CN103630690B - Hepatitis C virus antigen-antibody combined detection kit and detection method - Google Patents
Hepatitis C virus antigen-antibody combined detection kit and detection method Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/186—Hepatitis C; Hepatitis NANB
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- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
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Abstract
The invention discloses a hepatitis C virus (HCV) antigen-antibody combined detection kit. The kit comprises a microwell plate coated with an HCV chimeric antigen and an HCV monoclonal antibody, a sample diluent, an HCV antigen-antibody combined detection enzyme working fluid, an HCV abzyme working fluid, an HCV antigen enzyme working fluid, a substance A fluid, a substance B fluid, a 20-times concentrated washing fluid and a stopping fluid. The invention also discloses preparation and usage of the key components of the kit, such as the microwell plate of the HCV chimeric antigen and the HCV monoclonal antibody, the sample diluent, and the diluent of enzyme working fluid. The kit and detection method, disclosed by the invention, are able to be used for detecting the HCV antigen and antibody at the same time, or individually detecting the situation of the HCV antigen during the early hepatitis c or the acute infection period, or before the antibody is produced, or when an antigen-antibody compound is produced, or individually detecting the situation of the HCV antibody after the antibody is produced; the kit and detection method can be applied to early HCV detection and therapy evaluation, so as to provide important detection evaluation index for clinical guideline.
Description
Technical field
The invention belongs to technical field of immunoassay, relate in particular to a kind of hepatitis C virus (HCV) antigen-antibody combined detection kit and detection method thereof.
Background technology
Hepatitis C is a kind of global infectious disease being caused by hepatitis C virus (hepatitis C virus, HCV) infection.According to estimates, there are 1.7 hundred million hepatitis c virus infection persons in the whole world at present, and the third liver infection rate of China is approximately 3.2%, has at present 4000-6000 ten thousand third hepatopaths at least.The chronicity incidence of hepatitis C is apparently higher than hepatitis B, and easily there is in early days cirrhosis compared with hepatitis B, cause liver cancer, case fatality rate is higher, and especially the early diagnosis of HCV is of great importance for the infection sources, guiding clinical treatment and the prognosis judgement of examination HCV.Therefore, be necessary to find a kind of easily, accurately, method cheap and that can generally promote realizes the detection to HCV, especially true to developing country.
HCV virus belongs to flaviviridae, and its genome is single-stranded RNA, about 9.4kb, easily variation.HCV genome is comprised of a large open reading frame (ORF), and coding more than 10 is planted structure and non-structure (NS) albumen.3010～3033 amino acid whose polyproteins of encoding, putting in order is 5 ' UTR--C2--E1--E2--NS2--NS3--NS4--NS5--3 ' UTR, wherein C is core protein, E1 and E2 are envelope protein, NS is that non-structural protein .NS5B albumen is rna dependent rna polymerase, for HCV copies requiredly, it is the important target position of antiviral therapy.In open reading frame upstream, having 5 ' UTR region of 341 nucleotide is the most conservative regions in the genotypic genome of all HCV, and different genotype has 90% similarity, has become the research focus of HCV molecular diagnostic techniques.
The main detection method of hepatitis C all has certain limitation at present:
1, antibody to hepatitis C detects: be present most popular detection method, but its shortcoming one is to have " window phase ", after infecting, HCV also had one period of long term of about 40-70 days (average 66 days) before HCV antigen/antibody combination produces, now, blood donor is infected and have an infectivity, applying current antibody test reagent can not detect, this stage is called window phase (the perseroconversion window phase infecting before rear seropositive conversion, PWP) existence of .PWP is one of important threat of transfusion safety, receptor is still had through inputting anti-HCV and screen negative blood and the danger of HCV infection.The blood transfusion postoperative infection of hepatitis C accounts for 70% of hepatitis case at present, and in hepatitis C infection person, more has 80-90% to belong to blood transfusion postoperative infection, and this to a great extent in view of the foregoing.
2, HCV RNA detects: the selection and the curative effect monitoring that are mainly used in antiviral therapy, but it needs strictly to press the operation of PCR working specification, testing staff need and obtain corresponding qualification through professional training, and the quality control requirement of sample is high, censorship under cryogenic conditions in requiring to take a blood sample latter 2 hours, aseptic extracting RNA, easily because instrument, reagent, personnel and environment etc. cause error, produces false positive and false negative.
