Salmonid Ig (H+L) Antibody – Rabbit Polyclonal

ELISA Method
1. Prepare a stock solution of 10-20 ug/ml of virus antigens in carbonate coating buffer, pH 9.6 and add 100ul/well (ie 1.0-2.0ug of antigen/well). Incubate at 4ºC overnight. (For bacteria, dilute 1.0 OD 660 nm of suspension 1/100 in water, add 100 ul per well and dry down overnight at 37ºC. If your antigen is fish serum, you must dilute 1/10-1/80 in carbonate buffer and add 100 ul per well.)
2. The next day, rinse plate 3 times quickly with PBS-0.05% tween 20 detergent, pH 7.4.
3. Block the plate (100 ul per well) with PBS, pH 7.4, containing 3% pre-immune goat serum. Incubate for 1 hour at room temperature.
4. Flick out the blocking buffer and rinse plate 3 times quickly with PBS-0.05% tween 20.
5. Add 100 ul per well of the salmon immune serum, diluted 1/10-1/80 in *PBS-0.05% tween 20. Incubate for 2 hours at room temperature or overnight at 4ºC for best results.
6. Wash plate 3 times with PBS-0.05% tween 20 quickly. Then wash 3 x 10 minutes with PBS-0.05% tween 20 (ie. fill up the wells and let plate sit for 10 minutes, repeat 2 times) This longer washing step will reduce background.
7. Do not let the plate sit dry for long before adding 1/2000-1/5000† dilution of CLF003AP in *PBS-0.05% tween 20 to all the wells. Incubate for 2 hours at room temperature.
8. Wash as in step 6.
9. Do not let the plate sit dry long before adding 100 ?l per well of goat anti-rabbit IgG-Alk. Phos. secondary antibody, diluted 1/2500 – 1/4000 in PBS-0.05% tween 20-1.0% pre-immune goat serum. Incubate for 2 hours at room temperature or 1 hour at 37ºC.
10. Again, wash as in step 6.
11. Add 100 ul per well of Alk. Phos. substrate (one Sigma 104-105 Alk. Phos. substrate tablet in 5ml Diethanolamine buffer, pH 9.6). Incubate for 30-60 minutes in the dark. Read plate at 405 nm.

If background is high, add 1.0% pre-immune goat serum to the buffer solution.