Enzyme-Linked Immunosorbent Assay (ELISA) for Rainbow Trout (Oncorhynchus mykiss) Vittellogenin

Abstract

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.

Vitellogenin isolation, purification and antigenic cross-reactivity in three teleost species

“…In general, polyclonal antibodies recognise cross-reactive epitopes between related species (Nilsen et al, 1998). For example anti-rainbow trout vtg antiserum was 53% reactive with estrogenised serum from brown trout (Salmo trutta), 80% reactive with estrogenised brook charr (Salvelinus fontinalis) serum, 60% reactive with estrogenised Chinook salmon (Oncorhynchus tshawytscha) serum and 34% reactive with estrogenised Atlantic salmon serum (Mourot and LeBail, 1995 ). This relationship has also been demonstrated with monoclonal antibodies (Nilsen et al, 1998).…”

“…Because some regions of the gene are highly conserved it is reasonable to expect that antibodies may be reactive across species. Most commonly polyclonal antisera are prepared by inoculation of native vtg (Heppell and Sullivan, 1999;Mosconi et al, 1998;Nilsen et al 1998;Mourot and Lebail 1995) and monoclonal antibodies have also been developed (Nilsen et al, 1998;Heppel et al, 1995). However, the activity of these antibodies does not appear to follow a predictable pattern, as polyclonal antisera are not cross-reactive with all species which have been tested, despite being directed towards multiple epitopes (Yao and Crim, 1996;Mourot and Lebail 1995;Heppell et al, 1995), and certain monoclonals are cross-reactive, even though they recognise a single antigenic epitope (Nilsen et al, 1998;Heppel et al, 1995).…”

Gender determination in the Paiche or Pirarucu (Arapaima gigas) using plasma vitellogenin, 17β-estradiol, and 11-ketotestosterone levels

“…Vtg, a pubertal female specific glycolipophosphoprotein, is correlated to oocyte development in sexually mature fish during the reproductive cycle (Nuñez Rodriguez et al 1989;Kishida et al 1992;Kishida and Specker 1993;Mañanos et al 1994a;Mourot and Le Bail 1995;Nuñez Rodriguez et al 1995;Sherry et al 1999;Nilsen et al 2004;Ndiaye et al 2006). A non-invasive sexing technique for A. gigas using both the Vtg detection in maturing females by enzyme imuno assay (EIA) and the sexual steroids technique based on 17b-estradiol (E 2 ) and 11-ketotestosterone (11KT) plasma levels in mature and immature individuals of both sexes was then developed.…”

Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas)

“…Nevertheless, upon stimulation with estrogenic substances, blood plasma VTG protein levels in male fish can attain the same range as in mature females (Purdom et al, 1994;Korte et al, 2000). As methods to detect VTG synthesis both at the protein (Mourot and Le Bail, 1995;Monteverdi and Di Giulio, 1999;Parks et al, 1999;Tyler et al, 1999;Folmar et al, 2000) and at the mRNA level (Flouriot et al, 1996;Folmar et al, 2000;Korte et al, 2000) have been developed in recent years, this model offers a sensitive and specific means to detect estrogenic influences in male fish.…”