3, hepatitis C antigen detects: in the 1-2d after HCV RNA occurs, occur hepatitis C virus HCV antigen, and parallel with the level of HCV RNA, can be used as the sign that HCV copies.There are some researches show, HCV antigen detects and compares with antibody test, and the detection of HCV antigen can make the window phase detecting on average shift to an earlier date 49 days, shortens the risk that window phase HCV infects.
The blood source examination that the antibody combined detection kit of hepatitis C antigen can be used for HCV detects hepatitis C antigen and antibody simultaneously, both can improve with blood and transfusion safety, has also improved work efficiency and cost-saving simultaneously; In addition this kit and detection method thereof can be distinguished the existing disease infection of HCV or previous infection, and the early detection and the treatment that can be used for HCV are evaluated, for the clinical important detection evaluation index that provides is provided.
At present as follows for the Patents of the antibody combined detection of hepatitis C antigen:
(1) hepatitis C virus antigen-antibody time-resolved fluoroimmunoassay joint-detection and detection kit (application number: 201210385472.4).
(2) the direct labelled antigen of enzyme immunoassay (EIA) detects hepatitis C virus antigen-antibody and detection kit (application number: 201210391183.5).
(3) the direct labelled antigen of chemoluminescence method detects hepatitis C virus antigen-antibody and detection kit (application number: 201210376965.1).
(4) a kind of method of hepatitis C virus antigen-antibody combined detection (application number: 200810143274.0).
（5）Methods?for?the?simultaneous?detection?of?HCV?antigens?and?HCVantibodies(PCT/US02/19958).
Above-mentioned patent has all proposed the method for the antibody combined detection of hepatitis C antigen, and all proposes to adopt mutual nonreactive HCV monoclonal antibody and HCV recombinant antigen as coated raw material, can realize the object of the antibody combined detection of hepatitis C antigen.Wherein application number is that 201210385472.4,201210391183.5 and 201210376965.1 patent has proposed respectively to adopt time-resolved fluoroimmunoassay, enzyme immunoassay (EIA) and chemoluminescence method to detect, but the concrete grammar how to implement is not introduced in detail.Application number has been 200810143274.0 patent Introduction how for the preparation of HCV monoclonal antibody and HCV chimeric antigen and detecting step coated and that detect; and 8 primers have been mentioned; and HCV monoclonal antibody and the HCV chimeric antigen of these 8 primers and preparation are thus protected, but also in detail introduction how to prepare the concrete grammar of kit.
The introduction that United States Patent (USP) PCT/US02/19958 is detailed for the preparation of HCV monoclonal antibody and HCV chimeric antigen coated and that detect, and 37 different primers have been designed, prepare a plurality of HCV monoclonal antibodies and HCV chimeric antigen, be combined into antigen and antibody combination that dozens of can be used for joint-detection, and mentioned a kind of hepatitis C antigen and detected sample diluting liquid, describe its compound method and effect in detail.
But on the basis of above-mentioned patent, still have following problem unresolved:
1, coated problem: even do not react between corresponding antigen and antibody being coated on microwell plate mutually simultaneously, just because an albumen can cover another albumen or preponderate, and another kind of capture ability is wherein weakened greatly, the target that hepatitis C antigen antibody is detected is simultaneously affected greatly.Again because albumen is coated on microwell plate main by hydrophobic bond, ionic link etc.Envelope antigen and antibody, because the two isoelectric point differs far, are difficult to accomplish the two equal q.s and stable being coated on micropore simultaneously.Although above-mentioned patent all has, introduce coated mode, be all still the coated mode of traditional single species albumen, cannot accomplish that the two takes into account.
United States Patent (USP) PCT/US02/19958 has all introduced its coated mode to the use of its each combination raw materials, but it is coated that the method for its introduction is on microballoon, the mode more different microballoons being used in combination, does not mention and how antigen of the same race and antibody is coated in to the mode on this solid phase carrier of polystyrene micropore plate simultaneously.
2, c-hepatitis antibody content in blood is many, and c-hepatitis antibody detect because of for detection of core space antigen have in very strong body or external homology and allos binding ability, easily cause that background is high until false positive (Mastumoto, M. etc., Virology, 218:P43-51,1996; Kunkel, M. etc., Virology, 294:P239-245,2002; Majeau, N. etc., Journal of General Virology, 85:P971-981,2004).The sample size requirements of using during therefore with detection c-hepatitis antibody is considerably less, and in current common c-hepatitis antibody detection kit, sample size is generally 10ul; And hepatitis C antigen content in blood is considerably less, it is very many to the requirement of sample size detecting sample, and in current common hepatitis C antigen detection kit, sample size is generally 100ul.Therefore when the two detects simultaneously to there is certain contradiction on sample size requirements.
