Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin.
An enzyme-linked immunosorbent assay (ELISA) for goldfish gonadotropin (GTH) was developed with the intent of devising a simple, reliable and nonradioisotopic assay for the measurement of GTH in goldfish biological samples. In this assay, soluble GTH of the standards or samples competes with carp GTH (cGTH) immobilized on a solid support (96-well microplate) for the fixation on antibodies to the beta-subunit of carp gonadotropin. The immobilized antigen-antibody complexes are then revealed by the peroxidase-antiperoxidase (PAP) technique. After revelation of the peroxidase activity, the absorbance value of each well is measured with a microplate reader. The cGTH concentration used for coating the wells is 2 ng/ml and the final dilution of the specific antibody is 1:80,000. The assay can be performed within 24 h and can be used over a range of 0.125-4 ng/ml. At about 50% binding, the intra- and interassay coefficients of variation are 5% and 9% respectively. The displacement curves generated by goldfish plasma or pituitary perifusion fractions were strictly parallel to the standard cGTH. In addition, the stimulation by salmon gonadotropin-releasing hormone of pituitary fractions perifused in vitro caused an immediate increase in the GTH measured in the collected fractions, strongly reinforcing the assumption that this assay indeed measures GTH.
Kah
O
O
Laboratoire de Physiologie des Interactions Cellulaires, UA CNRS 339, Talence, France.
Pontet
A
A
Nunez Rodriguez
J
J
Calas
A
A
Breton
B
B
D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism; D002863:Chromogenic Compounds; D003530:Cyprinidae / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006054:Goldfish / Q000378:metabolism*; D006062:Gonadotropins / Q000032:analysis*; D007124:Immunoenzyme Techniques
D000936:Antigen-Antibody Complex; D002863:Chromogenic Compounds; D006062:Gonadotropins