An immunoassay designed to quantitate different molecular forms of rat urinary epidermal growth factor with equimolar...

Different molecular weight forms of epidermal growth factor (EGF) are present in vivo and this makes quantitation of EGF difficult. Most immunoassays employ antibodies against 6-kDa EGF, and such assays are likely to underestimate the amount of high molecular weight forms of EGF. The purpose of the present study was to develop a processing-independent ELISA which is able to quantitate different molecular forms of rat EGF with equimolar potency. Our "old ELISA" used two polyclonal antibodies against rat submandibular gland EGF as catching and detecting antibodies, and 6-kDa EGF purified from rat urine as calibrator. This assay was modified to a processing-independent ELISA by converting the different forms of EGF in the samples as well as the calibrator to the same immunoreactive form of EGF prior to analysis. This could be achieved by trypsinization because trypsin cleaved the different molecular forms of rat urinary EGF to a single immunoreactive form. We applied both the "old ELISA" and the processing-independent ELISA on different molecular forms of EGF and demonstrated that the "old ELISA" underestimated high molecular weight forms by two thirds. The relative amounts of high and low molecular weight forms of EGF in urine have been debated, since different results have been obtained by different techniques. In order to address the problem it is important to quantitate the different molecular forms with equimolar potency. Employing the processing-independent ELISA we find that high molecular weight forms of EGF constitute 40% and 6-kDa EGF 60% of EGF in fresh rat urine.