Due to patient infect hepatitis C virus product after about a period of time (average 49 days) can produce antibody, form antigenantibody complex, after producing antigenantibody complex, because free antigen reduces, the recall rate of hepatitis C antigen is reduced greatly.
Although United States Patent (USP) PCT/US02/19958 has introduced a kind of hepatitis C antigen sample diluting liquid, from its patent, can find out that it can make the recall rate of antigen greatly improve.But it contains a large amount of scaling agents and protein denaturant, as a kind of PB that is combined as 0.1M pH7.2 that releases liquid that it exemplifies, 0.01M EDTA, 0.5M NaCL, 2%SB-16,1.10%CTAB, 1.8%Triton-x100,2.5M Urea, 1%BSA, 2%mouse serum. SB-16 wherein, 1.10%CTAB etc. have all reached or have approached the upper solubility limit in solution, may cause and be difficult to dissolve.And it is applicable to hepatitis C antigen and detects, whether be applicable to that c-hepatitis antibody detects and not mentioned.And CORE district antigen has in very strong body or external homology and this binding ability of allos binding ability can use CORE district antigen or CORE chimeric antigen when detecting sample because allos is in conjunction with causing the even false-positive situation of high background.
If 3 to detect sample positive, because confirming the end, be that the hepatitis C antigen positive or c-hepatitis antibody are positive or the two is two positive, and need be equipped with again in addition other independently detection kit detect.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of hepatitis C virus (HCV) antigen-antibody combined detection kit and detection method thereof.
Hepatitis C virus antigen-antibody combined detection kit of the present invention, comprise following component: the microwell plate that is coated with HCV chimeric antigen and HCV monoclonal antibody, sample diluting liquid, enzyme working fluid, it is characterized in that: coated with the coated damping fluid that contains ionic surface active agent and reductive agent when prepared by described microwell plate, wherein ionic surface active agent is selected from dodecyl alanine, dodecyl-dimethyl amine second lactone or cetrimonium bromide (CTAB), reductive agent is selected from halfcystine, dithiothreitol (DTT) (DTT), reduced glutathione or beta-mercaptoethanol, described sample diluting liquid is to contain reductive agent, the solution of mouse IgG and protein deposition agent ammonium sulfate, wherein reductive agent is selected from halfcystine, dithiothreitol (DTT) (DTT), reduced glutathione or beta-mercaptoethanol, described enzyme working fluid is to include HCV antigen-antibody joint-detection enzyme working fluid, HCV antigen detects three independent packagings of enzyme working fluid and HCV antibody test enzyme working fluid.
In above-mentioned hepatitis C virus antigen-antibody combined detection kit: described ionic surface active agent is cetrimonium bromide (CTAB), and working concentration is 0.01wt%～1.3wt%; Described reductive agent is dithiothreitol (DTT) (DTT), and working concentration is 0.01wt%～2wt%.
Further preferred embodiment is: described ionic surface active agent is cetrimonium bromide (CTAB), and working concentration is 0.08wt%～0.5wt%; Described reductive agent is dithiothreitol (DTT) (DTT), and working concentration is 0.05wt%～1.0wt%.
Most preferred embodiment is: described ionic surface active agent is cetrimonium bromide (CTAB), and working concentration is 0.1wt%; Described reductive agent is dithiothreitol (DTT) (DTT), and working concentration is 0.1wt%.
In above-mentioned hepatitis C virus antigen-antibody combined detection kit: the reductive agent in described sample diluting liquid is halfcystine, working concentration is 20mM.
In above-mentioned hepatitis C virus antigen-antibody combined detection kit: the working concentration of described sample diluting liquid small mouse IgG is 1mg/ml.
In above-mentioned hepatitis C virus antigen-antibody combined detection kit: in described sample diluting liquid, the working concentration of protein deposition agent ammonium sulfate is 25wt%.
The most preferred formula of above-mentioned sample diluting liquid is: 5mM EDTA, 1%BSA, 1mg/ml mouse IgG, 1%SB-12,1%CTAB, 1%Triton-X114, the halfcystine of 20mM, 25%(NH
4)
2sO
4, 2.5M Urea, 0.01Proclin300,1 * PBS, PH5.8.
In above-mentioned hepatitis C virus antigen-antibody combined detection kit: the diluted of following formula for described enzyme working fluid: 5% lowlenthal serum, 1% thimerosal, 1%PVP, 1%BSA, 5% glucosan T2000,0.5% casein, 10mg/ml gentamicin, 0.01M pH7.4PBS.
