CN102175853A - ELISA (enzyme linked immunosorbent assay) kit for detecting pig progenitive and respiratory syndrome (PRRS) antibody - Google Patents
ELISA (enzyme linked immunosorbent assay) kit for detecting pig progenitive and respiratory syndrome (PRRS) antibody Download PDFInfo
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Abstract
The invention provides an ELISA (enzyme linked immunosorbent assay) kit for detecting PRRS antibody and application thereof, belonging to the field of biotechnology. The kit comprises an ELISA slat with recombinant GST-GP5 (glutathione transferase-glycoproteins) protein as coating antigen, a PBS (polydiethylene glycol sebacate) solution, a blood serum diluted solution, a rabbit anti-pig enzyme-labelled bi-antibody, a colour-developing substrate, a terminating liquor, PRRS positive blood serum, PRRS negative blood serum and the like. The kit provided by the invention is used for detecting PRRS antibody; after the indexes like specificity, sensitivity, repeatability and the like of a detection method are inspected, the method is used for detecting serum samples; and the detection coincidence rate with the ELISA kit in the prior art is 90%. The kit provided by the invention has the advantages that the operations are convenient, the repeatability is good, and the kit is suitable for being popularized and applied; a reliable measure for fast detecting PRRSV (pig progenitive and respiratory syndrome virus) antibody is supplied clinically.
Description
Technical field
The present invention relates to a kind of ELISA kit, be specifically related to detect the ELISA kit and the application thereof of porcine reproductive and respiratory syndrome antibody, belong to biological technical field.
Background technology
Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS) mainly cause breeding difficultys such as in-pig premature labor, miscarriage, stillborn foetus, mummy tire and the weak piglet of product, piglet and growing and fattening pigs mainly show as cough, expiratory dyspnea.First in U.S.'s outburst (Keffaber K K., 1989), the countries and regions of rapid each pig industry prosperity of extend over the entire globe brought enormous economic loss for the pig industry in the world to this disease subsequently in 1987.This sick cause of disease is porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV), belong to shell type virales Arteriviridae (Arteriviridae) arteritis virus member (Cavanagh, 1997; Snijderand Meulenberg, 1998).According to the difference of viral genome nucleotide sequence, it is divided into North America type and two genotype of Europe class, PRRSV comprises 8 open reading frame.Membrane glycoprotein GP5 by the ORF5 gene code approximately is made up of 200 amino acid, and the molecular weight size is about 25kD, is the main immunogenic protein of virus.People have not only carried out a large amount of explorations on the immunological characteristic of this albumen of research in recent years, and have also given very big concern on to the research of its antigenic characteristic always.
There is the generation of this disease in China in reported first in 1996, and proves (Guo Baoqing, 1996 due to the type PRRSV virus strain infection of North America; Yang Hanchun, 1997)." high-pathogenicity blue ear disease " that summer in 2006 occurred causes great harm and great economic loss for the pig industry of China.Morbidity this time and classical strains in the past are very different, and the pig of different days, different cultivars all can infect, and break with tremendous force, fall ill suddenly, propagate rapidly, mortality ratio is high, cure rate is low, has had a strong impact on the production safety of increasing peasant income and pig industry.
Use various immune serums learn a skill to eqpidemic disease make accurately, diagnosis rapidly is prevention and controls one of these sick important measures.At present, what be most widely used at the diagnostic method of PRRS is the ELISA method of setting up as envelope antigen with totivirus, as: the indirect ELISA reagent kit that American I DEXX company produces.Because virus needs to obtain and purified virus truly is very difficult by breeding in a large number in the cell culture.The testing cost of this kit is too high, and expensive testing cost makes many grass-roots units be difficult to accept, and makes it be unfavorable for the promotion and application of basic unit.Along with the appearance of subunit vaccine and nucleic acid vaccine research, can provide the kit of corresponding protein bag quilt to also become necessary.
