Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #4900: Detection of Infection in salmonids - by EIA for salmonid IgG

Background

The anti-salmonid IgG can be used to detect salmonid parasites (bacteria or viruses) indirectly by detecting raised immunoglobulin levels in serum from Salmo, Oncorhynchus and Salvelinus salmonids. The naturally occurring salmon antibodies (present in increased concentration in infected fish) are used, instead of using specific antibodies for each fish parasite.

If it is desired to identify the infecting parasite, plates can be coated with known fish parasites and screened with the infected salmon serum to determine which parasite specific immuno-globulins are present.

In addition the levels of serum immunoglobulins can be used to measure the effectiveness of salmonid vaccines.

EIA Method

1) For indirect assay, dilute pre-immune salmon serum 1/10 to 1/80 in carbonate buffer, pH 9.6, add 100 µl per well and dry overnight.

For detecting specific virus, coat virus antigens in carbonate coating buffer, pH9.6, 100 ml per well, at a concentration of 1.0-2.0 mg per well (i.e. a stock solution of 10-20 mg per ml) to make sure they stick to the plate. Leave at 4°C overnight.

For detecting bacteria, dilute a bacterial suspension of 1.0 OD at 660 nm to 1/100 –1/200 in distilled or Milli-Q water. Add 100 µl of this diluted suspension per well and dry down overnight at 37°C.

2) Next day, rinse plates 3 x quickly with PBS pH 7.4 / 0.05% Tween 20.

3) Block plate with 100 µl per well with PBS pH 7.4 containing 3% pre-immune goat serum. Incubate for 1 hour room temperature.

4) Remove blocking buffer and rinse plate 3 x quickly with PBS pH 7.4 / 0.05% Tween 20.

5) Add 100 µl per well of the salmon immune serum, diluted 1/10-1/80 in PBS pH 7.4 / 0.05% Tween 20. Incubate for 2 hours at room temperature or overnight at 4°C for best results.

6) Wash plate 3 x quickly with PBS pH 7.4 / 0.05 % Tween 20.

7) Wash plate 3 x 10 min with PBS pH 7.4 / 0.05 % Tween 20 i.e. fill up the wells and let the plate sit for 10 min before pouring off. Repeat twice. This longer washing will reduce background.

8) Add 100 µl of a 1/6,000 to 1/10,000 dilution of the rabbit anti-salmonid IgG in PBS pH 7.4 / 0.05 % Tween 20 to all wells. Incubate at room temperature for 2 hours.

9) Wash as in step #6.

10) Add 100 µl per well of goat-anti-rabbit IgG Alkaline Phosphatase secondary antibody, diluted 1/3,000 in PBS pH 7.4 / 0.05 % Tween 20. Incubate 2 hour at room temperature or 1 hour at 37°C.

11) Wash as in step #6.

12) Add 100 µl per well of Alkaline Phosphatase substrate (5 ml Di-ethanolamine buffer Ph 9.6 or 1 Alkaline Phosphatase substrate tablet (Sigma)). Incubate for 30-60 min in the dark. Read plate at 405 nm.

If the background is found to be too high add 0.5 % pre-immune goat serum to the PBS washing buffer solution.


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