In the application of hepatitis C virus antigen-antibody combined detection kit of the present invention: the HCV antigen-antibody joint-detection enzyme working fluid in described enzyme working fluid, HCV antigen detect enzyme working fluid and three independent packagings of HCV antibody test enzyme working fluid, under joint-detection HCV antigen and antibody, c-hepatitis antibody and three kinds of different situations of hepatitis C antigen detection, use respectively.
The invention provides hepatitis C virus antigen-antibody combined detection kit prepared by a kind of new coated technique, it can make to catch the third liver chimeric antigen and the equal q.s of monoclonal antibody and stable being coated on micropore of use; The applicable antigen-antibody joint-detection of sample diluting liquid provided by the invention, it both can improve the sensitivity that antigen detects, required blood plasma (or serum) amount while reducing antigen detection, the background in the time of can greatly reducing antibody test again; The invention provides in addition and a kind ofly use a kit random combine to realize the object that arbitrarily detectable antigens, antibody or antigen-antibody detect simultaneously.
Kit disclosed by the invention and detection method thereof both can detect HCV antigen and antibody simultaneously, also can detect separately that hepatitis C is early stage, acute infection period and the situation of HCV antigen before antibody produces or while having formed antigen-antibody complex, or the situation of HCV antibody after detecting separately antibody and producing.The blood source examination that kit of the present invention and detection method thereof can be used for HCV detects hepatitis C antigen and antibody simultaneously, both can improve with blood and transfusion safety, has also improved work efficiency and cost-saving simultaneously; In addition kit disclosed by the invention and detection method thereof can be distinguished the existing disease infection of HCV or previous infection, and the early detection and the treatment that can be used for HCV are evaluated, for the clinical important detection evaluation index that provides is provided.
Embodiment
The preparation of the microwell plate of embodiment 1 hepatitis C virus chimeric antigen and hepatitis C virus monoclonal antibody
(1) by the chimeric antigen to the sequential analysis of HCV, chimeric design reconfiguration HCV-cAg and other non-structural protein, clone the monoclonal antibody of different epi-positions, while making simultaneously envelope antigen antibody, both can not mutually combine, and avoid affecting the combination of corresponding HCV antibody and HCV-cAg in testing sample.After sequential analysis and chimera design, the preparation method of antigen and monoclonal antibody operates with reference to the conventional method on < < molecular cloning > >.
The preparation of A, antigen and Purification
1. extract total RNA of HCV virus;
2. RT-PCR amplification minute fragment is cloned NS-3, the corresponding gene fragment of NS-4 and Core;
3. be cloned on expression vector pRSET;
4. the abduction delivering of foreign gene in host cell, adopts affinitive layer purification target antigen;
5. Western blot method identified activity.
The foundation of B, hybridoma
1. immune animal;
2. Fusion of Cells and cloning;
3. ascites preparation;
4. the CHARACTERISTICS IDENTIFICATION of mAb (ELISA method).
(2) because albumen is coated on microwell plate main by hydrophobic bond, ionic link etc.Envelope antigen and antibody are because the two isoelectric point differs far, because albumen is coated on microwell plate main by hydrophobic bond, ionic link etc. simultaneously.Envelope antigen and antibody, because the two isoelectric point differs far, are difficult to accomplish the two equal q.s and stable being coated on micropore simultaneously.
The present invention, by introduce ionic surface active agent and reductive agent in coating buffer, adopts the mode of neutral buffered liquid to solve the problems referred to above.
Because ionic surface active agent can dissociate negative ion and kation in coating buffer, form two kinds of compounds with coated recombinant antigen and monoclonal antibody respectively, make the electric charge of compound considerably beyond recombinant antigen and monoclonal antibody, thereby make the two and be better coated on microwell plate.
Ionic surface active agent described in embodiment of the present invention includes but not limited to dodecyl alanine, dodecyldimethylammonium hydroxide inner salt, cetrimonium bromide (CTAB).The concentration of CTAB in the present invention in coating buffer is 0.01%～1.3%, is preferably 0.05～1.0%, and more excellent is 0.08～0.5%, most preferably is 0.1%.
The impact of CTAB on the sensitivity of HCV antigen-antibody combined detection kit in table 1 coating buffer
Reductive agent changes protein conformation by the disulfide bond changing in albumen, in the present invention, by add reductive agent in coating buffer, the folded state of coating protein is opened, and becomes flattened state, is adsorbed on microwell plate better more closely.Also make detection site better expose because being opened, detection sensitivity is improved greatly simultaneously.
