A better method for standardizing vitellogenin content of fish tissues.

Vitellogenin (vtg) concentrations were measured in plasma and liver samples from 12 hybrid Tilapia oreochromis niloticus x O. aureus to compare concentrations in these tissues. The results were calculated under two different normalizations: volume per gram of sample used (similar to normalization usually published in the literature and typically used for ELISA) and volume per total protein (similar to normalization used in polyacrylamide gel electrophoresis; PAGE). It was observed that the normalization procedure used in PAGE (per gram total protein) minimized the method detection limit by about 1000 and 2500 times in plasma and liver respectively, compared to the normalization usually reported in the literature. It was also observed that normalizing per gram total protein makes it possible to eliminate a potential problem of accidental dilution of plasma samples during sample collection. Moreover, the normalization on a per gram of total protein makes it possible even to compare results from the two different methods namely PAGE and ELISA. It also allows comparison between different tissues. Using the normalization procedures as used in PAGE (per gram total protein) for liver and the normalization method as reported in literature for ELISA (per volume of sample used), it was observed that liver samples had higher vtg levels (mean: 62 microg vtg/g) compared to the corresponding plasma samples (mean: 0.24 microg vtg/ml). However, when both results were normalized per gram total protein all but one liver sample were lower (62 microg vtg/g) than the corresponding plasma concentrations (mean = 246 microg vtg/g).

Enzyme-linked immunosorbent assay (ELISA) for rainbow trout (Oncorhynchus mykiss) vitellogenin.

1. A specific and simple enzyme-linked immunoassay for rainbow trout (Onchorynchus mykiss) vitellogenin (Vtg) is described. This assay is performed using a rabbit antiserum for Vtg purified from trout plasma. 2. This assay is based upon the competition between soluble Vtg and Vtg adsorbed on microtiter plates, for the rabbit anti-Vtg antibody binding sites. 3. The adsorbed Vtg-antibody complexes are revealed through the peroxidase-antiperoxidase antibody, which is colored by o-phenylendiamin. This assay can be performed in a day and a night. 4. Under our conditions, 90-20% of binding gave a sensibility range of 33-1473 ng/ml. With almost a 50% binding yield (335 ng/ml) the intra-assay coefficient of variation (CV) was 5.2% (n = 26) and the inter-assay CV was 12.5% (n = 5). 5. There was low immunological cross-reactivity with sera from other salmonids and with ovary extracts. Extracts of liver from oestrogenized male rainbow trout yielded displacements parallel to the vitellogenin standard and to mature female serum or oestrogenized male serum. 6. This enzyme immunoassay is simple and easy to use. Its great specificity allows its use only for the rainbow trout species.