Summary of the invention
At above-mentioned deficiency and needs, the invention provides a kind of ELISA kit and application thereof that detects porcine reproductive and respiratory syndrome antibody, be used for detection of antibodies.This kit is simple to operate, stability and good reproducibility, detect with low cost, good market prospects.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of detection porcine reproductive and respiratory syndrome (porcine reproductive andrespiratory syndrome, PRRS) the ELISA kit of antibody comprises following component:
(1) elisa plate bar: as envelope antigen, each kit is equipped with 4 blocks of laths through 1% (M/V) bovine serum albumin(BSA) (BSA) sealing, packs in 4 ℃ of preservations with packaging bag with the reorganization GP5 albumen of porcine reproductive and respiratory syndrome virus GP5 protein gene;
(2) cleansing solution: pH7.4 phosphate buffer (PBS) solution of 1 times of concentration;
(3) serum dilution: 100 milliliters of 1% (M/V) BSA;
(4) chromogenic substrate: added H
2O
2100 milliliters of tetramethyl benzidine (TMB) solution, contain 30% (M/V) concentration H among wherein per 10 ml soln TMB
2O
215 μ L;
(5) the anti-pig ELIAS secondary antibody of rabbit: used serum dilution 1% (M/V) BSA to be diluted to 100 milliliters of the anti-pig ELIAS secondary antibody of 1: 800 rabbit of working concentration;
(6) stop buffer: 2M H
2SO
4100 milliliters of solution;
(7) PRRS positive serum: 5 milliliters;
(8) PRRS negative serum: 5 milliliters.
ELISA kit of the present invention can be used for detecting porcine reproductive and respiratory syndrome antibody, and it is as follows mainly to detect step:
(1) adds blood serum sample
After with serum dilution 1% (M/V) BSA tested serum being done 1: 50 times of dilution, every hole adds 100 μ L, and each blood serum sample adds two holes, sets up PRRS positive and negative serum simultaneously in contrast, and each adds two holes; Act on 45min under the room temperature, discard the liquid in the reacting hole; The cleansing solution of each hole with about 200 μ L fully cleaned 3-5 time, continue 4-5min at every turn, after after the washing liquid in the reacting hole being removed at every turn, being removed cleansing solution for the last time, remove residual liquid;
(2) add the anti-pig ELIAS secondary antibody of rabbit:
Every hole adds the anti-pig ELIAS secondary antibody of 100 μ L rabbits, and room temperature effect 45min washs 3-5 time;
(3) add chromogenic substrate:
Every hole adds 100 μ L and has added H
2O
2TMB solution, act on 15min under the room temperature;
(4) cessation reaction:
In every hole, add 100 μ L 2M H
2SO
4The stop buffer cessation reaction;
(5) measure the OD value with microplate reader
Under the 450nm wavelength, measure each hole absorbance OD value, result of determination subsequently;
(6) result judges
Positive when sample OD450nm 〉=0.21,＜0.21 sample is negative.
The ELISA kit envelope antigen that the present invention detects porcine reproductive and respiratory syndrome antibody is the prokaryotic expression product of porcine reproductive and respiratory syndrome virus GP5 albumen, its advantage GP5 that is to recombinate is protein stabilized, be easy to obtain and purifying, with this albumen of purifying as envelope antigen, its concentration is easy to determine and control, for a large amount of productions of detection kit provide strong guarantee; Kit of the present invention compared with prior art, under the prerequisite that guarantees specificity, susceptibility, with the detection coincidence rate of prior art (as the ELISA kit of IDEXX company) be 90%.Kit of the present invention is used for detection of antibodies, it is simple to operate, stability and good reproducibility, detect with low cost, prior art detects the expense of every part of blood serum sample about 50 yuan, and testing cost of the present invention greatly reduces the detection cost about 30 yuan, be suitable for promoting, application prospect is good.
Description of drawings
Fig. 1 GST-GP5 melts the expression of albumen at Escherichia coli BL-21.