In addition the Main Antigenic Region section NS3 antigen of HCV contains a plurality of cysteine residues (only a.a1196-1473 just has 7 Cys), when recombinant expressed, this albumen may form the disulfide bond of mispairing, thereby change natural structure tin, cause NS3 antigen active to reduce, CORE epitope also causes activity decreased because exposure is insufficient.We add the DTT of variable concentrations in coating buffer, result shows that reductive agent improves the activity of HCV antigen in certain degree, show that recombinant HCV antigen exists mispairing disulfide bond, and the processing of going back original reagent can reopen mispairing disulfide bond, allow the original conformation of HCV antigen recovery.
Reductive agent described in embodiment of the present invention includes but not limited to halfcystine, dithiothreitol (DTT) (DTT), reduced glutathione, beta-mercaptoethanol.The working concentration of DTT in the present invention in coating buffer is 0.01%～2%, and preferred concentration is 0.05%～1%, most preferably is 0.1%.
The impact of DTT on the sensitivity of HCV antigen-antibody combined detection kit in table 2 coating buffer
The selection of surfactant, reductive agent and consumption and coated concrete technology are as follows:
(2) preparation of preparation antigen-antibody coating buffer: 1 * PBS(PH7.2) by the HCV of the anti-HCV monoclonal antibody of 5ug/ml purifying and 100ng/ml (1 * PBS for chimeric antigen, 0.1% CTAB, 0.1%DTT) after dilution, with 200ul/ hole, add in microwell plate, 4 ℃ of placements are spent the night, again through 1 * PBS(PH7.2) wash 2 times, add confining liquid (2% trehalose, 1%BSA, 1% casein sodium salt and 1% tryptophane, 1 * PBS PH7.2) 250ul/ hole, 4 ℃ of placements discard after spending the night in hole after liquid in room temperature, humidity <30% is dried 4h ± 0.5h, 2～8 ℃ of preservations of dry complete final vacuum encapsulation.
The experimental result contrast of two kinds of coated PROCESS FOR TREATMENT of difference is in Table 3.
The experimental result contrast of the coated PROCESS FOR TREATMENT of two kinds of differences of table 3
A group plate coordinates corresponding ELIAS secondary antibody through 37 ℃ of examinations 3 days with B group plate, and inspection HCV national standard dish, the results are shown in Table 4.
Table 4 and the comparison of the HCV of Nat'l Pharmaceutical & Biological Products Control Institute national standard dish
| ? | A group | B group |
| Negative reference material coincidence rate | 30/30 | 30/30 |
| Positive reference material coincidence rate | 30/30 | 26/30 |
| Precision | 3.34% | 6% |
| Result | Up to specification | Against regulation |
The preparation of embodiment 2 sample diluting liquids
C-hepatitis antibody content in blood is many, and c-hepatitis antibody detect because of for detection of core space antigen have in very strong body or external homology and allos binding ability, easily cause that background is high until sample size requirements false-positive so that use when detecting c-hepatitis antibody is considerably less, in current common c-hepatitis antibody detection kit, sample size is generally 10ul; And hepatitis C antigen content in blood is considerably less, it is very many to the requirement of sample size detecting sample, and in current commercial hepatitis C antigen detection kit, sample size requirements is 100ul.Therefore when the two detects simultaneously to there is certain contradiction on sample size requirements.
The present invention better comes out hepatitis C antigen by add protein deposition agent and also source agent, scaling agent etc. in sample diluting liquid, improves sensitivity, reduces the effect of background simultaneously, and blood demand reduces when hepatitis C antigen is detected.Due to IgG abnormal in human serum, the Aschoff body during as illnesss such as systemic loupus erythematosus, Huppert's diseases or in serum and other abnormal IgG can cause that background raises or false positive.The present invention adds mouse IgG in sample diluting liquid.
Table 5 adds the impact of mouse IgG concentration on background control and negative sample
| Sample | Basis sample dilution adds mouse IgG | Basis sample dilution does not add mouse IgG |
| N1 | 0.023 | 0.123 |
| N2 | 0.021 | 0.144 |
| N3 | 0.029 | 0.156 |
| N4 | 0.015 | 0.102 |
| N5 | 0.051 | 0.256 |
| N6 | 0.023 | 0.223 |
| N7 | 0.021 | 0.119 |
| N8 | 0.010 | 0.092 |
| N9 | 0.017 | 0.267 |
| N10 | 0.021 | 0.227 |
Note: above-mentioned sample is all negative interference samples of rheumatoid factor or lupus erythematosus positive sample but hepatitis C antigen.
