African Swine Fever Virus Antibody Detection Kit (Sandwich Method) Instruction Manual
Name:African Swine Fever Virus Antibody Test Kit
Intended Use:This kit is used to detect African swine fever virus (ASFV) antibody in pig serum.
Experimental Principle:
The African swine fever virus antibody detection kit is the first to use the double antigen sandwich method to determine whether the sample contains African swine fever virus (ASFV) antibody. The recombinant ASFV antigen was used to coat the microplate to make a stationary phase, and the sample was added to the microwells coated with the antigen, and then combined with the HRP-labeled ASFV antigen to form an antigen-antibody-enzyme-labeled antigen complex, which was washed and then colored with substrate TMB. TMB is catalyzed by HRPase to blue and eventually to yellow by acid. There was a positive correlation between the shade of color and the amount of antibodies to African swine fever virus (ASFV) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the OD value was calculated to determine whether the sample contained antibodies to African swine fever virus (ASFV).
Kit Composition:
Reagent Preparation:
1.1. Preparation of washing solution: After mixing well, 20 concentrated washing solution is diluted 20 times in deionized water.
2.1. Preparation of sample diluent: take the 20 sample dilution after mixing and dilute it 20-fold in deionized water.
3.Sample preparation: Dilute the sample 10-fold with 1 sample dilution before use (20 μl serum, 180 μl sample dilution recommended).
4.Preparation of substrate solution (ready-to-use): Before use, mix chromogenic solution A and chromogenic solution B according to the volume ratio of 1:1, and protect from light.
Procedure:
Equilibrate all components of the kit and the sample to be tested to room temperature before use.
1.Prepare various reagents, positive and negative reference materials and samples to be tested according to the above-mentioned reagent preparation items;
2.Calculate the enzyme label required for the detection sample, take the enzyme label out of the aluminum foil bag, put the remaining enzyme label back into the aluminum foil bag, seal the bag mouth, and store at low temperature;
3.Sample incubation: add negative control, positive control and samples to be tested respectively, and make records, 100 l wells, mix well; Then the enzyme-labeled antigen was added to the microplate, 50 l wells, mixed wells, and then laminated, 37 incubated for 30 minutes;
4.Wash the plate: discard the liquid in the wells, add 1 wash solution (300 l wells) to wash the plate five times, pat dry;
5.Chromogenesis: Add the pre-prepared substrate solution to the microplate, 100 l wells, mix well, and incubate for 10 min at 37 l in the dark;
6.Termination: Add 100 l of well termination solution to the microplate and gently shake the microplate until the color is uniform;
7.Reading: Read the absorption value at 450nm in 20 minutes.
Verdict:
1.The OD value of the ASFV negative reference is denoted as ODN; The OD value of the ASFV-positive reference is denoted as ODP; The OD value of the sample is denoted as ODS;
2.Calculation of cut off value (threshold): cut off value = ODN + 015；
3.Test establishment condition: ODN value 015 and an ODP value of 05. The test result is valid, otherwise the test will be re-conducted;
4.Result: The OD value of the sample is cut off, and the result is negative; The OD value of the sample was cut off, and the result was judged to be positive.
Notes:
1.Please follow the instructions strictly.
2.This kit should be used by adequately trained personnel.
3.Kits with different lot numbers cannot be mixed.
4.Each component of this kit can only be used once and cannot be reused.
5.The whole experiment should be carried out in a clean and dust-free environment as much as possible.
6.The kit must be equilibrated to room temperature with the sample to be tested before use.
7.The concentrated washing solution needs to be diluted 20 times before use, and if the washing solution crystallizes, it can be dissolved by placing 37.
8.In order to avoid any potential biological hazards in the sample, the test sample should be treated as infectious and avoid contact with ** and mucous membranes.
9.The microplate washer should be used every day to correct the amount of liquid injection and residue, and pay attention to whether the pipeline is unobstructed. When washing, confirm that each well is filled with wash solution. After each wash, pat the droplets in the micropores on dust-free absorbent paper. The next step should be carried out immediately after the plate is washed, and the plate should not be allowed to dry. Avoid interrupting the protocol for long periods of time to ensure uniform conditions from well to well.
10.If the measured value of the control is not within the expected range, the experiment is invalid and the experiment should be repeated.
11.All reagents in this kit should be stored in 2-8 months and have an expiration date of 12 months. If the slats are not used up at one time, the remaining slats should be sealed in a plastic bag and sealed for storage.
12.This kit is intended for in vitro diagnostic use only.