CN105004856A - Porcine circovirus II antibody detection ELISA kit - Google Patents
Porcine circovirus II antibody detection ELISA kit Download PDFInfo
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Abstract
The invention provides a porcine circovirus II antibody detection ELISA kit. The kit comprises an ELISA plate coated with a purified escherichia coli-expressed porcine circovirus II Cap protein as an antigen, positive contrast serum, negative contrast serum, a sample diluent, an enzyme labeling conjugate, a washing solution, a substrate solution A, a substrate solution B and a stopping solution. An indirect ELISA method is adopted, the coated plate is coated with the prokaryotic expressed Cap protein as an antigen and antigen purity is high. The ELISA detection kit prepared from the coated plate with the antigen has high sensitivity, specificity and repeatability.
Description
Technical field
The invention belongs to vaccine detection technique field, be specifically related to a kind of porcine circovirus 2 type antibody and detect ELISA kit.
Background technology
Porcine circovirus desease 2 type can cause inhibitive ability of immunity disease.This virus can bring out multiple virus, the mixed infection of bacterium and scabies secondary infection, causes huge economic loss, particularly just day by day receive publicity to the research of its vaccine to the research of this disease to pig industry.Vaccine is as the Main Means of prevention and control PCV2, and domestic and international researcher is is researching and developing various vaccine, and up to the present, part vaccine obtains successfully in clinical testing, and the application of input had, other candidate vaccines are also just in active research.
But, due to vaccine preparation batch on difference, cause the vaccine of different batches to there is difference in effect.At present in the evaluation of vaccine, fine jade expansion titration is carried out after vaccine is dissociated, the rear antibody test of immunity is carried out to vaccine exactly in the most direct aspect, but due to SPF pig or the more difficult acquisition of the negative pig of PCV2 antigen-antibody, be necessary to provide a kind of effective alternative animal to improve the sensitivity of detection.
Summary of the invention
The object of this invention is to provide a kind of porcine circovirus 2 type antibody and detect ELISA kit, the present invention adopts indirect ELISA, wrap wrap by plate quilt antigen be the Cap protein of prokaryotic expression, antigen purity is high, bag antigen coated thus the ELISA detection kit set up by plate, there is higher sensitivity, specificity, repeatability.
Porcine circovirus 2 type antibody of the present invention detects ELISA kit, includes after the carrying Cap gene of porcine circovirus type 2 purifying of Bacillus coli expression as antigen coated elisa plate, positive control serum, negative control sera, sample diluting liquid, enzyme labeling bond, cleansing solution, substrate solution A, substrate solution B and stop buffer.
Wherein positive control serum expands with the fine jade that porcine circovirus 2 type inactivated vaccine SH strain inoculation healthy guinea pig is made the serum that antibody titer is not less than 1:32;
Negative control sera is the serum of healthy guinea pig;
Described sample diluting liquid is the phage solution of the bacillus coli BL21 (E.coli BL21) of the PBS solution dilution using 0.01M pH7.2, and its total protein content is 1.2mg/ml.
Enzyme labeling bond is (example: get 10 μ l enzyme labelled antibodies, add enzyme labelled antibody dilution 80ml) that the goat-anti guinea pig antibodies enzyme labelled antibody diluted 8000 of horseradish peroxidase-labeled is doubly made.
Described cleansing solution is by 14.9g KH
2pO
4, 79.1g K
2hPO
43H
2o, 50.0mlTritonx-100,1.0g NaN
3, 8.0g NaCl is dissolved in 500ml water, adjusts pH 7.3, is finally settled to 1L.
Described substrate solution A is by 4.2g citric acid, 13.6g sodium acetate, 0.5g peroxide is crossed urea and is dissolved in 100ml deionized water, is settled to 1L, and adjust pH is made to 5.0;
Substrate solution B, is be dissolved in 50ml dimethyl sulfoxide (DMSO) by 1g TMB, adds 100ml formaldehyde again, 16.0g polyvinylpyrrolidone, be settled to 1L complete preparation after dissolving with deionized water after dissolving.
Described stop buffer is the sulfuric acid solution of 2mol/L.
