CN109444424A - A kind of HCV NS3 antibody detection method based on MIX antigen coat - Google Patents

A kind of HCV NS3 antibody detection method based on MIX antigen coat Download PDF

Info

Publication number
CN109444424A
CN109444424A CN201811287913.0A CN201811287913A CN109444424A CN 109444424 A CN109444424 A CN 109444424A CN 201811287913 A CN201811287913 A CN 201811287913A CN 109444424 A CN109444424 A CN 109444424A
Authority
CN
China
Prior art keywords
hcv
antibody
detection method
distilled water
method based
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201811287913.0A
Other languages
Chinese (zh)
Inventor
乔伟振
李另另
乔简
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuxi Peoples Hospital
Original Assignee
Wuxi Peoples Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuxi Peoples Hospital filed Critical Wuxi Peoples Hospital
Priority to CN201811287913.0A priority Critical patent/CN109444424A/en
Publication of CN109444424A publication Critical patent/CN109444424A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Invention proposes a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat, coating: microwell plate is coated with after respectively diluting HCV NS3 (1 type and 6 types) MIX antigen with 0.01mol/LPBS (pH 7.2), 100 holes μ l/, place 4 DEG C of refrigerator overnights, secondary daily 1 × PBST liquid washs 3 times, dry seal is detained, 4 DEG C store for future use；Sample-adding: enzyme mark hole is added in diluted ELIAS secondary antibody (goat-anti people-IgG), 100 holes μ l/ sequentially add 1 μ l serum specimen, are divided into blank control group, negative control group and positive controls, test serum, pat mixing；It incubates: setting 37 DEG C and incubate 1 hour；Based on the still incomplete status of current country's HCV antibody test reagent, in conjunction with the characteristics of the distribution of China domestic HCV genotype, a kind of HCV NS3 enzyme immunoassay antibody detection method with MIX antigen coat is established, to improve the recall rate of HCV antibody, to reduce the missing inspection of HCV infection person.

