RECOMBINANT SOMATOLACTIN AS A STABLE AND BIOACTIVE PROTEIN IN A CELL-CULTURE BIOASSAY - DEVELOPMENT AND VALIDATION OF A SENSITIVE AND REPRODUCIBLE RADIOIMMUNOASSAY

A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggre gate immediately after pituitary isolation, the majority of recombinan t sole SL (rsSL) remained as a monomeric protein after long-term stora ge, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respe ctively. Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism be tween rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activit y of rsSL, the ability of this recombinant product to prime gilthead s ea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 mu g/ml), similar to that r eported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which re tains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.