The wherein preparation method of elisa plate, that the Cap protein antigen pH 9.5 of purifying, the carbonate buffer solution of 0.05mol/L are diluted to 25 μ g/ml, be added to 96 hole bags by plate, 100 μ l/ holes, put 4 DEG C and spend the night, after washing 3 times with cleansing solution, add 1%BSA 0.01mol/LpH 7.2PBS close spend the night, get rid of deblocking liquid, under room epidemic disaster 40%, natural drying 24-48 hour completes preparation.
Embodiment
Embodiment 1 porcine circovirus 2 type guinea pig antibodies detects the preparation and application of ELISA kit
1. the preparation method of porcine circovirus 2 type guinea pig antibodies detection kit:
The preparation of PCV2 antigen and purifying: the dust Xi Shi colibacillus engineering of expressing restructuring carrying Cap gene of porcine circovirus type 2 is inoculated 2000 ml shake flasks by 1% ~ 2% inoculum concentration and cultivates, nutrient culture media is prepared, with 1mol/L sodium hydroxide solution adjust ph to 7.0 ~ 7.2 by volume 35%.Add the kanamycin sulfate injection liquid of 10% in the ratio of cumulative volume 0.1%, 35 ~ 36 DEG C of cultivations, treat the OD of bacterium liquid
600nmwhen value reaches between 0.8-1.0, add the 0.5mol/L alpha-lactose solution of sterilizing, make its final concentration reach 0.03mol/L, abduction delivering 5 hours.Collected by centrifugation thalline.By resuspended for the thalline physiological saline collected, ultrasound wave breaks bacterium, first carry out ammonium sulfate precipitation, then desalting column chromatography is carried out, the PCV2 protein liquid of redissolution is splined on SephadexG25 chromatographic column, with 0.05mol/L phosphate buffer wash-out, collects and spill protein peak, finally carry out ion-exchange chromatography separation, collect the eluting peak of antigen protein.
Bag is by the preparation of plate: the PCV2 Cap protein antigen pH 9.50.05mol/L carbonate buffer solution of purifying is diluted to 25 μ g/ml, be added to 96 hole bags by plate, 100 μ l/ holes, put 4 DEG C to spend the night, after washing 3 times with cleansing solution, add 1%BSA 0.01mol/L pH 7.2PBS close spend the night, get rid of deblocking liquid, under room epidemic disaster 40%, natural drying 24-48 hour, packaging.
The preparation of the enzyme labelled antibody (No. 1 liquid) of working concentration: the goat-anti guinea pig antibodies enzyme labelled antibody diluted 8000 times (example: get 10 μ l enzyme labelled antibodies, add enzyme labelled antibody dilution 80ml) of getting the horseradish peroxidase-labeled that BETHYL company produces.
The preparation of cleansing solution (No. 2 liquid): be by 14.9g KH
2pO
4, 79.1g K
2hPO
4.
3H2O, 50.0ml Tritonx-100,1.0g NaN
3, 8.0g NaCl is dissolved in 500ml water, adjusts pH 7.3, is finally settled to 1L.
The preparation of substrate nitrite ion: by 4.2g citric acid, 13.6g sodium acetate, 0.5g peroxide is crossed urea and is dissolved in 100ml deionized water, is settled to 1L, and adjust pH, to 5.0, is substrate A liquid (No. 3 liquid); 1g TMB is dissolved in 50ml dimethyl sulfoxide (DMSO), after dissolving, adds 100ml formaldehyde again, 16.0g polyvinylpyrrolidone, after dissolving, be settled to 1L with deionized water, be substrate B liquid (No. 4 liquid)
The preparation of sample diluting liquid (No. 5 liquid): picking bacillus coli BL21 (E.coli BL21) single bacterium colony from LB culture plate, transfer in the test tube containing LB nutrient solution, 37 DEG C of overnight incubation, prepare saturated bacterium liquid.Saturated bacterium liquid is transferred in the LB nutrient culture media of 250ml, inoculum concentration about 5%, cultivates 8 hours.6000 revs/min centrifugal 15 minutes.Thalline is dissolved in 15mlPBS.The bacterium lyolysis of dissolving is frozen, ultrasonic treatment.Put 6000 revs/min, centrifugal 15 minutes, collect supernatant.Protein content (detection method sees appendix 1) is measured by BCA protein content detection kit.Then broken bacterium liquid 0.01M pH7.2PBS being diluted to total protein content is 1.2mg/ml.
The preparation of stop buffer (No. 6 liquid): preparation 2mol/L sulfuric acid solution.
