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

尝试通过非竞争性测定（异源双位点酶免疫测定）来测量可替宁。通过可替宁与乙烯亚胺反应制备氨乙基可替宁，然后通过N-琥珀酰亚胺基-6-马来酰亚胺己酸酯、葡萄糖醛酸和N-羟基琥珀酰亚胺生物素的连续反应间接生物素化。最后，通过非竞争性试验（异源双位点酶免疫测定法）测定生物素基-氨基乙基可替宁。在这些步骤中，过量的标记物，部分未反应和部分与待测物质以外的物质结合，应被消除，为了消除这些过量的化学物质，氨基乙基可替宁被抗氨基乙基可替宁IgG-Sepharose 4 B柱捕获并洗脱，然后生物素化。氨基乙基可替宁生物素化后，通过SEP-PAK柱捕获并洗脱，随后通过抗氨基乙基可替宁IgG包被的聚苯乙烯珠捕获并洗脱。最后，生物素基-氨基乙基可替宁可通过异源双位点酶免疫测定法用链霉亲和素包被的聚苯乙烯珠和抗氨基乙基可替宁Fab '-过氧化物酶缀合物来测量。可替宁的检测限为1fmol，但条件是可替宁的生物素化反应在抗体IgG-Sepharose 4 B柱上洗脱的氨基乙基可替宁的阿摩尔水平上有效进行，并制备出特异性的可替宁抗血清，高特异性和高亲和力的可替宁抗血清尚待制备。

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.