CN104330558B - The method of Porcine epidemic diarrhea virus Specific IgA antibody in detection pig blood - Google Patents
The method of Porcine epidemic diarrhea virus Specific IgA antibody in detection pig blood Download PDFInfo
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Abstract
The invention discloses and a kind of detect the method for Porcine epidemic diarrhea virus specific antibody IgA in pig blood.Method step is: 1) preparation and the 96 hole polystyrene Sptting plates of Porcine epidemic diarrhea virus antigen are coated；2) gather the blood of sow to be measured, obtain serum one after process and resist；3) by after anti-for serum one dilution, adding on antigen coated Sptting plate by number, 37 DEG C of saturated humidities are reacted 1 hour, washing；4) after the goat-anti pig IgA antibody of horseradish peroxidase-labeled being diluted, being sequentially added on Sptting plate, 37 DEG C of saturated humidity lucifuges are reacted 2 hours, washing；5) by TMB developer, being sequentially added on Sptting plate, lucifuge is reacted 25 minutes, adds stop buffer；6) put in microplate reader, read OD650 data, it is determined that result.The present invention is applicable to the investigation of swinery large-area porcine epizootic diarrhea IgA antibody.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method background technology detecting Porcine epidemic diarrhea virus Specific IgA antibody in pig blood.
Background technology
Porcine epizootic diarrhea is by Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea
Virus, PEDV) a kind of acute infectious intestinal disease of pig of causing, with watery diarrhea, vomit and dehydration is characterized.Its pathological characters shows as dilatation of intestine, content is thin, in yellow, cystose, intestinal wall relaxes, and lacks flexibility, and thinning have transparent feel, intestinal mucosa fine hair severe atrophy, gastric mucosa flushing is congested or mottled hemorrhage, and gastric content is foresythia and is mixed with a large amount of milky ziega (or cotton-shaped small pieces), very harmful to pig industry.
Porcine epidemic diarrhea virus per os and nose enter small intestinal after infecting, can in swinery sustainable existence, the pig at various ages is the most susceptible.The sickness rate of suckling pig, feeder pig and growing and fattening pigs is up to 100%, and especially serious with suckling pig and prognosis mala, majority was not resistant to and dead.Although growing and fattening pigs sickness rate is higher, but mortality rate is relatively low.The sickness rate of sow is 15%～90%.Primary disease is the most multiple, and summer also can occur.It is strong that this disease has infectiousness, and morbidity is rapid, piglet mortality rate high.Within 1973, occur in China Shanghai first.2006, successively repeatedly in China's many ground eruption and prevalence, cause heavy economic losses to pig industry.
Pig is after infecting PEDV, and the immunoglobulin played a major role in immunology is IgG and IgA.First PEDV excites mucosa Local Humoral Immunity to react, and can stimulate the most again general immunity；Mucosa local immunity is based on IgA, and systemic humoral immune is based on IgG.The molecular weight of IgG is bigger, it is not easy to entering enteric cavity through intestinal wall, they are poor to the resistance of gastric acid and digestive enzyme.IgA is also secretory antibody, molecular weight, and they play a major role in mucosa-immune, can resist the digestion of gastric acid and digestive enzyme, and have affinity interaction with intestinal mucosa so IgA plays an important role in intestinal.
Without immunoglobulin in body during pig birth, by colostrum adaptive immune antibody after birth.Immunity Globulin of Colostrums is mainly IgG, also has the IgA of volume, and newborn piglet enters circulation by enteric epithelium after sucking colostrum, so provides serum antibody as sow for newborn piglet.So, at Piglet at Lactation Period, entering gastral virus is to play main protective effect by constantly neutralizing in intestinal with the antibody in breast milk.And the antibody originally entering body-internal-circulation works the most secondary by enteric cavity and the virus entered in digestive tract again, this immune mechanism is exactly passive immunity, also referred to as milk immunity.
At present, owing in week old, the immune system of piglet own is the most fully grown, so immune pregnancy sow can only be passed through, to making piglet obtain maternal antibody by milk, reach the purpose preventing newborn piglet to infect PEDV.Gather in-pig blood sample after immunity, separate serum, and detect specific serum IgA antibody, being to prevent the means main with monitoring newborn piglet epidemic diarrhea at present, to in-pig epidemic diarrhea vaccine immunity effect evaluation, suckling pig immune programme for children is formulated, popular status monitoring is respectively provided with significance.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of detect the method for Porcine epidemic diarrhea virus Specific IgA antibody in pig blood.
