|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1997 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1996 : ¥1,300,000 (Direct Cost : ¥1,300,000)
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.