The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.
A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.
Martínez-Barberá
J P
JP
Sección Departamental de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cádiz, Spain.
Pendón
C
C
Martí-Palanca
H
H
Calduch-Giner
J A
JA
Rodríguez
R B
RB
Valdivia
M M
MM
Pérez-Sánchez
J
J
D000818:Animals; D000906:Antibodies; D001667:Binding, Competitive; D003001:Cloning, Molecular; D013006:Growth Hormone / Q000235:genetics* / Q000276:immunology / Q000592:standards*; D006801:Humans; D007457:Iodine Radioisotopes; D010473:Perciformes; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity
D000906:Antibodies; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013006:Growth Hormone