A kind of detect the method for Porcine epidemic diarrhea virus Specific IgA antibody in pig blood, comprise the steps:
1) preparation of Porcine epidemic diarrhea virus antigen and antigen coated 96 hole polystyrene Sptting plates；
2) by tested animal Baoding, at its vena cava anterior, take blood, obtain serum one after the blood treatment that will collect and resist, save backup；
3) serum one is resisted, after the PBST solution 1:5 dilution of the defatted milk powder of mass percent concentration 3%, adding by number in the reacting hole of 96 antigen coated hole polystyrene Sptting plates, each reacting hole adds 100ul, 37 DEG C of saturated humidities are reacted 1 hour, standby with PBST washing；
4) by the goat-anti pig IgA antibody of horseradish peroxidase-labeled, after the PBST solution 1:2000 dilution of the defatted milk powder of mass percent concentration 3%, it is sequentially added in the reacting hole of 96 hole polystyrene Sptting plates, each reacting hole adds 100ul, 37 DEG C of saturated humidity lucifuges are reacted 2 hours, standby with PBST washing；
5) being sequentially added into by TMB developer in the reacting hole of 96 hole polystyrene Sptting plates, each reacting hole adds 100ul, and lucifuge is reacted 25 minutes, and the most each reacting hole adds 100ul 1M concentrated sulphuric acid, terminates reaction；
6) put in microplate reader, read OD650 data, it is determined that result.
Described step 1) is: dilute Porcine epidemic diarrhea virus purifying antigen to 5 × 10 with the carbonate buffer solution of pH 9.66TCID50/ml, is coated 96 hole polystyrene ELISA Sptting plates with the concentration in 100ul/ hole, and closes with the PBST solution of the defatted milk powder of mass percent concentration 5%, it is thus achieved that the antigen substrate of ELISA reaction, be placed in 4 DEG C standby.
Described step 3) is: after being diluted by Porcine epidemic diarrhea virus Specific IgA antibody 1:5 contained in porcine blood serum to be measured with the PBST solution of the defatted milk powder of mass percent concentration 3%, add in the reacting hole of 96 antigen coated hole polystyrene Sptting plates by number with the concentration in 100ul/ hole, 37 DEG C of saturated humidities are reacted 1 hour, standby with PBST washing；
Described step 4) is: after goat-anti pig IgA antibody 1:2000 of horseradish peroxidase-labeled being diluted with the PBST solution of the defatted milk powder of mass percent concentration 3%, join in the reacting hole of 96 hole polystyrene Sptting plates in order with the concentration in 100ul/ hole, 37 DEG C of saturated humidities are reacted 2 hours, standby with PBST washing；
Described step 6) is: 96 hole polystyrene Sptting plates after terminating, put in the microplate reader of preheating, read the OD650 numerical value of each reacting hole, and according to below equation result of determination: OD value≤0.3 of known negative sample, and OD value >=0.8 of known positive；In the case of above-mentioned condition is set up, if blood serum sample to be checked and ratio S/P >=0.25 of known positive OD value, then it is judged to the positive；Otherwise it is judged to feminine gender.
The present invention provide Porcine epidemic diarrhea virus serum IgA antibody detection method, have the advantage that 1) can to whole swinery carry out large area sampling, it is to avoid swinery butcher sampling；2) blood sample to temperature, environment, pollutant resistance higher, transport and preserve more convenient；3) quantify suckling pig obtain maternal immunity level, the data obtained the most accurate objective comprehensively；4) detection of Porcine epidemic diarrhea virus blood IgA antibody more can represent suckling pig than IgG antibody detection and obtain maternal immunity level；5) the porcine epizootic diarrhea immunity activity of in-pig, the immune trend of precognition suckling pig group and management can be monitored；6) detection of Porcine epidemic diarrhea virus serum IgA antibody and the coincidence rate of colostrum IgA antibody detection are more than 94%.
Accompanying drawing explanation
Fig. 1 is the process schematic of the method for Porcine epidemic diarrhea virus specific antibody in detection pig blood.
Detailed description of the invention
In detection pig blood, the method for Porcine epidemic diarrhea virus Specific IgA antibody comprises the steps:
1) preparation of Porcine epidemic diarrhea virus antigen and antigen coated 96 hole polystyrene Sptting plates；
2) by tested animal Baoding, at its vena cava anterior, take blood, obtain serum one after the blood treatment that will collect and resist, save backup；
3) serum one is resisted, after the PBST solution 1:5 dilution of the defatted milk powder of mass percent concentration 3%, adding by number in the reacting hole of 96 antigen coated hole polystyrene Sptting plates, each reacting hole adds 100ul, 37 DEG C of saturated humidities are reacted 1 hour, standby with PBST washing；
4) by the goat-anti pig IgA antibody of horseradish peroxidase-labeled, after the PBST solution 1:2000 dilution of the defatted milk powder of mass percent concentration 3%, it is sequentially added in the reacting hole of 96 hole polystyrene Sptting plates, each reacting hole adds 100ul, 37 DEG C of saturated humidities are reacted 2 hours, standby with PBST washing；
5) being sequentially added into by TMB developer in the reacting hole of 96 hole polystyrene Sptting plates, each reacting hole adds 100ul, and lucifuge is reacted 25 minutes, and the most each reacting hole adds 100ul 1M concentrated sulphuric acid, terminates reaction；
6) put in microplate reader, read OD650 data, it is determined that result.