The processing of reductive agent is opened the disulfide bond of mispairing, recovers the conformation of HCV antigen, improves the sensitivity detecting, and we add also reductive agent and have also obtained same effect in sample diluting liquid.The reductive agent that we use in sample diluting liquid is halfcystine.
In order to verify whether prepared sample dilution is applicable to the joint-detection of hepatitis C antigen antibody simultaneously, the present invention's positive sample in embodiments all adopts 10 parts of antigen positive samples to add 10 parts of antibody positive samples, calculates the mode of the mean value of 20 samples and carries out statistical.
The impact of halfcystine on the sensitivity of HCV antigen-antibody combined detection kit in table 6 sample diluting liquid
Note: the positive sample detecting is that 10 parts of antigen positive samples add 10 parts of antibody positive samples, the mean value of 20 samples of calculating
In order further to reduce the use of sample size, we study and find to add (NH in samples
4)
2sO
4and urea (Urea), can make albumen coagulation in sample and better be coated in catching albumino reaction and improve sensitivity on polystyrene micropore plate, the use of sample size is reduced.
(NH in table 7 sample diluting liquid
4)
2sO
4impact on combined detection kit sensitivity
Note: the positive sample detecting is that 10 parts of antigen positive samples add 10 parts of antibody positive samples, the mean value of 20 samples of calculating
The impact of Urea on combined detection kit sensitivity in table 8 sample diluting liquid
Note: the positive sample detecting is that 10 parts of antigen positive samples add 10 parts of antibody positive samples, the mean value of 20 samples of calculating
The present invention relatively finds the damping fluid of different PH in addition, and the sample diluting liquid of slant acidity, to the rare middle SB-12 of this sample, 25%(NH
4)
2sO
4, the solubleness of 2.5M Urea, 1%CTAB is better, but do not affect, does not detect effect.
Specifically being formulated as follows of final definite sample diluting liquid: 5mM EDTA, 1%BSA, 1mg/ml mouse IgG, 1%SB-12,1%CTAB, 1%Triton-X114, the halfcystine of 20mM, 25%(NH
4)
2sO
4, 2.5M Urea, 0.01Proclin300,1 * PBS, PH5.8.
By using new sample diluting liquid, while finally detecting, when required sample size is 50ul, can realize hepatitis C antigen antibody and detect also simultaneously and all can reach good effect.
The preparation of embodiment 3 enzyme working fluids and detection operating process
(1) preparation of enzyme mark albumen: get appropriate horseradish peroxidase (adding 1mg HRP by 1mg albumen measures), be dissolved in 1.0ml NaHCO by 5mg HRP
3solution, fully dissolves HRP.Add appropriate 0.06M NaIO
4room temperature lucifuge stirs 30 minutes.Move into bag filter, by 0.01M pH9.6 carbonate buffer solution dialysed overnight.Get purifying albumen to be marked appropriate, use 0.01M NaHCO
3solution fully dissolves, and mixes with enzyme liquid, packs bag filter into, and with 4 ℃ of lucifuge dialysis of 0.01M pH9.6 carbonate buffer solution 20 hours, liquid was changed 3 times in centre.Get after dialysis in conjunction with liquid, add 0.4%NaBH
4(by 5mg HRP, being dissolved in 0.2ml NaBH4 solution) stirred after 30 minutes, placed 4 hours for 4 ℃.Pack enzyme conjugates into bag filter, with PH7.20.01M PBS dialysis 48 hours.Sephadex G-200 gel filtration: by Sephadex G-200 chromatographic column on dislysate, with PH7.2 0.01M PBS wash-out, collect first eluting peak, then measure concentrate volume by the concentrated matter of PEG2000, get sample 0.1ml, censorship.All the other concentrates add equal-volume glycerine, and-20 ℃ save backup.The enzyme labelled antibody titration of tiring: anti-human IgG enzyme labeling thing adopts square formation titration, with indirect elisa method measure tire be not less than 1:5000 doubly can be for the preparation of kit.
In this kit, detect enzyme labeling monoclonal antibody and the enzyme labeling recombinant antigen difference mark of use.
(2) preparation of enzyme dilution: in order to prepare the enzyme dilution that can be applicable to hepatitis C antigen enzyme mark albumen+c-hepatitis antibody enzyme mark albumen, c-hepatitis antibody enzyme mark albumen and hepatitis C antigen enzyme mark albumen simultaneously, prepare HCV antigen-antibody joint-detection enzyme working fluid, HCV antigen detection enzyme working fluid and HCV antibody test enzyme working fluid.We prepare optimization two groups of dilutions compare.