The preparation of positive control serum and negative control sera:
Positive control serum: the healthy guinea pig inoculating about 250g with porcine circovirus 2 type inactivated vaccine (SH strain), every leg muscle vaccinate 0.2ml.One exempts from latter 14 days, carries out two exempt from same vaccine same dose, and two exempt from blood sampling in latter 21 days, and the fine jade measuring Porcine circovirus type 2 Cap expands antibody titer.Take a blood sample when fine jade expands when antibody titer is not less than 1:32, separation of serum.
Negative control sera: with the healthy guinea pig (PCV2-ELISA antibody and PCV2-AGP negative antibody) of about 250g, blood sampling, separation of serum.
2. porcine circovirus 2 type guinea pig antibodies detects the using method of ELISA kit:
(1) from refrigerator, take out each reagent, return to room temperature (20 ~ 25 DEG C).
(2) with serum dilution, serum to be checked is 1:400 dilution (get serum 5 μ l+95.0 μ l sample diluting liquid, get 6 μ l+114 μ l sample diluting liquids after mixing).
(3) adding 100 μ l does not need the negative serum diluted in A1 and A2 hole.
(4) adding 100 μ l does not need the positive serum diluted in A3 and A4 hole.
(5) adding 100 μ l serum dilutions in A5, A6 hole, is blank.
(6) blood serum sample that 100 μ l have diluted is added to bag by plate hole.
(7) ELISA Plate is mounted 37 DEG C of incubations 30 minutes.
(8) wash: added cleansing solution 2 in plate hole to bag, with distilled water cyclic washing 5 times, thieving paper pats dry.
(9) added enzyme labeling bond 2 in plate hole to bag, placed 30 minutes for 37 DEG C.
(10) wash: repeat step (8)
(11) develop the color: add substrate solution A 2, substrate solution B 2, shakes up gently, 37 DEG C are reacted 10 minutes.
(12) every hole adds only whole liquid 2 cessation reactions.
(13) ELISA Plate is placed in microplate reader, reads the absorbance value under 450nm wavelength.
1, computing method
2, the positive criterion of testing sample
Criterion positive control serum OD
450> 0.5, negative control sera OD
450< 0.15, blank well OD
450< 0.08, experiment is set up, otherwise invalid.
Computing formula: S/N value=sample OD450nm/ negative control OD450nm average to be checked criterion:
S/N >=2.5 are positive;
2.0 < S/N < 2.5 are suspicious;
S/N≤2.0 are negative.
Embodiment 2 pig circular ring virus guinea pig antibodies detects specificity, the susceptibility of ELISA kit and carries out contrast test with fine jade expanding method
1. specific test: the PCV2 ELISA antibody assay kit of this research development, for detection kit is to the specific detection of PCV-II, detect 10 kinds of single-factor positive serums such as porcine circovirus 2 type, porcine reproductive and respiratory syndrome virus, swine fever, pseudorabies (by obtained after vaccine immunity cavy) respectively with 3 batches of kits to detect, result other positive serums except PCV2 positive serum are feminine gender, refer to table 1, show that kit has good specificity.
Table 1:3 criticizes kit detection specificity test findings
Note: 1=porcine reproductive and respiratory syndrome positive serum; 2=swine fever positive serum; 3=pseudorabies positive serum; 4=Schweineseuche positive serum; 5=pig vesicular stomatitis positive serum; 6=atrophic rhinitis positive serum; 7=pig pleuropneumonia positive serum; 8=haemophilus parasuis positive serum; 9=pig Infection of Toxoplasma Gondii positive serum; 10=swine C.psittaci positive serum; 11,12=PCV2 positive serum; 13,14=PCV2 negative serum.
2. sensitivity test: the detect titre of PCV2 ELISA antibody assay kit to 3 parts of PCV2 positive serums (fine jade expansion is tired as 1:32) of this research development is 1:12800 ~ 1:25600, test findings shows, development the sensitivity that expands than fine jade of PCV2 ELISA kit high 400 ~ 800 times, there is higher susceptibility.The results detailed in Table 2.
Table 2: kit sensitivity tests result
Note: the criterion of 1.YEBIO kit is: during S/N value >=2.5, be judged to the positive;
Be judged to suspicious during 2.0 < S/P < 2.5; Feminine gender is judged to during S/P value≤2.0.
