1997 Fiscal Year Final Research Report Summary

Studies for the Practical Use of Novel and Ultrasensitive Enzyme Immunoassay (Immune Complex Transfer Enzyme Immunoassay) of Anti-HTLV-I IgG Using Synthetic Peptides as Antigens

Research Project

Project/Area Number	08670154
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	General medical chemistry
Research Institution	Miyazaki Medical College
Principal Investigator	HASHIDA Seiichi Miyazaki Medical College Faculty of Medicine Associate professor, 医学部, 助教授 (10156268)
Co-Investigator(Kenkyū-buntansha)	NISHIKATA Ichiro Miyazaki Medical College Faculty of Medicine Research Associate, 医学部, 助手 (50253844)
Project Period (FY)	1996 – 1997
Keywords	Noncompetitive assay / Hetero-two-site enzyme immunoassay / Hapten / Biotinylation / Cotinine / Streptavidin
Research Abstract	Attempt were made to measure cotinine by a noncompetitive assay (hetero-two-site enzyme immunoassay). Aminoethylcotinine was prepared by reacting cotinine with ethyleneimine, and subsequently was indirectly biotinylated by successive reactions with N-succinimidy1-6-maleimidohexanoate, giutathione and N-hydroxysuccinimidobiotin. Finally, biotinyl-aminoethylcotinine was measured by a noncompetitive assay (hetero-two-site enzyme immunoassay). In these steps, the excess of the labels, partly unreacted and partly bound to substances other than cotinine to be measured, should be eliminated. In order to eliminate the excess of these chemicals, aminoethylcotinine was trapped by anti-aminoethylcotinine IgG-Sepharose 4B column and eluted, and then was biotinylated. After biotinylation of aminoethylcotinine, was trapped by SEP-PAK column and eluted, and subsequently was trapped by anti-aminoethylcotinine IgG-coated polystirene beads and eluted. Finally, biotinyl-aminoethylcotinine can be measured by hetero-two-site enzyme immunoassay with streptavidin-coated polystirene bead and anti-aminoetylcotinine Fab'-peroxidase conjugate. The detection limit of cotinine was 1 fmol, provided that the chemical processes for biotinylation proceed efficiently at attomole levels of aminoethylcotinine eluted from antibody IgG-Sepharose 4B column and that specific anti-cotinine serum is prepared. The production of antisera to cotinine with high specificity and affinity still remain to be made.