ELISA materials and methods (detection of swine epidemic diarrhea virus antibodies)
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ELISA materials and methods (detection of swine epidemic diarrhea virus antibodies)
1 Material
(1) Polystyrene plastic micro tissue culture plate: 4 × 10 wells, produced by Shanghai No. 3 Plastic Factory.
(2) Coating solution (pH 96): NaHCO3 293g, Na2CO3 195g, distilled water was added to 1 000ml, and stored at 4 ℃.
(3) Washing solution (001mol / L, pH 74PBS): NaCl 80g, KH2PO402g, Na2HPO4 • 12 H2O 29g, KCl 02g, Tween20 05ml, add distilled water to 1000ml.
(4) Incubation solution: bovine serum albumin (BSA) 01g, 005% PBSTween20 100ml, stored at 4 ℃.
(5) Substrate solution (OPDH2O2)
Phosphate citrate buffer (pH 50): 02mol / L Na2HPO4 (284g / L) 257ml, 01mol / L citric acid (192g / L) 243ml, distilled water 50ml.
Substrate solution: Phosphate citrate buffer 100ml, o-phenylenediamine (OPD) 40mg, 30% H2O2 015ml, now used.
(6) Stop solution (2mol / L H2SO4): 222ml of concentrated sulfuric acid and 1778ml of distilled water.
(7) Porcine epidemic diarrhea (PED) antigen: PEDV pig fetal intestinal primary cell culture, repeatedly freeze-thaw, inactivated at 65 ° C for 30 min. Finally, centrifuge at 3 000 r / min for 30 min, take the supernatant and aliquot it, and store at -20 ℃ until use. The protein concentration of the antigen is 300 μg / ml.
(8) Enzyme-labeled rabbit anti-pig antibody conjugate: prepared according to Guo Chunxiang's method. The mol / L ratio is 196 and the enzyme conjugate titer is 1:10 000.
(9) Serum of tested pigs: collected from pigs in sick areas and sick pigs. PEDV immunized pig serum and healthy pig serum were used as positive and negative controls, respectively.
(10) ELISA tester: produced by Nanjing Huadong Electron Tube Factory.
2 ELISA procedure (indirect method)
(1) Antigen coating: PEDV antigen was diluted 1: 5 with the coating solution to coat the reaction plate, 100 μl per well, and negative cell culture control wells were set at 4 ℃ overnight.
(2) Washing: Wash twice with washing solution, 2min each time, drain.
(3) Add test serum: Dilute the test serum 1: 100 with the warming solution, add it to the two adjacent wells of the reaction plate, 100μl per well. At the same time as negative and positive standard serum control, set at 37 ℃ incubation for 1h.
(4) Wash 3 times: the method is the same as above.
(5) Add enzyme conjugate: Dilute the enzyme conjugate 1: 4 000 with the warming solution, add it to the reaction well, 100 μl per well, and incubate at 37 ° C for 1 hour.
(6) Wash 3 times: the method is the same as above.
(7) Add substrate: 100μl per well, let stand in the dark box at room temperature for 20min.
(8) Add stop solution: 50μl per well, let stand for 5min.
(9) Measure the OD value: Use a detector at 492nm wavelength, adjust the instrument according to the requirements of the instrument and the standards, measure the OD value of each reaction well, and record the result.
As a result, it was judged as positive if the serum P / N value exceeded 14 in the test. Visual inspection shows that the color of reaction wells is darker than that of normal cell control wells and normal serum control wells.
Precautions
1 The polystyrene plastic board is the most used in each carrier. The adsorption performance of reaction plates of different brands and even different batches is often very different, so they need to be selected before use. The method is: measure the same specimen on the whole plate, find the average OD value of each pair of holes, and compare this value with the total average value of the plate, the difference should be within ± 10%.
2 In order to increase the adsorption of polystyrene plates to certain antigens or antibodies, try tannic acid treatment, bovine serum albumin treatment, change the surface charge of the carrier, and introduce chemical groups to treat the reaction plate.
3 If the non-specific adsorption is strong, it should be excluded by blocking and other methods. (1) Blocking: 4% to 10% bovine serum albumin, 10% calf serum, 10% horse serum, 01% to 25% gelatin, etc. can be used.
(2) Detergent: It can prevent non-specific adsorption without affecting the specific antigen-antibody reaction. Commonly used are Tween 20, Tween 80, 01% NP40 and so on.
(3) High salt concentration buffer: such as phosphate buffer (pH 80) containing 05mol / L NaCl and 005% Tween.
(4) Shorten the incubation time: add a final concentration of 4% polyethylene glycol (PEG, MW6 000) to the warming solution
Can shorten the incubation time of antigen-antibody reaction (within 20min).
4 Due to the possible edge effect of the reaction plate, the average value of at least two wells should be taken when determining the specimen.
5 The performance of various commonly used enzyme-labeled reaction colorimeters is different, and the threshold value should be determined according to their respective instruments when determining.