Claims (9)
1. a porcine circovirus 2 type antibody detects ELISA kit, it is characterized in that, described kit includes after the carrying Cap gene of porcine circovirus type 2 purifying of Bacillus coli expression as antigen coated elisa plate, positive control serum, negative control sera, sample diluting liquid, enzyme labeling bond, cleansing solution, substrate solution A, substrate solution B and stop buffer.
2. kit as claimed in claim 1, is characterized in that, described positive control serum, is to expand with the fine jade that porcine circovirus 2 type inactivated vaccine SH strain inoculation healthy guinea pig is made the serum that antibody titer is not less than 1:32.
3. kit as claimed in claim 1, it is characterized in that, described negative control sera is the serum of healthy guinea pig.
4. kit as claimed in claim 1, is characterized in that, described sample diluting liquid is the phage solution of the bacillus coli BL21 of the PBS solution dilution using 0.01M pH7.2, and its total protein content is 1.2mg/ml.
5. kit as claimed in claim 1, it is characterized in that, described enzyme labeling bond is that the goat-anti guinea pig antibodies enzyme labelled antibody diluted 8000 of horseradish peroxidase-labeled is doubly made.
6. kit as claimed in claim 1, is characterized in that described cleansing solution is by 14.9gKH
2pO
4, 79.1g K
2hPO
43H
2o, 50.0ml Tritonx-100,1.0g NaN
3, 8.0g NaCl is dissolved in 500ml water, adjusts pH 7.3, is finally settled to 1L.
Described substrate solution A is by 4.2g citric acid, 13.6g sodium acetate, 0.5g peroxide is crossed urea and is dissolved in 100ml deionized water, is settled to 1L, and adjust pH is made to 5.0;
7. kit as claimed in claim 1, is characterized in that, described substrate solution B, 1gTMB is dissolved in 50ml dimethyl sulfoxide (DMSO), add 100ml formaldehyde again after dissolving, 16.0g polyvinylpyrrolidone, be settled to 1L with deionized water after dissolving and complete preparation.
8. kit as claimed in claim 1, it is characterized in that, described stop buffer is the sulfuric acid solution of 2mol/L.
9. kit as claimed in claim 1, it is characterized in that, the preparation method of described elisa plate, is that the Cap protein antigen pH 9.5 of purifying, the carbonate buffer solution of 0.05mol/L are diluted to 25 μ g/ml, be added to 96 hole bags by plate, 100 μ l/ holes, put 4 DEG C and spend the night, after washing 3 times with cleansing solution, add 1%BSA 0.01mol/L pH 7.2PBS close spend the night, get rid of deblocking liquid, under room epidemic disaster 40%, natural drying 24-48 hour completes preparation.
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| CN201510405952.6A CN105004856A (en) | 2015-07-10 | 2015-07-10 | Porcine circovirus II antibody detection ELISA kit |
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Cited By (5)
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| CN106841609A (en) * | 2017-01-12 | 2017-06-13 | 江苏南农高科技股份有限公司 | A kind of porcine circovirus 2 type blocking ELISA antibody assay kits and preparation method thereof |
| CN107037212A (en) * | 2017-04-20 | 2017-08-11 | 江苏省农业科学院 | Porcine circovirus 2 type antigen immue quantitative detection reagent box |
| CN110208542A (en) * | 2019-05-09 | 2019-09-06 | 江苏南农高科技股份有限公司 | A kind of the ELISA antigen detection kit and its detection method of porcine circovirus 2 type |
| CN112345767A (en) * | 2020-10-23 | 2021-02-09 | 军事科学院军事医学研究院军事兽医研究所 | Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody |
| CN113791216A (en) * | 2021-08-31 | 2021-12-14 | 石家庄和亚生物技术有限公司 | ELISA detection method for detecting human serum candida glabrata enolase IgG antibody |
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| CN110208542A (en) * | 2019-05-09 | 2019-09-06 | 江苏南农高科技股份有限公司 | A kind of the ELISA antigen detection kit and its detection method of porcine circovirus 2 type |
| CN112345767A (en) * | 2020-10-23 | 2021-02-09 | 军事科学院军事医学研究院军事兽医研究所 | Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody |
| CN113791216A (en) * | 2021-08-31 | 2021-12-14 | 石家庄和亚生物技术有限公司 | ELISA detection method for detecting human serum candida glabrata enolase IgG antibody |
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