Carp (Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay.
Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp.
Fukada
Haruhisa
H
Division of Marine Biosciences, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611, Hokkaido, Japan.
Fujiwara
Yumi
Y
Takahashi
Takayuki
T
Hiramatsu
Naoshi
N
Sullivan
Craig V
CV
Hara
Akihiko
A
D000818:Animals; D015153:Blotting, Western; D002347:Carps / Q000097:blood* / Q000276:immunology; D003429:Cross Reactions; D004032:Diet; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements; D008297:Male; D011498:Protein Precursors / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D012015:Reference Standards; D015203:Reproducibility of Results; D013025:Glycine max; D014819:Vitellogenins / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification*
D007106:Immune Sera; D011498:Protein Precursors; D014819:Vitellogenins