THE DEVELOPMENT AND CHARACTERIZATION OF AN ELUTED-STAIN-ASSAY (ESTA) FOR THE INSULIN-LIKE GROWTH-FACTORS

An Eluted Stain Assay System (ESTA) has been adapted for the bioassay of the insulin-like growth factors, IGF-I and IGF-II. This ESTA is bas ed on the Fischer Rat Thyroid cell line FRTL-5 which was grown as unif orm, adherent microcultures on 96-well microtitre plates, The cells we re stimulated with the growth factors for 48 h in hormone and serum fr ee conditions. Responses were determined by the addition of the tetraz olium salt MTT which was reduced to a purple formazan product in a dos e related manner. This was directly eluted from the cells and measured with a microtitre plate reader. The signal generated was solely depen dant on metabolic activation of the cells, since no increase in cell n umbers was detected during the bioassay. The advantages of using this method are its sensitivity, precision, specificity, rapidity and high sample throughout. This bioassay, which is based on a colorimetric met hod, is technically convenient compared with other systems including t he earlier cytochemical bioassays and the radioisotopic methods. We ha ve demonstrated that this MTT ESTA provides a useful method for the st udy of complex interactions between IGF-I and IGF-II and their related binding proteins and that IGF bioactivity in serum may also be invest igated using this ESTA bioassay.