GST-GP5 behind Fig. 2 purifying melts albumen
, it should be understood that described embodiment only is for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
Embodiment
1. the suitableeest bag of antigen determining by concentration and serum optimal dilution
With coating buffer with the PRRSV of purifying reorganization GP5 albumen made gradient dilution 1: 160 (20 μ g/mL), 1: 320,1: 640,1: 1280, bag is by 96 hole ELISA Plate, every hole adds 100 μ L.Serum was diluted to 1: 200 from 1: 25,1: 50, formed square formation, operated with EL ISA method.Spectrophotometric determination OD450nm value.Select positive serum OD450nm value about 1 and positive OD450nm value/antigen coated concentration and antibody dilution during negative OD450nm value (being P/N) ratio maximum is best effort concentration.
The square formation titration results shows, when the bag of reorganization GP5 albumen is 1: 640 (5 μ g/mL) by concentration, when serum dilution is 1: 50, the P value was about 1, and P/N ratio is 15.667 to the maximum.Therefore the bag of determining reorganization GP5 albumen is 5 μ g/mL by optium concentration, and the optimum dilution degree of serum is 1: 50.
Table 1ELISA square formation titration results
2. the selection of confining liquid
Use 5% skimmed milk, 1%BSA, 1.5% gelatin and 5% calf serum (FCS) as confining liquid respectively, in room temperature sealing 2h, under the identical situation of other conditions and method of operating, known positive and negative serum is carried out ELISA to be detected, the serum of every kind of confining liquid dilution repeats four holes, estimates its sealing effect by the OD value.The result determines that 1%BSA is as confining liquid.
3. sealing condition determines
Under the identical situation of other conditions, respectively at 37 ℃ and room temperature sealing 1h, 2h, 3h, 4h, the back is used known positive and negative serum and is detected, to determining off-period and temperature with selected confining liquid.The result shows: no matter room temperature or 37 ℃, sealing 2h just can reach good effect.
4. the selection of serum dilution
Respectively with PBST, 1%BSA, 3% calf serum as the known feminine gender of diluted, positive serum, each concentration dilution liquid repeats four holes, under the identical situation of other conditions, carrying out ELISA measures, calculate and respectively organize the P/N value, hang down with this value maximum and negative serum OD value and select serum dilution.The serum dilution of determining is 1%BSA.
5. serum action time determines
With selected serum dilution with known positive and negative serum do be diluted to optimum diluting multiple at 1: 50 after, respectively at room temperature and 37 ℃ of following 15min, 30min, 45min, 60min of acting on, under the identical situation of other conditions and method of operating, carry out ELISA and detect, get lower one group of P/N maximum and negative serum background and be defined as the best use of time.The result determines that the time of serum effect is room temperature and 37 ℃ of following 45min.
6. ELIAS secondary antibody best effort concentration determines
With antigen diluent is 5 μ g/mL bag by the polystyrene micro-reaction plate, the PRRS positive serum is by dilution in 1: 50, the anti-pig ELIAS secondary antibody of rabbit was according to 1: 200,1: 400,1: 800,1: 1600 totally 4 gradient dilutions, 37 ℃ of 45min carry out indirect ELISA and detect.Measure the OD450nm value, the P/N value is the suitableeest working concentration of ELIAS secondary antibody when maximum.
The result shows: when the ELIAS secondary antibody dilutability was 1: 800, positive serum OD450nm value was near 1, and negative serum OD450nm value is lower, and the P/N value is maximum.Therefore determine that ELIAS secondary antibody best effort concentration is 1: 800.
