87919-07-1Relevant articles and documents
Studies on the Enzyme Immunoassay of Bio-active Constituents Contained in Oriental Medicinal Drugs. II. Enzyme Immunoassay of Glycyrrhizin
Kanaoka, Matao,Yano, Saburo,Kato, Hiromi,Nakano, Naoko,Kinoshita, Eiko
, p. 1866 - 1873 (2007/10/02)
In order to develop an enzyme immunoassay of glycyrrhizin (GL), N-(glycyrrhizinyl)-ε-aminohexanoic acid and -trans-4-aminomethylcyclohexanecarboxylic acid were synthesized as haptens from GL via 6',6''-di-tert-butyl GL (VI) as a key intermediate, which was obtained by the selective tert-butylation of GL with O-tert-butyl-N,N'-dicyclohexylisourea or by the hydrogenolysis of 6',6''-di-tert-butyl-30-benzyl GL (V) over palladium carbon.Coupling of the hapten with bovine serum albumin (BSA) (carrier protein) and β-galactosidase (labelled enzyme) was carried out by the N-hydroxysuccinimide ester method.Anti-GL serum was elicited in rabbits by immunization with N-(glycyrrhizinyl)-ε-aminohexanoic acid-BSA conjugate (XIV).Separation of bound and free fractions was perfomed by a double antibody method using a goat antiserum to rabbit IgG. 7-β-D-Galactopyranosyl-4-methylcoumarin was used as the substrate for the fluorometric assay of β-galactosidase activity.A satisfactory standard curve for GL was obtained in the range of 0.2-20 ng/ml.Keywords --- enzyme immunoassay; glycyrrhizin; ε-aminohexanoic acid; trans-4-(aminomethyl)cyclohexanecarboxylic acid; N-hydroxysuccinimide ester method; O-tert-butyl-N,N'-dicyclohexylisourea; O-benzyl-N,N'-dicyclohexylisourea; β-galactosidase; 7-β-D-galactopyranosyloxy-4-methylcoumarin