Evaluation of High-molecular Weight Epidermal Growth Factor for Its Distribution in Human and Animal Tissues

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To assess in detail the physiological distribution of epidermal growth factor, attempts were made to measure the contents of high molecular weight human EGF (HMW-hEGF) in a variety of tissues in man and animals by establishing an enzyme immunoassay (EIA) system, and to immunohistochemically detect its localization in the tissues.(1) Preparation of EIA system for HMW-hEGF: In the amino acid sequence of HMW-hEGF lying at 828-1023 on the NH2-terminal side of the molecular structure of HEGF precursor, two segments of polypeptide (amino acids 858-873 and 952-969) were synthesized and coupled to bovine serum albumin or hemocyanin.These four substances were emulsified with Freund's adjuvant and injected sc into separate groups of the New Zealand albino rabbits. Eighteen weeks later, two anti-HMW-hEGF antisera to the polypeptides were obtained and their r-globulin fractions prepared. Using these antibodies and anti-hEGV- antibody which we had already possessed, sandwich enzyme immunoassay syst … More ems comprising the antibody (Fab')2- immobilized solid-phase and the antibody Fab' fractions labeled with 3-D-galactosidase were prepared.Standard HMW-hEGF was provided by Wakunaga Pharmaceuticals Co., in Japan. Contrary to expectation, the maximal intensity of fluorescence was limited to 3 times the control. For the time, further studies are planned to establish an EIA system using synthesized polypeptides composing of higher molecules than those used in the present study.(2) Purification of HMW-hEGF: We purified about 300 ng of HMW-hEGF from 290 g of HMW-hEGF-containing urine material eluted and concentrated from 330 1 of human urine (provided by Hitachi Chemical Co.).However, HMW-hEGF over 10-20 times as much amount as obtained in the present study is required for the preparation of an EIA system. Since the collection of urine needed for the purification of such a lot of HMW-hEGF is difficult, the attempt to purify HMW-hEGF was judged to be impossible.(3) Histochemistry using HMW-hEGF Antibodies: Using the 858-872 and 952-969 antibodies, HMW-HEGF was assessed for its distribution in human and animal tissues by employing the ABC method. Both the 858-872 and 952-969 antibodies showed negative staining in all the rat tissues tested: the submandibular gland, pancreas, liver, kidney, stomach, duodenum and the colon. However, the 952-969, but not the 858-872, antibody resulted in positive staining in interstitial and intimal cells and uriniferous tubule cells of the kidney and in ductal and interstitial cells of the submandibular gland in man. A study for identifying the substances stained by the antibody with HMW-hEGF is in progress. Less