Table 2 ELIAS secondary antibody best effort concentration is determined
7. the selection of substrate and stop buffer
Under the identical situation of other conditions, in using TMB (OD respectively under the room temperature and under 37 ℃
450), OPD (OD
490) as substrate, detect known positive and negative serum, add 2M H respectively
2SO
4, 2M HCL, 0.5%HF be as stop buffer, every 10min value of reading on microplate reader, calculates its P/N ratio in stopping back 30min, selects suitable substrate.The result determines that TMB is a chromogenic substrate, and stop buffer is 2M H
2SO
4
8. the selection of substrate-function time
Adopt known positive and negative serum, use the substrate of determining, respectively at behind room temperature and 37 ℃ effect 10min, 15min, 20min, 25min, the 30min with the stop buffer cessation reaction of determining, in stopping the value of reading on the inherent microplate reader of back 30min, with the time and the operative temperature of definite substrate-function.Be room temperature 15min the action time of determining substrate TMB at last.
9. criterion determines
With 10 parts of known after testing PRRS negative serums collecting according to dilution in 1: 50 after, detect according to the ELISA program of having set up, measure every part of serum OD450nm value.According to Principle of Statistics, during the OD450nm of sample 〉=negative serum OD mean value (X)+3 * standard variance (SD), be judged to be the positive, on the contrary negative.
The X=0.1695 of 10 parts of negative serum OD450nm values as calculated, SD=0.0143.So ELISA negative and positive critical value=0.1695+3 * 0.0143=0.2125.Therefore, positive when sample OD450nm 〉=0.21,＜0.21 sample is negative.
10. reorganization GP5 albumen---determining of ELISA running program
Operate by above every definite optimal conditions, obtain the process optimization program of this method: take out to have wrapped and also sealed good elisa plate bar, with sample diluting liquid tested serum is done dilution in 1: 50, in the adding sample well, room temperature effect 45min discards the liquid in the reacting hole; The cleansing solution of each hole with about 200 μ L fully cleaned 3-5 time, after each washing, the liquid in the reacting hole is removed; After removing cleansing solution for the last time, on thieving paper, pat, remove residual liquid; Every hole adds the anti-pig ELIAS secondary antibody of 100 μ L rabbits, and room temperature effect 45min washs 3-5 time.Add 100 μ L tmb substrate solution in every hole, room temperature lucifuge effect 15min, the stop buffer of adding 100 μ L in every hole, cessation reaction.Measure each hole absorbance OD value with microplate reader under the 450nm wavelength, the value of reading is calculated and result of determination.
11. determining of envelope antigen storage life
Purifying protein is sealed with packaging bag with best effort concentration bag quilt and after sealing, places 4 ℃ of preservations, takes out every other month later on and detects according to the method for being set up with known negative, positive serum.Detect altogether 6 times.The result shows that the reorganization GP5 albumen preservation of wrapping quilt still can be used for detecting in 6 months.
Obtaining of embodiment 2 reorganization GP5 albumen
Design a pair of primer (upstream primer: TTCGAAT TCAGCAACAACAACAGCTCTCAT according to 32-200 amino acid in the PRRSV GP5 albumen; Downstream primer: TCGCTCGAGGAGACGACCCCATTG) amplification PRRSV GP5 protein gene sequence (concrete sequence is seen sequence table), and at its 5 ' end and 3 ' hold and to introduce EcoRI and Xho I restriction enzyme site respectively, clone in cutting among the prokaryotic expression carrier pGEX-6p-1 that carries glutathione transferase of processing through same enzyme, identify correct expression vector through order-checking, with IPTG it is carried out abduction delivering, adopt 12% polyacrylamide gel to carry out the SDS-PAGE electrophoresis observation, the results are shown in Figure 1.The albumen disposal route of a large amount of abduction deliverings is with reference to GST amalgamation and expression system operation handbook, and the GST-GP5 recombinant protein SDS-PAGE electrophoresis result behind the purifying is seen Fig. 2.Embodiment 3 detects the ELISA kit of porcine reproductive and respiratory syndrome antibody, comprises following component:
(1) elisa plate bar: as envelope antigen, each kit is equipped with 4 blocks of laths through 1% (M/V) BSA (bovine serum albumin(BSA)) sealing, packs in 4 ℃ of preservations with packaging bag with the reorganization GP5 albumen of porcine reproductive and respiratory syndrome virus GP5 protein gene; (2) cleansing solution: the pH7.4PBS of 1 times of concentration (phosphate buffer) solution; (3) serum dilution: 100 milliliters of 1% (M/V) BSA; (4) chromogenic substrate: added H
2O
2100 milliliters of TMB (tetramethyl benzidine) solution, contain 30% (M/V) concentration H among wherein per 10 ml soln TMB
2O
215 μ L; (5) the anti-pig ELIAS secondary antibody of rabbit: used serum dilution 1% (M/V) BSA to be diluted to 100 milliliters of the anti-pig ELIAS secondary antibody of 1: 800 rabbit of working concentration; (6) stop buffer: 2MH
2SO
4100 milliliters of solution; (7) known PRRS positive serum: 5 milliliters; (8) known PRRS negative serum: 5 milliliters.