Description

A kind of HCV NS3 antibody detection method based on MIX antigen coat
Technical field
The present invention relates to biotechnology diagnostic techniques field, specially a kind of HCV NS3 antibody based on MIX antigen coat Detection method.
Background technique
Enzyme immunoassay (EIA) detection anti-HCV is commonly used as hepatitis C (HC) diagnosis index, the country is at present Conventional screening is being carried out to blood donor's anti-HCV, so that the incidence of HC is greatly reduced after blood transfusion, but still is having part receptor hair HC after raw blood transfusion, this result confirm that HCV detection reagent domestic at present is perfect not enough；HCV easily morphs, gene Sequence has height heterogeneity.HCV is divided into 6 genotype at present, a gene hypotype more than 20, and the HCV of different genotype is wide General to be distributed in different parts of the world, in Asia, HCV has the distribution of 1,2,6 types, and in China based on 1b type, but mixed infection is obvious Higher than other countries and area, the variation of HCV genome sequence and the change of genome encoding protein antigenicity, biological property Out-phase is closed, this has a major impact the serology antibody test result of HCV.
The core of HCV antibody kit is antigenic component, and non-structural district 3 (NS3) antigen is current anti-HCV EIA detection One of core component in reagent.NS3 albumen has a very strong antigenicity, but area's gene order in HCV different genotype and Variability in hypotype is larger, therefore detects other type (hypotype) HCV infection marks with the HCV antigen of specific genotype (hypotype) This when, antigenicity will receive the restriction of this otherness and influence recall rate.However, detection examination provided by currently on the market NS3 antigen used is all from 1 type of HCV genotype in agent box, and 1 type antigenic agents are inevitable for the antibody test of the HCV of non-1 type It will receive the heterogeneous influence of gene order.
There are notable differences for different genotype HCV NS3 protein antigenicity, so developing HCV serodiagnosis reagent It is specifically contemplated that influence of the different genotype NS3 antigenic specificity to antibody test.It is China domestic to be mainly with 1 type of genotype It is main, but there are still the mixed infections for having other genotype, therefore cannot equally have with the NS3 antigen that specific genotype is 1 type Detect the antibody of other genotype in effect ground.The characteristics of based on above and combination China domestic genotype distribution, comparing 1 type On the basis of 6 type, two kinds of antigenic differences of different genotype NS3,1 type and 6 type NS3 recombinant antigens are mixed into (MIX in proportion Antigen) it is applied in antibody test as envelope antigen, the antibody test of HCV is applied it to, so that than applying 1 type merely The efficiency of NS3 antigen detection is improved.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the existing defects, provides a kind of HCV based on MIX antigen coat NS3 antibody EIA detection method, based on the still incomplete status of current country's HCV antibody test reagent, in conjunction with China domestic The characteristics of HCV genotype is distributed, establishes a kind of HCV NS3 antibody EIA detection method with MIX antigen coat, to improve HCV The recall rate of antibody can effectively solve the problems in background technique to reduce the missing inspection of HCV infection person.
To achieve the above object, the present invention proposes: a kind of detection side HCV NS3 antibody EIA based on MIX antigen coat Method,
(1) it is coated with: being wrapped after respectively diluting HCV NS3 (1 type and 6 types) MIX antigen with 0.01mol/LPBS (pH 7.2) By microwell plate, 4 DEG C of refrigerator overnights are placed in 100 holes μ l/, and secondary daily 1 × PBST liquid washs 3 times, detain dry seal, 4 DEG C of storages are standby With；
(2) it is loaded: enzyme mark hole is added in diluted ELIAS secondary antibody (goat-anti people-IgG), 100 holes μ l/ sequentially add 1 μ L serum specimen is divided into blank control group, negative control group and positive controls, test serum, pats mixing；
(3) it incubates: setting 37 DEG C and incubate 1 hour；
(4) it washs: discarding liquid in hole, PBST liquid washs 4 times, and button is dry；
(5) it develops the color: adding substrate to every hole of blank control group, negative control group and positive controls, test serum respectively Each 50 μ l of A liquid, substrate B liquid, pats mixing, sets 37 DEG C and incubates 10 minutes；
(6) 50 μ l of terminate liquid is added, pats mixing；
(7) measure: microplate reader is returned to zero with blank control wells, and each hole OD450 value, printing measurement are measured at wavelength 450nm As a result；
