration degerming, with the dilution of 9ml sample diluting liquid, abundant mixing, in the packing standard vial, every bottle of 1ml is labeled as " negative control product ", preserves down for 2～8 ℃.
4. the result calculates and judges
Qualitative analysis
The result calculates: ratio=sample OD
450/ weak positive control OD
450
The result judges:
Ratio＜0.95 feminine gender
0.95＜ratio≤1.0 critical values, suggestion redeterminates
Ratio＞1.0 positives
Quantitative test
Drawing standard curve: with 5 OD that proofread and correct product
450Value is ordinate, and each self-corresponding concentration is horizontal ordinate, draws out typical curve.
The result calculates: calculate the OD that sample is measured
450Mean value is read respective concentration (RU/ml) then on typical curve.
The result judges:
〉=12RU/ml the positive
＜12RU/ml feminine gender
Sequence table
＜110〉ShangHai RongSheng Biology Pharmacy Co., Ltd
＜120〉composition of vitro detection hepatitis B C antibody
<130>BRS.HF.9008
<160>1
<170>PatentIn?version?3.3
<210>SEQ?ID?No?1
<211>183
<212>PRT
＜213〉hepatitis B C antigen
<400>1
Met?Asp?Ile?Asp?Pro?Tyr?Lys?Glu?Phe?Gly?Ala?Ser?Val?Glu?Leu?Leu?Ser?Phe?Leu?Pro?Ser?Asp
1 5 10 15 20
Phe?Phe?Pro?Ser?Ile?Arg?Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Arg?Glu?Ala?Leu?Glu?Ser?Pro
25 30 35 40 45
Glu?His?Cys?Ser?Pro?His?His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu?Met?Asn
50 55 60 65
Leu?Ala?Thr?Trp?Val?Gly?Ser?Asn?Leu?Glu?Asp?Pro?Ala?Ser?Arg?Glu?Leu?Val?Val?Ser?Tyr?Val?Asn
70 75 80 85 90
Val?Asn?Met?Gly?Leu?Lys?Ile?Arg?Gln?Leu?Leu?Trp?Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Gly?Arg
95 100 105 110
Glu?Thr?Val?Leu?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg?Pro?Pro
115 120 125 130 135
Asn?Ala?Pro?Ile?Leu?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Arg?Gly?Arg?Ser?Pro?Arg
140 145 150 155
Arg?Arg?Thr?Pro?Ser?Pro?Arg?Arg?Arg?Arg?Ser?Gln?Ser?Pro?Arg?Arg?Arg?Arg?Ser?Gln?Ser?Arg?Glu
160 165 170 175 180
Ser?Gln?Cys
Claims (19)
1. the composition of a vitro detection hepatitis B C antibody comprises the labelled antibody of the antigen with two above antibody identification meter positions, the check-out console that is coated with antibody binding proteins, label mark and the IgG albumen of serum or purifying.
2. the composition of vitro detection hepatitis B C antibody according to claim 1, the antibody binding proteins amount that it is characterized in that described check-out console pan coating is 0.1ng/cm
2-9,000ng/cm
2
3. the composition of vitro detection hepatitis B C antibody according to claim 1 is characterized in that described serum use amount 〉=1 μ l/100 μ l reaction system.
4. the composition of vitro detection hepatitis B C antibody according to claim 1 is characterized in that the IgG albumen use amount 〉=50 μ g/100 μ l reaction systems of described purifying.
5. the composition of a vitro detection hepatitis B C antibody, mainly comprise antibody binding proteins bag quilt check-out console, have the hepatitis B C antigen of two above antibody identification meter positions, healthy human serum or purifying human IgG albumen, be combined with the hepatitis B C antibody and the chromogenic substrate of label.
6. the composition of vitro detection hepatitis B C antibody according to claim 5, the antibody binding proteins amount that it is characterized in that the pan coating of described check-out console is 1ng/cm
2-5,000ng/cm
2
7. the composition of vitro detection hepatitis B C antibody according to claim 5, the antibody binding proteins amount that it is characterized in that the pan coating of described check-out console is 1ng/cm
2-500ng/cm
2
8. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described serum use amount 〉=1 μ l/100 μ l reaction system.
9. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that the IgG albumen use amount 〉=50 μ g/100 μ l reaction systems of described purifying.
10. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described antibody binding proteins is selected from one or more of Protein A, Protein G, Protein A/G, Protein L and anti-human IgG antibody.
11. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described antigen is hepatitis B C antigen.
12. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described hepatitis B C antigen is recombinant hepatitis B virus C antigen.
13. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described hepatitis B C antigen amino acid sequence is selected from SEQ ID No:1.
14., it is characterized in that described hepatitis B C antigen is recombinant expressed by red complete yeast according to the composition of the described vitro detection hepatitis B of one of claim 5-13 C antibody.
15. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described hepatitis B C antigen is for to be connected in the resulting antigen of high molecular polymer with two identical or different antibody identification meter positions by chemical mode.
16. the composition of vitro detection hepatitis B C antibody according to claim 15, it is characterized in that covalent bond between described antibody identification meter position and the described high molecular polymer be selected from amido link, urine key, ester bond,, thioether bond, hydrazone key, oxime key and disulfide bond one or more.
17. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described hepatitis B C antigen for selecting plural antibodies epi-position from SEQ ID No:1, the antigen that obtains after connecting by chemical mode.
18. the composition of vitro detection hepatitis B C antibody according to claim 5, it is characterized in that described hepatitis B C antigen for from the plural antibodies epi-position of SEQ ID No:1 selection, by chemical mode and the antigen that obtains after high molecular polymer is connected.
19. the composition of vitro detection hepatitis B C antibody according to claim 5 is characterized in that described label is selected from fluorescent marker, isotope labeling, enzyme labeling thing or chemiluminescent labels.
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| CN108226479A (en) * | 2017-12-21 | 2018-06-29 | 林美琼 | A kind of composition of vitro detection antibody |
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
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Application publication date: 20110518 |