The main method of operating that the ELISA kit that embodiment 4 detects porcine reproductive and respiratory syndrome antibody is used to detect is as follows:
(1) add blood serum sample: with serum dilution tested serum is done dilution in 1: 50, add in the sample well, every hole 100 μ L add two holes altogether, discard the liquid in the reacting hole behind room temperature effect 45min; The cleansing solution of each hole with about 200 μ L fully cleaned 5 times, after each washing, the liquid in the reacting hole is removed; After removing cleansing solution for the last time, remove residual liquid; (2) add the anti-pig ELIAS secondary antibody of rabbit: every hole adds the anti-pig ELIAS secondary antibody of 100 μ L rabbits, and room temperature effect 45min washs 4 times; (3) add chromogenic substrate: add 100 μ L tmb substrate solution, room temperature lucifuge effect 15min in every hole; (4) cessation reaction: in every hole, add the stop buffer of 100 μ L, cessation reaction; (5) measure the OD value with microplate reader: under the 450nm wavelength, measure each hole absorbance OD value and result of determination.(6) result judges: positive when sample OD450nm 〉=0.21,＜0.21 sample is negative.
The test of embodiment 5 specificitys
Under identical conditions, with the indirect ELISA method of having set up (kit of embodiment 3, the detection method of embodiment 4) the known positive serum of known PRRS positive serum, PRRS negative serum, swine fever, pig circular ring virus II type, pig epidemic diarrhea, pig parvoviral, the common eqpidemic disease of pseudoabies 6 boars is carried out blind check; The result that will draw compares with known results again.
The result shows that the ratio that has only PRRS positive serum sample OD450nm value and PRRS negative serum sample OD450nm is much larger than 0.21, and the ratio of all the other sample OD450nm values and PRRS negative serum sample OD450nm is all less than 0.21.This presentation of results kit of the present invention is used to detect and has good specificity.
Embodiment 6 replica tests
Revision test in batch: get 5 parts of positives and 5 parts of PRRS serum that negative antibody horizontal is different, the ELISA method of setting up according to the present invention in a collection of test (kit of embodiment 3, the detection method of embodiment 4) is tested, and every duplicate samples repeats 5 times.
The result shows that the OD450nm coefficient of variation of 10 parts of serum is 3.1%～6.3%, and degree of variation is very little, has better repeatability.
Revision test between batch: get 5 parts of positives and 5 parts of different PRRS serum of negative antibody horizontal, 10 discontinuous working days, carry out the repetition ELISA test (kit of embodiment 3, the detection method of embodiment 4), the result shows, the OD450nm coefficient of variation of same sample is 5.6%～9.9%, and degree of variation is little, has better repeatability.
Embodiment 7 contrast tests
IDEXX detection kit with GP5ELISA kit test method of setting up among the present invention (kit of embodiment 3, the detection method of embodiment 4) and u s company detects 40 parts of porcine blood serum simultaneously, relatively both coincidence rates.
The result shows, 22 parts of serum of GP5ELISA test positive, IDEXX kit testing result is also all positive, and remaining 18 parts of serum sample is negative with the result that the GP5ELISA kit detects, and 16 parts of the results that the IDEXX kit detects are negative, and coincidence rate is 90%.
