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

为了详细评估表皮生长因子的生理分布，我们尝试通过建立酶免疫测定（EIA）系统来测定人和动物各种组织中高分子量人表皮生长因子（HMW-hEGF）的含量，并通过免疫组织化学方法检测其在组织中的定位。(1) HMW-hEGF EIA体系的制备：在HMW-hEGF前体分子结构nh2端828-1023氨基酸序列中，合成了858-873和952-969两个氨基酸片段，并偶联到牛血清白蛋白或血青素上。将这四种物质与弗氏佐剂乳化后分别注射到各组新西兰白化兔体内。18周后，获得两种抗hmw - hegf多肽的抗血清，并制备其r-球蛋白组分。利用这些抗体和我们已经获得的抗hegv抗体，制备了包含抗体（Fab’）2固定固相和抗体Fab’标记三维半乳糖苷酶的三明治酶免疫分析系统。标准HMW-hEGF由日本Wakunaga制药公司提供。与预期相反，最大荧光强度限制在对照的3倍。目前，进一步的研究计划是利用合成的比本研究中使用的分子组成更高的多肽来建立一个EIA系统。(2) HMW-hEGF的纯化：我们从330 1人尿液（日立化学公司提供）中洗脱浓缩的290 g含HMW-hEGF的尿液物质中纯化了约300 ng的HMW-hEGF。然而，制备环评系统所需的HMW-hEGF量是本研究的10-20倍以上。由于纯化如此大量的HMW-hEGF所需的尿液收集是困难的，因此纯化HMW-hEGF的尝试被认为是不可能的。(3) HMW-hEGF抗体组织化学：采用ABC法，以858-872和952-969抗体检测HMW-hEGF在人和动物组织中的分布。858-872和952-969抗体在大鼠颌下腺、胰腺、肝脏、肾脏、胃、十二指肠和结肠等组织中均呈阴性。然而，952-969抗体在人的肾间质细胞和内膜细胞、尿小管细胞以及颌下腺导管细胞和间质细胞中呈阳性染色，而858-872抗体则没有。用HMW-hEGF鉴定抗体染色物质的研究正在进行中。少

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