As a preferred technical solution of the present invention: coating dilution is by PBS, NaCl, KCl, Na2HPO4·12H2O、 KH2PO4Distilled water composition, 0.01mol/L PBS, NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 3.58g, KH2PO40.2g adds distilled water to 1L, adjusts pH to 7.2.
As a preferred technical solution of the present invention: carbonate buffer solution is by Na2CO3、NaHCO3、Na2N3, distilled water Composition, Na2CO31.5g、NaHCO32.9g、Na2N30.2g adds distilled water to be adjusted to pH 9.6 to 1000ml.
As a preferred technical solution of the present invention: ELIAS secondary antibody dilution I by 20% glycerol, 30% calf serum, 50%0.01mol/L PBST composition.
As a preferred technical solution of the present invention: ELIAS secondary antibody dilution II by NaCl, thimerosal, Tween-20, Dimethyl sulfoxide, calf serum, phosphate buffer composition, 1.31g NaCl, 0.005g thimerosal, 45 μ l Tween-20, 135 μ l dimethyl sulfoxides, 5ml calf serum, 45ml 0.01mol/L pH7.4 phosphate buffer (PB) mixing.
As a preferred technical solution of the present invention: cleaning solution is by NaCl, KCl, Na2HPO4·12H2O、 KH2PO4、 Tween-20, distilled water composition, NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 3.58g, KH2PO40.2g, Tween- 200.5ml adds distilled water to 1L, adjusts pH to 7.2.
As a preferred technical solution of the present invention: substrate solution A is by tetramethyl benzidine, dehydrated alcohol, distilled water group At, tetramethyl benzidine 200mg and dehydrated alcohol 100ml plus distilled water to 1L.
As a preferred technical solution of the present invention: substrate solution A is made of tetramethyl benzidine, DMSO, tetramethyl connection Aniline 200mg and DMSO 100ml, add distilled water to 1L.
As a preferred technical solution of the present invention: substrate solution B is by Na2HPO4, citric acid, 0.75% hydrogen peroxide urine Element, distilled water composition, Na2HPO414.6g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml add distilled water to 1L, adjust PH to 7.2.
As a preferred technical solution of the present invention: terminate liquid is the H2SO4 solution of 2mol/L.
Compared with prior art, the beneficial effects of the present invention are: it is still inadequate based on current country HCV antibody test reagent It is anti-to establish a kind of HCV NS3 with MIX antigen coat in conjunction with the characteristics of the distribution of China domestic HCV genotype for perfect status Body EIA detection method, to improve the recall rate of HCV antibody, to reduce the missing inspection of HCV infection person.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described reality Applying example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field Those of ordinary skill's every other embodiment obtained without making creative work, belongs to guarantor of the present invention The range of shield.
The present invention the following technical schemes are provided:
A kind of HCV NS3 antibody EIA detection method based on MIX antigen coat,
(1) it is coated with: being wrapped after respectively diluting HCV NS3 (1 type and 6 types) MIX antigen with 0.01mol/LPBS (pH 7.2) By microwell plate, 4 DEG C of refrigerator overnights are placed in 100 holes μ l/, and secondary daily 1 × PBST liquid washs 3 times, detain dry seal, 4 DEG C of storages are standby With；
(2) it is loaded: enzyme mark hole is added in diluted ELIAS secondary antibody (goat-anti people-IgG), 100 holes μ l/ sequentially add 1 μ L serum specimen is divided into blank control group, negative control group and positive controls, test serum, pats mixing；
(3) it incubates: setting 37 DEG C and incubate 1 hour；
(4) it washs: discarding liquid in hole, PBST liquid washs 4 times, and button is dry；
(5) it develops the color: adding substrate to every hole of blank control group, negative control group and positive controls, test serum respectively Each 50 μ l of A liquid, substrate B liquid, pats mixing, sets 37 DEG C and incubates 10 minutes；
(6) 50 μ l of terminate liquid is added, pats mixing；
(7) measure: microplate reader is returned to zero with blank control wells, and each hole OD450 value, printing measurement are measured at wavelength 450nm As a result；
As a preferred technical solution of the present invention: coating dilution is by PBS, NaCl, KCl, Na2HPO4·12H2O、 KH2PO4Distilled water composition, 0.01mol/L PBS, NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 3.58g, KH2PO4 0.2g adds distilled water to 1L, adjusts pH to 7.2.
As a preferred technical solution of the present invention: carbonate buffer solution is by Na2CO3、NaHCO3、Na2N3, distilled water Composition, Na2CO3 1.5g、NaHCO32.9g、Na2N30.2g adds distilled water to be adjusted to pH 9.6 to 1000ml.
As a preferred technical solution of the present invention: ELIAS secondary antibody dilution I by 20% glycerol, 30% calf serum, 50%0.01mol/L PBST composition.
As a preferred technical solution of the present invention: ELIAS secondary antibody dilution II by NaCl, thimerosal, Tween-20, Dimethyl sulfoxide, calf serum, phosphate buffer composition, 1.31g NaCl, 0.005g thimerosal, 45 μ l Tween-20, 135 μ l dimethyl sulfoxides, 5ml calf serum, 45ml 0.01mol/L pH7.4 phosphate buffer (PB) mixing.
