Instruction manual of equine encephalitis virus ELISA kit

Instructions for use of equine encephalitis virus enzyme-linked immunoassay (ELISA) kit This kit is for research use only. Drug name: Generic name: Horse blue ear virus enzyme-linked immunoassay kit Purpose: This kit is used for the qualitative determination of Japanese encephalitis virus in horse serum, plasma and related liquid samples. Experimental principle This kit uses an indirect method to determine equine encephalitis virus in specimens. Coated microtiter plates with purified Japanese encephalitis virus antibodies to make solid phase antibodies. After incubation with Japanese encephalitis virus in samples, biotin-labeled anti-IgG antibodies were added, followed by streptavidin HRP binds to form immune complexes, and after thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm and compared with the CUTOFF value to determine the presence or absence of equine encephalitis virus in the specimen. The kit consists of 1 20 times concentrated washing solution 50ml × 1 bottle 8 sample diluent 6ml × 1 bottle 2 streptavidin-HRP 6ml × 1 bottle 9 negative control 0.5ml × 1 bottle 3 enzyme label coated plate 12 wells × 8 strips 10 positive control 0.5ml × 1 bottle 4 biotin-labeled anti-IgG antibody 6ml × 1 bottle 11 sealed bag 1 5 developer A solution 6ml × 1 bottle 12 3 sealing film 6 developer B 6ml × 1 / bottle 13 instructions 1 copy 7 Stopper 6ml × 1 vial sample requirements 1. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. 2. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be carried out immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing steps should be avoided. 1. Numbering: Number the microwells corresponding to the samples in sequence, each plate should be set with 2 wells for negative control and 2 wells for positive control 1. Blank control (without sample, biotin-labeled anti-IgG antibody, streptavidin-HRP, the rest of the operation is the same) 1 well 2. Add sample: add negative control and positive control to the negative and positive control wells respectively 50μl. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix, and incubate at 37 ℃ for 45 minutes. 3. Mixing solution: dilute 20-fold concentrated washing solution with distilled water 20-fold and reserve for use 4. Washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds and then discard , Repeat this 4 times, pat dry. 5. Add biotin-labeled anti-IgG antibody: Add 50 μl of biotin-labeled anti-IgG antibody to each well (except blank wells). Incubate at 37 ° C for 30 minutes. 6. Wash: operate the same as 4. 7. Add streptavidin-HRP: add 50 μl of streptavidin-HRP to each well (except the blank well), gently shake to mix, 37 Incubate at 30 ° C for 30 minutes. 8. Washing: The operation is the same as 4. 9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C for 15 minutes in the dark. 10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time, the blue color turns to yellow). 11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. Judgment of results: Test validity: average of positive control wells ≥1.00; average of negative control wells ≤0.10 cut-off value (CUT OFF) calculation: cut-off value = average of negative control wells +0.15 negative judgment: OD value of samples

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