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 based on the Fischer Rat Thyroid cell line FRTL-5 which was grown as uniform, adherent microcultures on 96-well microtitre plates. The cells were stimulated with the growth factors for 48 h in hormone and serum free conditions. Responses were determined by the addition of the tetrazolium salt MTT which was reduced to a purple formazan product in a dose related manner. This was directly eluted from the cells and measured with a microtitre plate reader. The signal generated was solely dependent on metabolic activation of the cells, since no increase in cell numbers 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 method, is technically convenient compared with other systems including the earlier cytochemical bioassays and the radioisotopic methods. We have demonstrated that this MTT ESTA provides a useful method for the study of complex interactions between IGF-I and IGF-II and their related binding proteins and that IGF bioactivity in serum may also be investigated using this ESTA bioassay.