A, lowlenthal serum (10%)
1% thimerosal
BSA(1%)
Casein (0.3%)
10mg/ml celebrating is large
0.01MPBS(PH7.4)
B, lowlenthal serum (5%)
1% thimerosal
PVP(1%)
BSA(1%)
Glucosan T2000 (5%)
Casein (0.5%)
10mg/ml celebrating is large
0.01MPBS(PH7.4)
Definite test of table 9 A group enzyme labeling thing dilution
| Sample | HCV antigen-antibody associating enzyme working fluid | HCV antigen enzyme working fluid | HCV antigen enzyme working fluid |
| P1 | 1.765 | 1.102 | 0.305 |
| P2 | 1.123 | 0.877 | 0.672 |
| P3 | 1.321 | 0.987 | 0.573 |
| P4 | 1.634 | 1.296 | 0.263 |
| P5 | 2.110 | 1.291 | 0.189 |
| N1 | 0.027 | 0.023 | 0.029 |
| N2 | 0.018 | 0.021 | 0.022 |
| N3 | 0.020 | 0.018 | 0.022 |
| N4 | 0.029 | 0.026 | 0.025 |
| N5 | 0.017 | 0.019 | 0.022 |
Note: P1-P5 sample is all positive samples of hepatitis C antigen antibody, N1-N5 is all negative samples of hepatitis C antigen antibody.
Definite test of table 10 B group enzyme labeling thing dilution
| Sample | HCV antigen-antibody associating enzyme working fluid | HCV antigen enzyme working fluid | HCV antigen enzyme working fluid |
| P1 | 2.765 | 2.102 | 0.805 |
| P2 | 2.345 | 1.830 | 1.072 |
| P3 | 2.861 | 1.987 | 1.031 |
| P4 | 3.034 | 2.296 | 1.263 |
| P5 | 3.011 | 1.291 | 0.189 |
| N1 | 0.030 | 0.033 | 0.029 |
| N2 | 0.028 | 0.031 | 0.032 |
| N3 | 0.030 | 0.028 | 0.032 |
| N4 | 0.039 | 0.036 | 0.035 |
| N5 | 0.047 | 0.049 | 0.052 |
Note: P1-P5 sample is all positive samples of hepatitis C antigen antibody, N1-N5 is all negative samples of hepatitis C antigen antibody.
According to experimental result, selection group B formula is best enzyme dilution.
(3) enzyme working fluid
If it is positive to detect sample, because confirming the end, be that the hepatitis C antigen positive or c-hepatitis antibody are positive or the two is two positive, and need be equipped with again in addition other independently detection kit detect.
We are bright by enzyme working fluid being divided into three independent packagings (being respectively HCV antigen-antibody joint-detection enzyme working fluid, HCV abzyme working fluid and HCV hepatitis antibody enzyme working fluid), when the antibody combined detection of hepatitis C antigen, c-hepatitis antibody and hepatitis C antigen detect, under three kinds of different situations, use respectively, thereby realized, use a set of kit can solve above-mentioned contradiction.
(4) detect operating process:
1. take out reagent, in equilibrium at room temperature 30 minutes, coated plate all needed to blank, positive and negative contrast each diplopore.Blank well adds 100 μ l blank liquid, and positive and negative control wells adds 50 μ l positive and negative control serums, and all the other holes add testing sample 50 μ l, and except blank well, every hole adds 50 μ l sample diluting liquids, mixes rear 37 ℃ of temperature and bathes 60 minutes.
2. with the cleansing solution after 1:20 dilution, wash plate 6 times, washroom, every controlling 30 seconds, pats dry for the last time.
3. except blank well, every hole adds 100 μ l HCV antigen-antibody joint-detection enzyme working fluids, and 37 ℃ of temperature are bathed 30 minutes, 4 ℃ of preservations of residual enzyme working fluid.
4. wash plate same 2..
5. first add developer A liquid 50 μ l to add developer B liquid 50 μ l, 37 ℃ of temperature are bathed lucifuge colour developing 10 minutes again.
6. every hole adds 50 μ l stop buffers.Stop surveying OD value in latter 10 minutes.
7. result is judged: during OD value >=reference value, result is positive; During OD value < reference value, result is negative.
