Studies on immunoassays of peptide factors. II. Fluorescence enzyme immunoassay for human little-gastrin.
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.
Moroder
L
L
Bovermann
G
G
Mourier
G
G
Göhring
W
W
Gemeiner
M
M
Wünsch
E
E
D000596:Amino Acids / Q000032:analysis; D001667:Binding, Competitive; D002918:Chymotrypsin; D005455:Fluorescent Antibody Technique; D005755:Gastrins / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D007124:Immunoenzyme Techniques
D000596:Amino Acids; D005755:Gastrins; C013550:gastrin 17; D002918:Chymotrypsin