For detection of Foot and Mouth Disease virus type A antibody in swine，cattle and sheep serum samples.
【Usage and Result Interpretation】
Reagent Preparation
Equilibration All reagents should be warm up to room temperature (approximately 30 minutes) before use. Before the test, the liquid reagents shake and mix gently. After usage, seal and store at 2 ～ 8 ℃ immediately.
Wash Buffer Preparation Dilute Wash Buffer Concentrate (20 x) into distilled water or deionized water to prepare wash buffer
Operation steps Numbering Numbering wells corresponding to the samples in sequence. Each plate should be set with 2 negative control (NC) wells, 2 positive control (PC) wells.
Adding Samples In each well, add 50ul diluted samples to be tested and 50ul into negative and positive controls respectively, mix gently.
Incubation Cover with the closure membrane provided. Incubate for 1 hour at 37 ° C.
Add enzyme Add 50ul HRP-conjugate reagent in each well without a wash, and incubate for 1 hour at 37 ° C.
Wash the plate Peel off the closure membrane carefully and wash the plate.Machine wash Repeat the wash process for a total of 5 times with an autowasher, and try to dry it the last time.
Manual wash Wash by filling each well 300ul of diluted wash buffer, and let it stand for 30 seconds, discard of the liquid. Repeat the wash process for a total of 5 times in this way, and try to dry it as much as possible the last time.
Coloration Add 50ul Substrate A and 50ul Substrate B to each well, mix well. Incubate at 37℃ in the dark for 15 minutes.
Termination Add 50ul stop solution to each well, gently tap the plate to ensure thorough mixing, and measure the result within 10 minutes.
Determination Set the microplate reader at 450nm, measure the OD value of each well.
Result
InterpretationValidity of the result The OD value of the negative control ≥1.5, and the OD value of the positive control≤0.5, otherwise the test is invalid.
Calculation method Blocking rate= (1- Sample OD Value/negative control mean OD)*100%.
Result When blocking rate ≥ 50% is positive, and when blocking rate < 50%is negative.
【Precautions】
All samples, waste liquid, positive control, etc. are detoxified according to infectious pollutants, 121℃ autoclaved for 30 minutes, or with 5.0g / L sodium hypochlorite disinfectants for 30 minutes.
Avoid the freeze-thaw cycle and contamination of the sample.
Do not mix reagents from different batches and different varieties, do not use expired reagents;
Reagents need to be mixed before use. If crystal has formed in some solutions (such as washing solution), warm up to room temperature, and mix gently until crystals have completely dissolved It does not affect the use of.
Please follow the instructions and control the reaction temperature and time strictly.
The unused antibody-coated plate can be put with the remaining desiccant in the sealed bag and store it at 2 ~ 8℃ for short-term; Do not reuse the closure plate membrane.
Avoid operating in the environment with volatile substances and hypochloric acid substances (such as 84 disinfectant)
Please use a calibrated pipette to ensure the accuracy of the test results; when adding different samples or different reagent components, the tip and the addition tank should be replaced to avoid contamination.
When washing the plate, each well should be filled with wash buffer to prevent the free enzyme in the well from being washed. When with autowasher, pay attention to the washing head and ensure no blockage of the suction head, which will not completely affect the detection result.
Add Substrate A first then add Substrate B to avoid result error; The stop solution is diluted sulfuric acid, used safely.
Be careful with the stop solution(dilute sulfuric acid).
The result calculation must be based on the microplate reader, and the reading should be taken within 10 minutes after termination; when reading the results, the bottom of the microplate should be kept clean and there are no air bubbles in the well. Fingerprints or scratches can affect plate readings
