DEVELOPMENT OF A HIGHLY SENSITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY BASED ON POLYCLONAL ANTIBODIES FOR THE DETECTION OF POLYCHLORINATED DIBENZO-P-DIOXINS

The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for the polychlorinated dibenzo-p-dioxins is described, We previously reported the synthesis of haptens and generat ion of antibodies for detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Antisera were screened with seven different coating antigens (hapten-protein conjugates), including trans-3-(7, chlorodibenzo-p-dio xin-2-yl)-cis-2-methylpropenoic acid (VII) and ibenzo-p-dioxin-2-yl)pe nta-trans,trans-2,4-dienoic acid (X). All inhibition screening and opt imization studies were conducted using a less toxic surrogate standard for TCDD [2,3, 7-trichloro-8-methyl-dibenzo-p-dioxin (TMDD; XVII)] wh ich responded similarly to 2,3,7,8-TCDD in the ELISA, The most sensiti ve assay from the screening studies [coating antigen VII-BSA, 0.1 mu g /mL, and antiserum 7598 (anti-X-LPH), 1:10000] was further optimized a nd characterized. It exhibited an IC50 value of 12 pg/well (240 pg/mL) , with working range from 2 to 240 pg/well (40 to 4800 pg/mL), The inf luence of various physical and chemical factors (time, soh ent, deterg ent) was investigated, The optimized assay was then used to assess cro ss-reactivity by congeners of halogenated dioxins and related structur es, DMSO up to concentrations of 37.5% decreased the IC50 value in the assay, whereas methanol to concentrations of 30% did not lead to impr oved IC50 values.