Moroder L, Bovermann G, Mourier G, Göhring W, Gemeiner M, Wünsch E
Biol Chem Hoppe Seyler. 1987 Jul;368(7):831-8. doi: 10.1515/bchm3.1987.368.2.831.
The fluorogenic chymotrypsin substrate N alpha-(4-carboxybutyryl)-L-phenylalanine (4-methyl-7-coumaryl)amide was converted to a thiol-containing compound via its condensation at the carboxyl function with cystamine followed by reduction of the resulting disulfide compound to a cysteamine derivative. By subsequent reaction of the thiol group with N alpha-maleoyl-beta-alanyl-human-little-gastrin-I-[2-17] a fluorogenic substrate-labeled gastrin, fully immunoreactive against antigastrin antisera, was obtained. This tracer was then applied for developing a fluorescence immunoassay based on separation of bound and free tracer followed by chymotryptic digestion of the fluorogenic substrate in the supernatant. The fluorescence intensity of the extracted fluorophore i.e., 7-amino-4-methyl-coumarin, was found to monitor gastrin concentrations in a reproducible manner. With the model peptide hormone human little-gastrin-I the sensitivity of this alternative immunoassay procedure was well documented.
荧光胰凝乳蛋白酶底物Nα-(4-羧基丁酰基)-L-苯丙氨酸(4-甲基-7-香豆基)酰胺通过其羧基功能与胱胺缩合，随后将所得二硫化合物还原为半胱胺衍生物，转化为含硫醇的化合物。通过硫醇基团与Nα-马来酰-β-丙氨酰-人-小胃泌素-I-[2-17]的后续反应，获得了对抗胃泌素抗血清具有完全免疫反应性的荧光底物标记胃泌素。然后将该示踪剂用于开发一种荧光免疫测定法，该方法基于结合和游离示踪剂的分离，随后对上清液中的荧光底物进行胰凝乳蛋白酶消化。发现提取的荧光团即7-氨基-4-甲基香豆素的荧光强度以可重复的方式监测胃泌素浓度。对于模型肽激素人小胃泌素-I，这种替代免疫测定方法的灵敏度得到了充分证明。
