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Avian Influenza Virus ELISA Kit Reagents
Volume
- Test plate,coat with monoclonal antibodies against AIV 1 plate
- Rabbit anti-AIV-NP 10 ml
- Goat anti rabbit IgG,labeled with HRP 10 ml
- Sample diluent 100 ml
- Positive control 1 ml
- Negative control 1 ml
- Substrate solution A 10 ml
- Substrate solution B 10 ml
- Stop solution 10 ml
- 20× Wash concentrate 30 ml
Preparation
Wash Solution
Determine the amount of 20× Wash concentrate needed for washing the test plates. Dilute the 20× Wash concentrate 1:20 with water (1 part concentrate with 19 parts water).
Sample Treatment
Chorioallantoic liquid : Certain volume of chorioallantoic liquid is collected and mixed with the sample treatment liquid of the same volume. The mixture is placed under 36 to 38℃ for 15 min, blended at an interval of 3 to 5 min, centrifuged at 1000rpm for 30 to 60 seconds. The supernatant is taken for test.
Organ or tissue specimen: An organ tissue of approximately 1g is weighed and placed in the homogenizer. 1 ml sample treatment liquid is added for homogenization. After freeze-and-thaw for 3 times, the homogenized tissue is placed under 36 to 38℃ for 15 min, and centrifuged at 10000rpm for 5 min. The supernatant is taken for test.
Throat swab and cloaca swab specimens: The transudate of throat or cloaca of a bird is collected by a sterile swab and soaked in 1ml of sample treatment liquid. After strongly shaving for 1min, the swab is removed and the liquid is placed under 36 to 38℃ for 15 min following 3 times of freeze and thaw, then centrifuged under 2 to 8℃ at 12000 rpm for 5 min. The supernatant is taken for test.
Test Protocol
All reagents must be allowed to come to room temperature (18~25℃) before use.
1. Wash appropriate wells of test plate with 200μl of wash solution. Aspirate liquid contents of all wells and tap residual wash fluid from each plate onto absorbent material.
2. Dispense 100μl of treated samples and controls into each wells of the test plate. Incubate the plate for 30 minutes at 36~38℃.
3. Wash appropriate wells of test plate with 200μl of wash solution five times. Following each wash, aspirate liquid contents of all wells and tap residual wash fluid from each plate onto absorbent material.
4. Dispense 100μl of Rabbit anti-AIV-NP into each wells. Incubate the plate for 30 minutes at 36~38℃.
5. Repeat Step 3.
6. Dispense 100μl of Goat anti rabbit IgG into each wells. Incubate the plate for 30 minutes.
7. Repeat Step 3.
8. Dispense 50μl substrate solution A and substrate solution B into each well. Incubate the plate at room temperature (18~25℃) for 10 minutes.
9. Stop the color reaction by adding 50μl Stop solution into each well.
10. Read the results using a photometer at a wavelength of 630nm.
Results
To validate the assay the optical density (OD) of the positive control must be≥0.8, and should not exceed 2.0. The OD of the negative control should not exceed 0.2.
Interpretation of Results
OD ＞0.3 ≤0.3
interpretation positive negative
Precautions and warning for users
? Reagents should be mixed by gentle swirling and vortexing.
? The reagents in different lot number cannot mix use.
? Do not open the sealing film on the antigen-coating plate that has not been used.
? Users should comply with the national laws or rules on avian influenza reporting, viral isolation and harmless disposal.
? All wastes should be properly decontaminated prior to disposal.
Pack Size 96 wells each kit
Storage Tests must be storaged at 2~8℃.
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