As a preferred technical solution of the present invention: cleaning solution is by NaCl, KCl, Na2HPO4·12H2O、 KH2PO4、 Tween-20, distilled water composition, NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 3.58g, KH2PO40.2g, Tween- 20 0.5ml add distilled water to 1L, adjust pH to 7.2.
As a preferred technical solution of the present invention: substrate solution A is by tetramethyl benzidine, dehydrated alcohol, distilled water group At, tetramethyl benzidine 200mg and dehydrated alcohol 100ml plus distilled water to 1L.
As a preferred technical solution of the present invention: substrate solution A is made of tetramethyl benzidine, DMSO, tetramethyl connection Aniline 200mg and DMSO 100ml, add distilled water to 1L.
As a preferred technical solution of the present invention: substrate solution B is by Na2HPO4, citric acid, 0.75% hydrogen peroxide urine Element, distilled water composition, Na2HPO414.6g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml add distilled water to 1L, adjust PH to 7.2.
As a preferred technical solution of the present invention: terminate liquid is the H2SO4 solution of 2mol/L.
Embodiment one:
HCV NS3 antibody test program optimization
One, the selection of NS3MIX envelope antigen optimum dilution degree and enzyme labelled antibody optimum dilution degree
1. method: the method titrated with square matrix, by the purified MIX antigen containing HCV NS3 (1 type), NS3 (6 type) Make doubling dilution coated elisa plate, while doubling dilution made to the goat anti-human igg of HRP label, the dilution of the two is from 1: 200 arrive 1:3200, detect 2 parts of ' negative ' specimens respectively and 2 parts of positive samples, sample-adding amount are 1 μ l, set up blank control wells, have Gymnastics makees that steps are as follows states, and finally selects positive serum OD450Value and negative serum OD450The highest dilution of the ratio between value is most Good dilution.
2. specific steps: being coated with microwell plate, 100 μ after NS3MIX antigen is diluted with 0.01mol/L PBS (pH 7.2) 4 DEG C of refrigerator overnights are placed in the hole l/；Secondary daily 1 × PBST liquid washs 3 times, and button is dry, and sealing, 4 DEG C store for future use.
(1) it is loaded: the diluted ELIAS secondary antibody goat-anti people-IgG of label (HRP) is added enzyme mark hole, 100 holes μ l/, then 1 μ l serum specimen (setting up blank control, negative control and positive control) is sequentially added, mixing is patted；
(2) it incubates: setting 37 DEG C and incubate 1 hour；
(3) it washs: discarding liquid in hole, PBST liquid washs 4 times, and button is dry；
(4) develop the color: including blank control wells, every hole adds each 50 μ l of substrate A liquid, B liquid, pats mixing, sets 37 DEG C and incubates 10 Minute.
(5) 50 μ l of terminate liquid is added, pats mixing；
(6) measure: microplate reader is returned to zero with blank control wells, and each hole OD is measured at wavelength 450nm450Value, printing measurement knot Fruit；
(7) result judgement standard: ratio representation is used: with the OD of measurement specimen hole450Hole is measured with ' negative ' specimens OD450(when ' negative ' specimens measure hole OD450When<0.06 in terms of 0.06) ratio (P/N) indicate, be judged to sun as P/N>2.1 Property, it is otherwise feminine gender.
3. result: determining envelope antigen and enzyme labelled antibody with the method that square matrix titrates, doubling dilution degree is respectively from 1:200 To 1:3200, the results show that when concentration of antigen (antibody) is fixed, with the increase of antibody (antigen) dilution, yin and yang attribute The OD value of sample is on a declining curve.When antigen coat concentration is in 1:1600, enzyme labelled antibody concentration is in 1:3200, two parts of positives Reaching highest with the ratio between ' negative ' specimens OD value is respectively 59.8 and 67.9 (as shown in the table), so determining above-mentioned two dilution Degree is respectively antigen coat and the diluted concentration of enzyme labelled antibody.
1 square matrix titration results of table (P/N ratio)
Two, the selection of NS3 MIX antigen coat liquid
1. method: use the carbonate buffer solution of the PBS and pH 9.6 of pH 7.2 to be coated with MIX antigen as dilution respectively, 5 parts of feminine genders and 5 parts of positive serum samples (setting multiple holes) are detected, two kinds of coating dilution detection sample OD are compared450Whether there is or not systems for value Meter learns difference.
2. specific steps: Detailed operating procedures are identical as one, 2 as method.
3. result: being coated with MIX antigen with PBS and carbonate buffer solution respectively, detect 5 parts of positive serum samples and 5 parts of yin Property serum specimen (shown in following table), with matched-pair design T inspection different, the P > that carries out two kinds of dilution OD value differences of statistical analysis 0.05, it is not statistically significant, this method selects conventional use of PBS buffer as coating buffer.
