The GreenSpring™Avian Infectious Bursal Disease virus VP2 antibody (IBDV-VP2 Ab) ELISA kitis developed to detect the IBDV-VP2 antibodies level in chicken serum sample and can be used to evaluate Infectious Bursal Disease vaccine status in chickens.
This kit is based on solid-phase enzyme-linked immunosorbent assay (ELISA) principle, composed by the reaction Micro-plate coated with high purity IBDV-VP2 antigen, horseradish peroxidase-labeled anti-chicken IgG and other reagents. The reaction mechanism is the coated antigen binding with IBDV-VP2-Ab in sample, and then with the enzyme-labeled anti-chicken IgG antibody to form a "coated antigen + IBDV-VP2-Ab + anti-chicken IgG HRP antibody" complex, add substrate, it will have coloration by the enzyme catalytic reaction. Color depth is proportional to the amount of IBDV-VP2-Ab, when the sample chromogenic reaction, the results detected by the microplate reader exceeds a set threshold value result judged as positive, indicating that the immune produced antibodies or natural infection exists.
1. The test sample is chicken serum, collecting sample without bacteria, store at 2℃~8℃ for less than a week, store at lower than -20℃ for long-term storage.
2. Avoid using sample of severe hemolysis, sediments,containing suspended long fibrin and pollution bacteria.
3. Samples with conventional dosage of EDTA, sodium citrate or sodium heparin anticoagulant do not affect the experiment.
1) Bring ELISA reagents to the room temperature (20-25 ℃) for 30 min to get best results.
2) Sample dilution: use the sample diluent to dilute the sample at 40 times, mix the diluted sample evenly can get better result.
3) Washing solution preparation: Dilute the 20×concentrated washing buffer with deionized water at 20 times.
7. Test procedure
Adding sample: Take out the required coated plates according to sample quantity (Can be detached) and record the sample position on a worksheet. Set 2 wells for negative control serum and 2 wells for positive control serum, add undiluted negative and positive control serum to its well accordingly, 100 μL/well. Others are sample wells, add diluted sample, 100μl/well(both single-well and double-well test is OK).
Incubation: cover with Adhesive Foil after adding sample, incubate at 37℃ for 30 min.
Remove adhesive foil. Pour the liquid out of the wells, add the diluted Washing solution into each well fully, be static for 10s, pour out directly. Repeat 3 times, at last time pat to dry on absorbent paper.
Add 100 μL enzyme conjugate into each well.
Cover with adhesive foil and incubate at 37℃ for 30 min.
Repeat step 3.
Add 100 μL substrate into each well, mix properly, Color for 10 min at 37℃ in the dark.
Add 50uL stop solution into each well, shake for 10s, and determine the result.
Read OD value of each well with ELISA Reader at double-wave length: 450/630nm.
Generally speaking, the average NDV-Positive control OD value should be ≥ 0.6, the average NDV-Negative control OD value should be less than 0.15, otherwise the experiment do not success, re-test it.
The result is judged by S/P value,
S/P=(Sample OD450/630- NCx(—))/(PCx(—)-NCx(—)), NCx(—) means Negative control’s average OD450/630 value, PCx(—) means Positive control’s average OD450/630 value
If S/P≥0.2, it is positive; less than 0.2, it is negative.
9. Interpretation of the result
1. Severe hemolysis, fiber protein in the serum separation is not sufficient, containing erythrocytes, a precipitate, a sample with bacteria may lead to false positive.
2.Negative results may occur on individual chickenafter vaccinesdue to individual differences or immune duration.
3. Positive results for serological diagnosis and epidemiological investigation of chicken to be combined with other
Specifications: 96 wells × 2.
Expiry date: 12 months.
Storage: at 2-8℃, don’t expose in strong light.
Production Date: On outer-packing of the test kit.
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