Embodiment 8 clinical practices
With the GP5ELISA method (kit of setting up of embodiment 3, the detection method of embodiment 4) (150 parts of sow serum wherein of 650 part serum in the serum sample that suburbs, Tangshan pig farm 2008-2009 is gathered, 500 parts of piglet serum) detect, the results are shown in Table 3.
Table 3 clinical practice result
Claims (2)
1. detect porcine reproductive and respiratory syndrome (porcine reproductive and respiratorysyndrome, PRRS) the ELISA kit of antibody comprises following component:
(1) elisa plate bar: as envelope antigen, each kit is equipped with 4 blocks of laths through 1% (M/V) bovine serum albumin(BSA) (BSA) sealing, packs in 4 ℃ of preservations with packaging bag with the reorganization GP5 albumen of porcine reproductive and respiratory syndrome virus GP5 protein gene;
(2) cleansing solution: pH7.4 phosphate buffer (PBS) solution of 1 times of concentration;
(3) serum dilution: 100 milliliters of 1% (M/V) BSA;
(4) chromogenic substrate: added H
2O
2100 milliliters of tetramethyl benzidine (TMB) solution, contain 30% (M/V) concentration H among wherein per 10 ml soln TMB
2O
215 μ L;
(5) the anti-pig ELIAS secondary antibody of rabbit: used serum dilution 1% (M/V) BSA to be diluted to 100 milliliters of the anti-pig ELIAS secondary antibody of 1: 800 rabbit of working concentration;
(6) stop buffer: 2M H
2SO
4100 milliliters of solution;
(7) PRRS positive serum: 5 milliliters;
(8) PRRS negative serum: 5 milliliters.
2. the application of the described ELISA kit of claim 1 in detecting porcine reproductive and respiratory syndrome antibody, main operational steps is as follows:
(1) adds blood serum sample
After with serum dilution 1% (M/V) BSA tested serum being done 1: 50 times of dilution, every hole adds 100 μ L, and each blood serum sample adds two holes, sets up PRRS positive and negative serum simultaneously in contrast, and each adds two holes; Effect is 45 minutes under the room temperature, discards the liquid in the reacting hole; The cleansing solution of each hole with about 200 μ L fully cleaned 3-5 time, continue 4-5 minute at every turn, after after the washing liquid in the reacting hole being removed at every turn, being removed cleansing solution for the last time, remove residual liquid;
(2) add the anti-pig ELIAS secondary antibody of rabbit:
Every hole adds the anti-pig ELIAS secondary antibody of 100 μ L rabbits, and room temperature effect 45 minutes is washed 3-5 time;
(3) add chromogenic substrate:
Every hole adds 100 μ L and has added H
2O
2TMB solution, under the room temperature effect 15 minutes;
(4) cessation reaction:
In every hole, add 100 μ L 2M H
2SO
4The stop buffer cessation reaction;
(5) measure the OD value with microplate reader
Under the 450nm wavelength, measure each hole absorbance OD value, result of determination subsequently;
(6) result judges
Positive when sample OD450nm 〉=0.21,＜0.21 sample is negative.
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| CN103044544A (en) * | 2011-10-17 | 2013-04-17 | 华中农业大学 | ELISA (enzyme-linked immunosorbent assay) kit for detecting highly pathogenic PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof |
| CN103336114A (en) * | 2013-06-28 | 2013-10-02 | 无锡同心塑料制品有限公司 | ELISA (Enzyme Linked Immuno-Sorbent Assay) kit for detecting porcine reproductive and respiratory syndrome |
| CN103364551A (en) * | 2012-04-01 | 2013-10-23 | 武汉中博生物股份有限公司 | ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as preparation method and application of ELISA detection kit |
| CN104459160A (en) * | 2014-12-30 | 2015-03-25 | 洛阳普莱柯万泰生物技术有限公司 | Kit and preparation method and application method thereof |
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