Two kinds of coating buffer coating microwell plate testing results compare
Three, the selection of enzyme labelled antibody dilution
1. method: with containing 0.5mol/LNaCl, 1 ‰ Tween-20,0.1g/L thimerosals, 0.3% dimethyl sulfoxide 7.4 phosphate buffer of 0.01mol/L pH (containing stabilizing solution), which is prepared, makees enzyme labelled antibody dilution I containing 10% calf serum, then It prepares the PBST containing 30% calf serum, 20% glycerol and makees sample diluent II, detect 5 parts of positives and 5 parts of negative serum samples (setting multiple holes) compares the OD detected with two kinds of enzyme-labelled antigen dilutions450Value has no difference of science of statistics.
2. specific steps: Detailed operating procedures are identical as one, 2 as method.
3. result: dilute ELIAS secondary antibody with two kinds of enzyme labelled antibody dilutions (I and II) respectively, detect respectively 5 parts of positives and The mean value of 5 parts of negative serum samples, the OD value mean value and negative (positive) group sample OD value that calculate separately multiple holes sample is (as follows Shown in table)；Statistical analysis, P > 0.05, no difference of science of statistics, since enzyme labelled antibody dilution I exists are carried out with paired t-test 4 DEG C of stability are preferable, so this method selects the dilution that it is enzyme labelled antibody.
Two kinds of enzyme labelled antibody dilution testing results (OD value) are compared
Four, serum sample dilution selects
1. method: according to the experiment condition optimized above, detecting 10 parts of positives respectively and 10 parts of negative serum samples (are set Multiple holes), each sample sets 1:200 (0.5 μ l serum/hole), 1:100 (1 μ l serum/hole), 1:50 (2 μ l serum/hole), 1 respectively: The detection of 20 (5 μ l serum/hole) sample dilutions, more each dilution recall rate and OD450The ratio of value selects recall rate High and positive serum and negative serum OD450The ratio of value is relatively high and the dilution that operates conveniently is optimal sample dilution Degree.
2. specific steps: Detailed operating procedures are identical as one, 2 as method.
3. result: the anti-HEV IgG positive and each 10 parts of ' negative ' specimens are taken respectively, by 1:200,1:100,1:50,1:20 Dilution after detect, it is last the results show that positive (yin) property recall rate of the sample of different sample dilutions is consistent； When the thinner ratio of sample is 1:100, positive serum and negative serum OD450The ratio of value is relatively high, with sample dilutions Reduction, the raising trend of ratio has not been apparent (as shown in the table)；So this method selects 1:100 for serum sample This dilution, i.e., every 1 μ l of hole increase serum.
Different sample dilutions testing results (OD value ratio) are compared
Embodiment two:
The repeatability and stability test of HCV NS3 antibody test
One, repetitive test
1. method: taking 3 parts of positive samples, while measuring 10 times, calculate variation within batch coefficient (CV), same 3 parts of samples are every Its measurement one-time continuous is surveyed 5 days, and interassay coefficient of variation is calculated.
2. specific steps: Detailed operating procedures are identical as one, 2 in embodiment 1 as method.
3. result: 3 parts of positive serum samples being measured 10 times in the same period, calculate variation within batch coefficient；It will be same 3 parts of positive serum METHOD FOR CONTINUOUS DETERMINATIONs 5 times, once a day, calculate interassay coefficient of variation；As a result the coefficient of variation is respectively less than 10%, shows Show the repeatability of this method preferably (shown in institute's following table).
Repetitive test result (CV)
Two, stability test
1. method: will be with a collection of reagent (pre-coated elisa plate, diluted ELIAS secondary antibody, developing solution A and B, terminate liquid) 4 DEG C of refrigerators and 37 DEG C of incubators are individually placed to, it includes HCV strong positive, middle positive, weakly positive and negative blood that different time, which detects 4 parts, Clear sample, comparing kit test result (OD value) when being stored in 4 DEG C and 37 DEG C has no difference of science of statistics.
2. specific steps: Detailed operating procedures are identical as one, 2 in embodiment 1 as method.
3. result: by kit (including pre-coated elisa plate, ELIAS secondary antibody dilution, enzyme conjugates substrate solution A and B, Terminate liquid) it is placed 4 days in 37 DEG C of incubators, with the 3 parts of positives of kit Parallel testing and 1 part of negative serum for being placed on 4 DEG C of refrigerators Sample carries out heat stabilization test (as shown in the table).It is analyzed through paired t-test, value=0.12 P, shows OD under two states The difference of value does not have statistical significance.It is placed by 37 DEG C and is equivalent within 1 day 4 DEG C of placements calculating in 1.6 months, which at least may be used To stablize 6 months.