When 8. the positive sample of testing result is checked, can adopt HCV antigen detection enzyme working fluid, the HCV antibody test enzyme working fluid of independent packaging in this kit to distinguish antigen positive or antibody positive, its detecting step with 1. above-mentioned-7., but in the 3. step, during detectable antigens, change into and add HCV antigen detection enzyme working fluid, while detecting antibody, change into and add HCV antibody test enzyme working fluid.
Claims (10)
1. a hepatitis C virus antigen-antibody combined detection kit, comprises following component: the microwell plate that is coated with HCV chimeric antigen and HCV monoclonal antibody, sample diluting liquid, enzyme working fluid, substrate A liquid, substrate B liquid, 20 times of concentrated cleaning solutions and stop buffer, is characterized in that: coated with the coated damping fluid that contains ionic surface active agent and reductive agent when prepared by described microwell plate, wherein ionic surface active agent is selected from dodecyl alanine, dodecyl-dimethyl amine second lactone or cetrimonium bromide (CTAB), reductive agent is selected from halfcystine, dithiothreitol (DTT) (DTT), reduced glutathione or beta-mercaptoethanol, described sample diluting liquid is to contain reductive agent, the solution of mouse IgG and protein deposition agent ammonium sulfate, wherein reductive agent is selected from halfcystine, dithiothreitol (DTT) (DTT), reduced glutathione or beta-mercaptoethanol, described enzyme working fluid is to include HCV antigen-antibody joint-detection enzyme working fluid, HCV antigen detects three independent packagings of enzyme working fluid and HCV antibody test enzyme working fluid.
2. hepatitis C virus antigen-antibody combined detection kit according to claim 1, it is characterized in that: the ionic surface active agent in described coated damping fluid is cetrimonium bromide (CTAB), working concentration is 0.01 wt %～1.3 wt %; Reductive agent in described coated damping fluid is dithiothreitol (DTT) (DTT), and working concentration is 0.01 wt %～2 wt %.
3. hepatitis C virus antigen-antibody combined detection kit according to claim 2, it is characterized in that: the ionic surface active agent in described coated damping fluid is cetrimonium bromide (CTAB), working concentration is 0.08 wt %～0.5 wt %; Reductive agent in described coated damping fluid is dithiothreitol (DTT) (DTT), and working concentration is 0.05 wt %～1.0 wt %.
4. hepatitis C virus antigen-antibody combined detection kit according to claim 2, is characterized in that: the ionic surface active agent in described coated damping fluid is cetrimonium bromide (CTAB), and working concentration is 0.1wt%; Reductive agent in described coated damping fluid is dithiothreitol (DTT) (DTT), and working concentration is 0.1wt %.
5. hepatitis C virus antigen-antibody combined detection kit according to claim 1, is characterized in that: the reductive agent in described sample diluting liquid is halfcystine, and working concentration is 20mM.
6. hepatitis C virus antigen-antibody combined detection kit according to claim 1, is characterized in that: the working concentration of described sample diluting liquid small mouse IgG is 1mg/ml.
7. hepatitis C virus antigen-antibody combined detection kit according to claim 1, is characterized in that: in described sample diluting liquid, the working concentration of protein deposition agent ammonium sulfate is 25 wt %.
8. hepatitis C virus antigen-antibody combined detection kit according to claim 1, is characterized in that: described Sample Dilution formula of liquid is: 5mM EDTA, 1%BSA, 1mg/ml mouse IgG, 1% SB-12,1%CTAB, 1%Triton-X 114, the halfcystine of 20mM, 25%(NH
4)
2sO
4, 2.5M Urea, 0.01Proclin300,1 * PBS, PH5.8.
9. hepatitis C virus antigen-antibody combined detection kit according to claim 1, is characterized in that: the diluted of following formula for described enzyme working fluid: 5% lowlenthal serum, 1% thimerosal, 1%PVP, 1%BSA, 5% glucosan T2000,0.5% casein, 10mg/ml gentamicin, 0.01M pH7.4 PBS.
10. the application of hepatitis C virus antigen-antibody combined detection kit described in claim 1, it is characterized in that: the HCV antigen-antibody joint-detection enzyme working fluid in described enzyme working fluid, HCV antigen detect enzyme working fluid and three independent packagings of HCV antibody test enzyme working fluid, under joint-detection HCV antigen and antibody, c-hepatitis antibody and three kinds of different situations of hepatitis C antigen detection, use respectively.
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| CN106093402A (en) * | 2016-05-31 | 2016-11-09 | 湖南康润药业有限公司 | Hepatitis C virus antigen-antibody combined detection kit |
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