An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (Pleuronectes vetulus): development, validation and cross-reactivity with other pleuronectids.
Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen-antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3',5,5'-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10-450 ng ml-1 (85-20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.
Lomax
D P
DP
Environmental Conservation Division, Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration, Seattle, WA 98112, USA. dan.lomax@noaa.gov
Roubal
W T
WT
Moore
J D
JD
Johnson
L L
LL
D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D003429:Cross Reactions; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D004958:Estradiol / Q000494:pharmacology; D004968:Estrogens, Non-Steroidal / Q000633:toxicity; D005260:Female; D005432:Flounder / Q000097:blood*; D008099:Liver / Q000378:metabolism; D008297:Male; D011817:Rabbits; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014819:Vitellogenins / Q000096:biosynthesis / Q000097:blood* / Q000276:immunology