Avian Influenza Virus antibody ELISA kit
1. Brief
Avian influenza, also known as true roup or European roup, is an acute, highly lethal infectious disease caused by Avian Influenza Virus (AIV) Type A, characterized by acute septic death to asymptomatic poisoning.
This ELISA kit is used to detect avian influenza virus type A antibody in poultry sample of serum, plasma or egg yolk from chicken, duck, turkey, quail, guinea fowl and goose etc. And can be used for evaluation of immune effect and auxiliary diagnosis.
2. Principle
This kit is composed by AIV antigen pre-coated microplate, Antibody working solution, Enzyme conjugate etc, to detect avian influenza virus type A antibody in poultry sample of serum, plasma or egg yolk by principle of Competitive enzyme - linked immunosorbent assay method (blocking ELISA). Add diluted sample and antibody working solution on pre-coated microplate wells when run the test, if there is AIV antibody in the sample, it will block the combination of antibody working solution with the AIV antigen pre-coated on microplate. Lead to no color reaction for later step, otherwise there is color reaction. The color depth of the sample was negatively correlated with the specific antibody content in the sample. After adding stop solution, color become yellow. Measure the absorbance value of each reaction well by microplate reader at 450nm. Then we can know whether there is Avian Influenza Virus (AIV) Type A antibody in the sample.
3. Components
| Code | Name | 1X96T | 2X96T |
| 1 | AIV antigen coated microplate | 96T×1 | 96T×2 |
| 2 | Enzyme conjugate (red cap) | 11ml×1 | 11ml×2 |
| 3 | Antibody working solution (blue cap) | 5.5ml×1 | 11ml×1 |
| 4 | 20x Concentrated washing buffer | 40ml×1 | 40ml×1 |
| 5 | Substrate A (white cap) | 6ml×1 | 11ml×1 |
| 6 | SubstrateB (black cap) | 6ml×1 | 11ml×1 |
| 7 | Stop solution (yellow cap) | 6ml×1 | 11ml×1 |
| 8 | Positive control(red cap) | 1.0 ml×1 | 1.5 ml×1 |
| 9 | Negative control(green cap) | 1.0 ml×1 | 1.5 ml×1 |
| 10 | Adhesive Foil | 1 piece | 2 piece |
| 11 | Instruction | 1 piece | 1 piece |
4. Materials Required But Not Provided
1 Microplate Reader (Including wave-length of 450/630 nm).
2 Adjustable micropipette
3 37℃Constant temperature equipment
5. Sample preparation
1) Return the 20x Concentrated washing buffer into room temperature (about 25℃) before use, shake to dissolve theprecipitated salt, then dilute it with distilled water or deionized water at 20 times.
2) Serum/Plasma: Take animal whole blood, get serum or plasma by regular method, the serum/plasma need to be clear, no hemolysis, no pollution. For short-term storage(within 1 week), the sample can store at 2～8℃, for long-term storage, at -20℃.
Egg yolk: Take 2ml fresh egg yolk, add 2ml saline, shake and mix evenly, centrifuge at 3000r/min for 15min, take up-layer liquid as sample.
3) Dilute the Serum, plasma or prepared egg yolk sample with diluted washing buffer at 5 times(e.g. 10ul sample add into 40ul diluted washing buffer, mix evenly). Positive Control and Negative Control do not need dilute.
6.Test procedure
1) Return the kit to room temperature for 30mins before use.
2) Take the needed quantity microplate well, set 2 wells for negative control, 2 wells for positive control, seal the unused plate, store at 2～8℃ soon.
3) Add Negative control to negative control well, 50ul/well, Positive control to positive control well, 50ul/well; for sample well, add diluted sample 50ul/well;
4) Add Antibody working solution to each well, 50ul/well. Mix evenly, Incubation at 37 ℃ for 30 minutes.
5) Discard liquid of the wells and fill all wells with washing solution, incubate for 30s and discard. Repeat washing procedure 5 times as above, pat to dry.
6) Add enzyme conjugate to each well, 100ul/well. Incubation at 37 ℃ for 30 minutes.
7) Wash as step 5).
8) Add Substrate A: 50ul/well, then Substrate B: 50ul/well to each well, mix evenly, incubation at 37 ℃ in dark for 15 minutes.
9) Add stop solution: 50ul/well to each well, mix evenly, use ELISA Reader to measure A value at 450nm (630nm as reference) of each well.
7.Results
1) In normal, A value of negative control well ≥1.0;
2) In normal, A value of positive control well ≤ A value of negative control well X 50%;
3) PI(blocking rate) = (1- Sample value/ Average value of Negative control) X 100%,
PI ≥ 50%: Positive; PI < 50%: Negative
4) No immune flocks: there maybe infected for positive results, need to be combined with clinical data for analysis.
5) Immunized flocks: monitor and record the antibody level of the sample at this time, analyze the distribution of antibody levels and the movement of the immune status of the flocks according to the results.
8. Limitation
The kit can only detect AIV antibody qualitatively. Make crude evaluation strong, medium and weak of antibody level based on PI value.
9.Notes
1) Wear gloves and work clothes when operate, strictly sound and perform disinfection and isolation system. Various experimental wastes should be treated as contaminants
2) The stop solution is corrosive, avoid touch skin and clothes, wash with tap water if touched.
3) Microplate removed from the refrigerated environment should be balance to dry at room temperature, and seal the unused microplate with desiccant.
4) Wash solution is easily crystallized at low temperature, return to room temperature when used to dissolve.
5) Add Washing buffer to each well fully, to prevent orifice free enzyme, which can not be washed
6) The test sample should be fresh.
7) Determination of the test results must be based on ELISA reader.
8) Never mix use reagents from different batches.
Specifications: 1X96 wells/kit or 2X96 wells/kit.
Expiry date:12 months.
Storage: Storing at 2-8℃, in the dark.
Store the microplate at 2-8℃, to avoid moisture, for 2 month after open the package.