Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio).
The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish (Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg x ml(-1) and the practical detection limit was 40 ng Vtg x g(-1) fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng x l(-1) of ethinylestradiol (EE2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE2 x l(-1) induced a 200-fold increase in Vtg levels.
Holbech
H
H
Danish Centre for Environmental Oestrogen Research, Odense University, Odense, Denmark. hol@biology.sdu.dk
Andersen
L
L
Petersen
G I
GI
Korsgaard
B
B
Pedersen
K L
KL
Bjerregaard
P
P
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004527:Egg Proteins; D015387:Egg Proteins, Dietary / Q000276:immunology / Q000302:isolation & purification; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004997:Ethinyl Estradiol / Q000494:pharmacology; D008297:Male; D015203:Reproducibility of Results; D014819:Vitellogenins / Q000032:analysis* / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification; D015027:Zebrafish / Q000378:metabolism*
D000906:Antibodies; D004527:Egg Proteins; D015387:Egg Proteins, Dietary; D014819:Vitellogenins; D004997:Ethinyl Estradiol; C001549:lipovitellin