ELISA test kits avian influenza 96wells AIV Antibody immunossay set 2plates LSY-30028

Avian Influenza Virus antibody ELISA kit

Catalog No. LSY-30028

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, 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

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) Washing buffer preparation: Return the 20x Concentrated washing buffer into room temperature (about 25℃) before use, shake to dissolve the precipitated salt, then dilute it with distilled water or deionized water at 20 times(Like: 1 part 20x Concentrated washing buffer + 19 parts distilled water or deionized water).

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) The Serum, plasma or prepared egg yolk sample need to return to room temperature, mix it evenly ready for use. 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 wells, firstly add diluted washing buffer 40ul/well, then add sample: 10ul/well;

4) Add Antibody working solution to each well, 50ul/well. Mix evenly, cover plate with adhesive foil, Incubate at 37℃(recommend water bath) for 30 minutes in dark.

5) Discard liquid of the wells and fill all wells with diluted washing buffer: 350ul/well, be static for 30s and discard. Repeat washing procedure 5 times as above, at last pat to dry.

6) Add enzyme conjugate to each well, 100ul/well. cover plate with adhesive foil, Incubation at 37 ℃ for 30 minutes in dark.

7) Wash as step 5).

8) Add Substrate A: 50ul/well, then Substrate B: 50ul/well to each well, mix evenly, cover plate with adhesive foil, incubation at 37 ℃ in dark for 15 minutes.

9) Add stop solution: 50ul/well to each well, mix evenly, use ELISA Reader to measure OD value at 450nm (630nm as reference) of each well.

7.Reference

For the test be valid, OD value of negative control≥1.0, OD value of positive control≤OD value of negative control X 50%.

8. Results

1) PI(blocking rate) = (1- Sample OD value/ Average OD value of Negative control) X 100%,

PI ≥ 50%: Positive; PI < 50%: Negative

2) No immune flocks: there maybe infected for positive results, need to be combined with clinical data for analysis.

3) 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.

9. Limitation

The kit can only detect AIV antibody qualitatively. Make crude evaluation strong, medium and weak of antibody level based on PI value.

10. Notes