Described step 1) is: dilute Porcine epidemic diarrhea virus purifying antigen to 5 × 10 with the carbonate buffer solution of pH 9.66TCID50/ml, is coated 96 hole polystyrene ELISA Sptting plates with the concentration in 100ul/ hole, and closes with the PBST solution of the defatted milk powder of mass percent concentration 5%, it is thus achieved that the antigen substrate of ELISA reaction, be placed in 4 DEG C standby.
Described step 3) is: after being diluted by Porcine epidemic diarrhea virus Specific IgA antibody 1:5 contained in porcine blood serum to be measured with the PBST solution of the defatted milk powder of mass percent concentration 3%, add in the reacting hole of 96 antigen coated hole polystyrene Sptting plates by number with the concentration in 100ul/ hole, 37 DEG C of saturated humidities are reacted 1 hour, standby with PBST washing；
Described step 4) is: after goat-anti pig IgA antibody 1:2000 of horseradish peroxidase-labeled being diluted with the PBST solution of the defatted milk powder of mass percent concentration 3%, join in the reacting hole of 96 hole polystyrene Sptting plates in order with the concentration in 100ul/ hole, 37 DEG C of saturated humidity lucifuges are reacted 2 hours, standby with PBST washing；
Described step 6) is: 96 hole polystyrene Sptting plates after terminating, put in the microplate reader of preheating, read the OD650 numerical value of each reacting hole, and according to below equation result of determination: OD value≤0.3 of known negative sample, and OD value >=0.8 of known positive；In the case of above-mentioned condition is set up, if blood serum sample to be checked and ratio S/P >=0.25 of known positive OD value, then it is judged to the positive；Otherwise it is judged to feminine gender.
Embodiment
1) with the Porcine epidemic diarrhea virus of commercialization, 107TCID50/ml(half cell infection amount/milliliter) concentration, vero cells infection, after cultivating 72 hours, collect virus liquid, 4000g is centrifuged 10min, takes supernatant；35000g is centrifuged 1h, it is thus achieved that purified virus, and titrates TCID50.Antigen is diluted to 5 × 10 with the carbonate buffer solution of pH 9.66TCID50/ml, is coated 96 hole polystyrene ELISA Sptting plates with the concentration in 100ul/ hole, under saturated humidity, hatches 16h for 4 DEG C.Discard subsequently and be coated liquid, pat dry elisa plate, add the 5% defatted milk powder 200ul/ hole diluted with PBST (pH7.3 PBS, 0.05% tween-20), close 1h in 22-28 DEG C.Discarding confining liquid, pat dry elisa plate, every hole adds 350 ul washing liquids (PBST), washs 1 min × 3 time, pats dry elisa plate, be placed in 4 DEG C standby, see Fig. 1.
2) pig is carried out Baoding, first enter at pin, to carry out partly sterilised to vena cava anterior with 75% cotton ball soaked in alcohol, then dry with dry cotton ball, push down vena cava anterior proximal part with thumb from start to finish, then with the anxious vena cava anterior stinging distal end of the syringe needle sterilized, after blood outflow first and second discards, connect blood, make blood flow into along bottle wall, after adopting 7 milliliters of blood, unclamping thumb, blood i.e. stops flowing out, and presses a lower pinhole i.e. to reach disinfecting hemostasis with 5% iodine tincture cotton balls.Give adopt blood indicates number, immediately stand make it solidify, be brought to laboratory, winter is placed in dark cold place warm place, summer, makes serum separate out.Through 10-12 hour, the serum of precipitation is poured in another sterilization empty bottle.After serum coding, for preliminary storage and transport (under room temperature, in 6 hours, or at 4 DEG C, be transported to laboratory in 72 hours).Saliva is centrifuged 15min with 3000g, takes supernatant, it is thus achieved that saliva one resists, and save backup with 4 DEG C (< 7d) or-20 DEG C (< 60d).
3) resist processing standby serum one, after 3% defatted milk powder solution (PBST is molten) 1:5 dilution, add on antigen coated Sptting plate by number, 100ul/ hole, the repetition of 3, each sample, negative and each 2 holes of positive antibody comparison are set simultaneously.Under saturated humidity, hatch 1h for 22-28 DEG C.Discarding subsequently and be coated liquid, pat dry elisa plate, every hole adds PBST 350 ul washing liquid (PBST), washs 1 min × 4 time, pats dry elisa plate, standby, sees Fig. 1.
