1. Usage
Avian Flu virus H5N1 subtype Antibody ELISA kit is used for detection antibody of Avian Flu virus in the chicken serum, assessment of immunity conditions against Avian Flu virus in the chicken farm and investigation of the epidemiology of the Avian Flu virus.
2. Principle
The test kit is the antigen coated microtiter plate made from the inactivated, purified and concentrated recombinant protein. In the test, the diluted control serum and sample are added, then incubate. If in the sample it exists Avian Flu virus H5N1 subtype antibodies, they will be bound with the antigens of the microtiter plate, the unbound antibodies and other components are removed by washing. Next, add enzyme conjugate (goat-anti-chicken IgG-HRP) to specifically bind with the compound of antibody-antigens of the microtiter plate, the unbound enzyme conjugate will be removed by washing, add the TMB substrate into the wells, react and generate a blue color. At last, end the reaction by adding stop solution, read the absorbance value at 630 nm.
3. Components
1 Antigen coated microtiter plate......................................... 2 pieces (96 wells)
2 Negative (green lid) control ................................................... 1 tube (1 mL)
3 Positive (red lid) control......................................................... 1 tube (1 mL)
4 Anti-chicken IgG-HRP conjugate (yellow lid).......................... 1 bottle (22 mL)
5 20×concentrated washing solution (white lid)......................... 1 bottle (30 mL)
6 Substrate A (red lid)/B (black lid)......................... 1 bottle each (12 mL/bottle)
7 Stop solution (blue lid)......................................................... 1 bottle (12 mL)
8 Sample diluent solution (transparent lid)............................... 1 bottle (50 mL)
4. Material Required But not Provided
1 50 μL-,100 μL-,1000 μL- micropipettes, adjustable.
2 Distilled water.
3 Constant temperature box or incubator.
4 Microtiter plate spectrophotometer (630nm).
5 Absorbent paper.
5. Warning and Precautions for the Users
1 All reagents should be adjusted to the room temperature before use.
2 Never mix up reagents from different test kits in batch number.
3 Prevent the microtiter plate to get damp.
6. Preparation of Washing Solution
Washing solution: dilute 20×concentrated washing solution with distilled water at 1:20 (1 μL concentrated washing solution + 19 μL distilled water), mix properly. It can be kept for 7 days at 4 ℃.
7. Test Procedure
1 Take out the pre-coated microtiter plate(just to the required quantity). Dilute the sample at 1:40 with the sample diluent solution, then add the diluted sample into appropriate wells, 100 μL each. Dilute the negative and positive control serum at 1:10. Set 2 wells for negative control serum, add negative control serum, 100 μL/well; 2 wells for positive control serum, add positive control serum, 100 μL/well. Set one blank control well, add 100 μL sample diluent solution. Mix gently by shaking the plate manually, incubate at 37 ℃ for 30 min.
2 Pour the liquid out of the wells, wash each well with washing solution, 300μL/well, place it for 3min. Repeat 3 times, then pat to dry on absorbent paper.
3 Add 100 μL of enzyme conjugate(anti-chicken IgG-HRP combination) to each well, incubate at 37 ℃ for 30 min.
4 Repeat step 2.
5 Add 50 µL substrate A and 50 µL substrate B into each well, mix gently by shaking the plate manually , incubate for 10 min at room temperature in the dark.
6 Add one drop (50 μL) of stop solution to each well, and determine the result within 10 min.
8. ELISA analysis
Set zero for the blank well and test the OD630 nm value on the Microtiter plate spectrophotometer. The conditions for the test to be tenable are that the positive control wells’ average OD630nm value is higher than or equal to 0.6, and the negative control wells’ average OD630 nm value is less than 0.2.
If the sample’s OD630 nm value is higher than 0.38, it is judged to be positive; between 0.30 - 0.38, skeptical, the sample should be retest. if the test result repeats, the animal should be sampled and tested at a later date; if less than 0.34, negative.
Specifications : 96 wells × 2.
Expiry date: 6 months.
Storage: Store at 2~8 ℃, in dark.
Production Date: See outer-packing of the test kit.