37 DEG C and 4 DEG C placement results compare (OD value)
Embodiment three:
The sensibility and specificity of HCV NS3 antibody test is tested
1. method and step
(1) 90 parts of anti-HEV IgG positive serums are detected.
(2) HCV antigen neutralization test: taking 10 parts of anti-HEV IgG positive serums to be separately added into the recombination MIX antigen of HCV, 37 DEG C incubate 1 hour；Control group takes same 10 parts of positive serums, but MIX antigen is not added, and same 37 DEG C incubate 1 hour； Then sample is detected with established detection method respectively.
(3) 6 parts of anti-HAV antibody positives, 9 parts of anti-HBV antibody positives, 20 parts of anti-HEV antibody positive blood are detected respectively Clear sample.
(4) the negative quality controlled serum of 200 parts of health examination serum specimens and 20 parts of visiting centers is detected respectively.
2. result
(1) with the serum specimen of 90 parts of anti-HEV IgG positives of detection, as a result have 65 parts is by HCV NS3 antibody test The positive, positive rate 72.2%.
The serum specimen of (2) 10 parts of anti-HEV IgG positives is separately added into the MIX antigen of HCV recombination, and 37 DEG C of incubations 1 are small The detection of Shi Houyong this method, as a result all negative, i.e., neutralization test is the positive；And control group this 10 parts of serum do not add Enter MIX antigen, testing result is all positive.
(3) it is anti-that 6 parts of anti-HAV antibody positives, 9 parts of anti-HBV antibody positives, 20 parts of anti-HEV are detected respectively with this method Body positive serum sample, it is as a result all negative, illustrate this method and A type, type and Hepatitis E patients serum without Cross reaction has preferable specificity.
The negative quality controlled serum this method at (4) 200 parts of healthy population physical examination serum specimens and 20 parts of visiting centers is through examining Survey all feminine genders of result.
The HCV RT-nested-PCR RNA of embodiment 4:HCV NS3 antibody test is verified
1. method and step
(1) design of primers: noncoding region is held to be designed for the universal primer that HCV is detected according to HCV 5 ':
5 '-GCC ATG GCG TTA GTA YGA GT-3 ' (- 260~-241) of HC1；
5 '-TTT CGC RAC CCA ACR CTA CT-3 ' (- 66~-85) of HC2；
5 '-AGT GTC RTR CAG CCT CCA GG-3 ' (- 243~-224) of HC3；
5 '-ACC CAA CRC TAC TMG GCT AG-3 ' (- 73~-92) of HC4；
(Y=C or T, R=G or A) outer primer HC1 and HC2 amplified fragments are 195bp, and inner primer HC3 and HC4 expand piece Section is 171bp, is synthesized by Shanghai Bio-engineering Corporation.
(2) extraction of RNA: taking 100 μ l of serum to add 250 μ l of Trizol, mixes, sets room temperature 5 minutes；80 μ of chloroform is added L high vibration 20 seconds, stands ice bath 5 minutes；4 DEG C 14000 revs/min, 15 minutes；Supernatant is shifted to process to through DEPC Eppendorf pipe in, add isometric isopropanol, be mixed by inversion, ice bath 15 minutes；4 DEG C 13000 revs/min, 10 minutes；It abandons Supernatant adds 75% ice ethyl alcohol, 300 μ l, is centrifuged after rinsing；Supernatant is abandoned, is inverted in drying at room temperature 15 minutes；DEPC is added to handle water 12 μ l sufficiently dissolve (RNasin containing 6U)；RNA95 DEG C is denaturalized 5 minutes, and ice bath 10 minutes, carry out reverse transcription immediately.
(3) reverse transcription
System is as follows:
42 DEG C water-bath 60 minutes, 95 DEG C 5 minutes, ice bath 10 minutes immediately, -20 DEG C of preservations.
(4) first time PCR reacts
System is as follows:
94 DEG C initial denaturation 2 minutes；94 DEG C 1 minute, 52 DEG C 1 minute, 72 DEG C 1 minute, 35 circulation；The last one circulation After 72 DEG C extend 8 minutes.
(5) second of PCR reaction
5 μ l of first time PCR product is taken, inner primer HC3 and HC4 and PCR reaction mixture is added, reaction condition is the same as first It is secondary；PCR amplification sets negative and positive control；Take last product 5 μ l, 2.5% agarose gel electrophoresis, testing goal item Band.
2. result: having detected the serum specimen of 30 parts of anti-HEV IgG positives in total, as a result have 16 parts is through PCR amplification The positive, this 16 parts of anti-HEV IgGs and RNA positive serum specimen is through HCV NS3 antibody test also all positives.
Benefit of the present invention: based on the still incomplete status of current country's HCV antibody test reagent, in conjunction with China domestic The characteristics of HCV genotype is distributed, establishes a kind of HCV NS3 antibody detection method with MIX antigen coat, to improve HCV antibody Recall rate, to reduce the missing inspection of HCV infection person.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding And modification, the scope of the present invention is defined by the appended.

