【Application】
For diagnosing subtype Universal influenza viruses. It is suitable for early diagnosis at the beginning of highly pathogenic avian influenza virus (HPAIV) outbreaks. It is also suitable for surveying poultry for Universal Avian influenza virus.
【Introduction】
The kit is composed of monoclonal antibody-coated microwell plates, rabbit serum, anti-rabbit IgG-HRP and other reagents. It applies the principle of indirect sandwich ELISA to detect subtype Universal Avian influenza virus in poultry.
【kit composition 】
1. Microwell plates coated with monoclonal antibody 1 plate (96-well)
2. Negative control sample 1 vial (1 ml/vial)
3. Rabbit serum 1 vial (10 ml/vial)
4. Rabbit serum dilution buffer 1 bottle (12 ml/vial)
5. Enzyme labeled 2°Ab (anti-rabbit IgG-HRP) 1 bottle (12ml/vial)
6. 10x concentrated wash buffer 1 bottle (50 ml/vial)
7. Substrate solution A and B 1 vial each (22 ml/ vial)
8. Stop Solution 1 vial (12 ml/ vial)
9. Sample Dilution Buffer 1 bottle (50 ml/ bottle)
【TECHNICAL HINTS/CAUTION】
1. Bring all reagents to room temperature before using the kit.
2. Do not mix or use kit components from different lot numbers.
3. Avoid humid environment or contact with water after opening the microwell plates.
【SAMPLE TREATMENT】
Mix 0.5 ml of Sample Dilution Buffer with ~ 1.0 g of tissue, put into a homogenizer. After complete homogenization, harvest the tissue fluid, freeze and thaw 3 times, spin at 10,000rpm for 10min at 4ºC, transfer supernatant to a new tube for detection.
【ASSAY PROCEDURE】
1 Take the pre-coated microwell plate needed (depends on number of samples, the wells can be divided to use in different times). Make 1x wash buffer from 10x concentrated wash with distilled water. Wash plates once with the 1x wash buffer (200 ml/well), let stand for 3 min. Complete removal of liquid at each step is essential for a good performance. It can be accomplished by blotting against clean paper towels.
2 Add the pretreated samples, 100 ml to each well, set up 2 negative control well on each plate and 1 blank control. Incubate at 37°C for 30 min.
3 Wash 4 times with 1x wash buffer as in step 1.
4 Dilute Rabbit serum IgG with its dilution buffer at 1:5000, add 100 ml to each well. Incubate at 37°C for 30 min.
5 Wash 4 times with 1x wash buffer as in step 1.
6 Add 100 ml Enzyme labeled secondary antibody (anti-rabbit IgG-HRP) to each well. Incubate at 37°C for 30 min.
7 Wash 5 times with 1x wash buffer as in step 1.
8 Add a drop of substrate solution A and B to each well and mix well. Incubate at room temperature for 10 min. Avoid light.
9 Add a drop of Stop Solution to each well, read results within 15 min.
【READING THE RESULT】
1. Judging with naked eyes
"++++" dark blue, no light can go through the liquid
"+++" blue, faint light can go through the liquid
"++", medium blue, light can go through the liquid
"+" light blue, the liquid is transparent
"-" colorless, the liquid is completely transparent.
Samples showing color reaction at "++" or above is considered as positive.
2. Judging with a microplate reader
Setting the blank well’s reading as zero. Measure the OD630 value of each well using a microplate reader. For the test to be valid, OD value of the negative control shall be below 0.2.
Positive OD630>0.40
Negative OD630<0.35
Suspect 0.35<OD630<0.40
【Shelf Life 】 6 months
【Storgae】 Store at 2-8 °C, avoiding exposure to light
【Date of Production】 Printed on the outside package of the kit