4) by the goat-anti pig IgA antibody of horseradish peroxidase (HRP) labelling, with 3% defatted milk powder solution (PBST is molten) as diluent, make 1:2000 times dilute after, join above-mentioned one by 100ul/ hole and resist in coated reacting hole, 22-28 DEG C/saturated humidity reaction 2h, discarding subsequently and be coated liquid, pat dry elisa plate, every hole adds PBST 350 ul washing liquid (PBST), wash 1 min × 5 time, pat dry elisa plate, standby, see Fig. 1.
5) TMB nitrite ion is joined in above-mentioned reacting hole by 100ul/ hole, room temperature lucifuge reaction 25min, it is subsequently added 100ul/ hole stop buffer (1M concentrated sulphuric acid), terminates reaction, see Fig. 1.
6) Sptting plate after terminating, puts in the microplate reader of preheating, reads the OD650 numerical value of each reacting hole, and according to below equation result of determination: OD value≤0.3 of known negative sample, and OD value >=0.8 of known positive；In the case of above-mentioned condition is set up, if blood serum sample to be checked and ratio S/P >=0.25 of known positive OD value, then it is judged to the positive；Otherwise it is judged to feminine gender.
Claims (3)
1. one kind is detected the method for Porcine epidemic diarrhea virus Specific IgA antibody in pig blood, it is characterised in that comprise the steps:
1) preparation of Porcine epidemic diarrhea virus antigen and antigen coated 96 hole polystyrene Sptting plates；
2) by tested animal Baoding, at its vena cava anterior, take blood, obtain serum one after the blood treatment that will collect and resist, save backup；
3) serum one is resisted, after the PBST solution 1:5 dilution of the defatted milk powder of mass percent concentration 3%, adding by number in the reacting hole of 96 antigen coated hole polystyrene Sptting plates, each reacting hole adds 100ul, 37 DEG C of saturated humidities are reacted 1 hour, standby with PBST washing；
4) by the goat-anti pig IgA antibody of horseradish peroxidase-labeled, after the PBST solution 1:2000 dilution of the defatted milk powder of mass percent concentration 3%, it is sequentially added in the reacting hole of 96 hole polystyrene Sptting plates, each reacting hole adds 100ul, 37 DEG C of saturated humidity lucifuges are reacted 2 hours, standby with PBST washing；
5) being sequentially added into by TMB developer in the reacting hole of 96 hole polystyrene Sptting plates, each reacting hole adds 100ul, and lucifuge is reacted 25 minutes, and the most each reacting hole adds 100ul 1M concentrated sulphuric acid, terminates reaction；
6) put in microplate reader, read OD650 data, it is determined that result.
The most according to claim 1 a kind of detect the method for Porcine epidemic diarrhea virus Specific IgA antibody in pig blood, it is characterised in that described step 1) is: dilute Porcine epidemic diarrhea virus purifying antigen to 5 × 10 with the carbonate buffer solution of pH 9.66TCID50/ml, is coated 96 hole polystyrene ELISA Sptting plates with the concentration in 100ul/ hole, and closes with the PBST solution of the defatted milk powder of mass percent concentration 5%, it is thus achieved that the antigen substrate of ELISA reaction, be placed in 4 DEG C standby.
The most according to claim 1 a kind of detect the method for Porcine epidemic diarrhea virus Specific IgA antibody in pig blood, it is characterized in that, described step 6) is: 96 hole polystyrene Sptting plates after terminating, put in the microplate reader of preheating, read the OD650 numerical value of each reacting hole, and according to below equation result of determination: OD value≤0.3 of known negative sample, and OD value >=0.8 of known positive；In the case of above-mentioned condition is set up, if blood serum sample to be checked and ratio S/P >=0.25 of known positive OD value, then it is judged to the positive；Otherwise it is judged to feminine gender.
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| CN103675274A (en) * | 2013-12-17 | 2014-03-26 | 广西大学 | Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody |
| CN103777010A (en) * | 2014-01-27 | 2014-05-07 | 深圳市宝舜泰生物医药股份有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof |
| CN103969450A (en) * | 2014-05-26 | 2014-08-06 | 江苏省农业科学院 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody |
| CN103995119A (en) * | 2014-04-14 | 2014-08-20 | 杭州贝尔塔生物技术有限公司 | Method for detecting classical swine fever virus specific antibody in pig saliva |
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|---|---|---|---|---|
| CN103675274A (en) * | 2013-12-17 | 2014-03-26 | 广西大学 | Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody |
| CN103777010A (en) * | 2014-01-27 | 2014-05-07 | 深圳市宝舜泰生物医药股份有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof |
| CN103995119A (en) * | 2014-04-14 | 2014-08-20 | 杭州贝尔塔生物技术有限公司 | Method for detecting classical swine fever virus specific antibody in pig saliva |
| CN103969450A (en) * | 2014-05-26 | 2014-08-06 | 江苏省农业科学院 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody |
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