Claims (10)

1. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat, which is characterized in that
(1) it is coated with: being coated with after respectively diluting HCV NS3 (1 type and 6 types) MIX antigen with 0.01mol/LPBS (pH 7.2) micro- 4 DEG C of refrigerator overnights are placed in orifice plate, 100 holes μ l/, and secondary daily 1 × PBST liquid washs 3 times, detain dry seal, 4 DEG C store for future use；
(2) it is loaded: enzyme mark hole is added in diluted ELIAS secondary antibody (goat-anti people-IgG), 100 holes μ l/ sequentially add 1 μ l serum Sample is divided into blank control group, negative control group and positive controls, test serum, pats mixing；
(3) it incubates: setting 37 DEG C and incubate 1 hour；
(4) it washs: discarding liquid in hole, PBST liquid washs 4 times, and button is dry；
(5) develop the color: respectively to every hole of blank control group, negative control group and positive controls, test serum add substrate A liquid, Each 50 μ l of substrate B liquid, pats mixing, sets 37 DEG C and incubates 10 minutes；
(6) 50 μ l of terminate liquid is added, pats mixing；
(7) measure: microplate reader is returned to zero with blank control wells, and each hole OD450 value is measured at wavelength 450nm, prints measurement result；
(8) it result judgement standard: uses ratio representation: measuring hole OD450 with the OD450 and ' negative ' specimens of measurement specimen hole The ratio (P/N) of (when ' negative ' specimens measure hole OD450<0.06 in terms of 0.06) indicates, is judged to the positive as P/N>2.1, no It is then feminine gender.
2. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: coating dilution is by PBS, NaCl, KCl, Na2HPO4·12H2O、KH2PO4Distilled water composition, 0.01mol/L PBS, NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 3.58g, KH2PO40.2g adds distilled water to 1L, adjusts pH to 7.2.
3. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: carbonate buffer solution is by Na2CO3、NaHCO3、Na2N3, distilled water composition, Na2CO3 1.5g、NaHCO3 2.9g、 Na2N30.2g adds distilled water to be adjusted to pH 9.6 to 1000ml.
4. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: ELIAS secondary antibody dilution I is made of 20% glycerol, 30% calf serum, 50%0.01mol/L PBST.
5. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: ELIAS secondary antibody dilution II is delayed by NaCl, thimerosal, Tween-20, dimethyl sulfoxide, calf serum, phosphate Fliud flushing composition, 1.31g NaCl, 0.005g thimerosal, 45 μ l Tween-20,135 μ l dimethyl sulfoxides, 5ml calf serum, 45ml 0.01mol/L pH7.4 phosphate buffer (PB) mixing.
6. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: cleaning solution is by NaCl, KCl, Na2HPO4·12H2O、KH2PO4, Tween-20, distilled water composition, NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 3.58g, KH2PO40.2g, Tween-20 0.5ml add distilled water to 1L, adjust pH extremely 7.2。
7. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: substrate solution A is made of tetramethyl benzidine, dehydrated alcohol, distilled water, tetramethyl benzidine 200mg and anhydrous second Alcohol 100ml, add distilled water to 1L.
8. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: substrate solution A is made of tetramethyl benzidine, DMSO, tetramethyl benzidine 200mg and DMSO 100ml plus double steamings Water is to 1L.
9. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: substrate solution B is by Na2HPO4, citric acid, 0.75% hydrogen peroxide urea, distilled water composition, Na2HPO414.6g, lemon Lemon acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml add distilled water to 1L, adjust pH to 7.2.
10. a kind of HCV NS3 antibody enzyme immunoassay detection method based on MIX antigen coat according to claim 1, Be characterized in that: terminate liquid is the H2SO4 solution of 2mol/L.
CN201811287913.0A 2018-10-31 2018-10-31 A kind of HCV NS3 antibody detection method based on MIX antigen coat Pending CN109444424A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811287913.0A CN109444424A (en) 2018-10-31 2018-10-31 A kind of HCV NS3 antibody detection method based on MIX antigen coat

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811287913.0A CN109444424A (en) 2018-10-31 2018-10-31 A kind of HCV NS3 antibody detection method based on MIX antigen coat

Publications (1)

Publication Number Publication Date
CN109444424A true CN109444424A (en) 2019-03-08

Family

ID=65549423

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811287913.0A Pending CN109444424A (en) 2018-10-31 2018-10-31 A kind of HCV NS3 antibody detection method based on MIX antigen coat

Country Status (1)

Country Link
CN (1) CN109444424A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020034759A (en) * 2000-11-03 2002-05-09 황규언 A detecting method of hepatitis C virus using recombinant mixed antigens derived from HCV and a diagnostic kit thereof
CN1670532A (en) * 2005-03-23 2005-09-21 北京科卫临床诊断试剂有限公司 Antibody diagnosing reagent kit for detecting HepatitisType C virus, its preparation and method for detection
CN101196518A (en) * 2006-12-07 2008-06-11 北京科美东雅生物技术有限公司 Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method
CN101551397A (en) * 2009-05-13 2009-10-07 郑州安图绿科生物工程有限公司 Reagent box for detecting nature of third type hepatitis virus antibody by using chemiluminescence method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020034759A (en) * 2000-11-03 2002-05-09 황규언 A detecting method of hepatitis C virus using recombinant mixed antigens derived from HCV and a diagnostic kit thereof
CN1670532A (en) * 2005-03-23 2005-09-21 北京科卫临床诊断试剂有限公司 Antibody diagnosing reagent kit for detecting HepatitisType C virus, its preparation and method for detection
CN101196518A (en) * 2006-12-07 2008-06-11 北京科美东雅生物技术有限公司 Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method
CN101551397A (en) * 2009-05-13 2009-10-07 郑州安图绿科生物工程有限公司 Reagent box for detecting nature of third type hepatitis virus antibody by using chemiluminescence method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
乔伟振: "丙型肝炎病毒不同基因型不同区域重组蛋白的抗原性分析及应用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *
乔伟振等: "两种不同基因型NS3 抗原在HCV感染者血清学诊断中的应用", 《中华医院感染学杂志》 *

Similar Documents

Publication Publication Date Title
Kassim et al. Use of dengue NS1 antigen for early diagnosis of dengue virus infection
CN113009154B (en) Novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof
CN111929433B (en) African swine fever virus antibody ELISA detection kit and preparation method thereof
CN101735319B (en) Monoclonal antibody against GP73 protein, preparation method and application thereof
CN110927390A (en) ELISA method and kit for detecting African swine fever CD2v protein antibody and application
CN105675866B (en) For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit
CN116023506B (en) ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof
CN115873079A (en) Canine infectious hepatitis virus hexon protein antigen, truncation and application thereof
Qiu et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections
US20210356464A1 (en) Serological assays for diagnosing or confirming covid-19 virus infections
BRPI0200986B1 (en) METHOD FOR DETERMINING HCV PRESENCE IN A SAMPLE
CN117229367A (en) African swine fever virus epitope polypeptide and ELISA antibody detection kit
CN106771218B (en) It is a kind of detect HIV-1 p24 antigen kit and its application
US20040152070A1 (en) Method of detection of HCV antibodies in combination assay or sole antibody assay
CN115078737B (en) Kit for detecting immune plasma titer of rabies, and preparation method and application thereof
CN109444424A (en) A kind of HCV NS3 antibody detection method based on MIX antigen coat
Kleemola et al. Evaluation of an antigen-capture enzyme immunoassay for rapid diagnosis of Mycoplasma pneumoniae infection
Kahane et al. Detection of IgG antibodies specific for measles virus by enzyme-linked immunosorbent assay (ELISA)
CN108303533A (en) A kind of pre-coated detection method of I type D antigens of poliovirus and its detection kit and application
CN104165998B (en) PRRSV genetic marker vaccine strain ELISA differential diagnosis kit and methods and applications
CN105866428A (en) Immunodetection kit for detecting EV71 virus IgG antibody and preparation method thereof
CN113721032B (en) Novel quantitative detection kit for neutralizing antibodies of coronaviruses and application of kit
CN117777259B (en) Antigen composition for detecting tuberculosis infection, kit and application thereof
CN112444626B (en) African swine fever virus antibody ELISA detection kit and preparation method thereof
CN114705857B (en) Microporous plate type chemiluminescence detection kit for pig foot-and-mouth disease virus O-type and A-type antibodies and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20190308

RJ01 Rejection of invention patent application after publication