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 generation 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-(7,8-dichlorodibenzo-p-dioxin-2-yl)-cis-2-methylpropeno ic acid (VII) and 5-(3,7,8-trichlorodibenzo-p-dioxin-2-yl)penta-trans,trans-2,4-dien oic acid (X). All inhibition screening and optimization studies were conducted using a less toxic surrogate standard for TCDD [2,3,7-trichloro-8-methyl-dibenzo-p-dioxin (TMDD; XVII)] which responded similarly to 2,3,7,8-TCDD in the ELISA. The most sensitive assay from the screening studies [coating antigen VII-BSA, 0.1 microgram/mL, and antiserum 7598 (anti-X-LPH), 1:10,000] was further optimized and 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 influence of various physical and chemical factors (time, solvent, detergent) was investigated. The optimized assay was then used to assess cross-reactivity by congeners of halogenated dioxins and related structures. DMSO up to concentrations of 37.5% decreased the IC50 value in the assay, whereas methanol to concentrations of 30% did not lead to improved IC50 values.
This paper reports the synthesis of haptens and the generation and
preliminary evaluation of
polyclonal antibodies for the detection of dioxins such as TCDD
(2,3,7,8-tetrachlorodibenzo-p-dioxin)
by ELISA (enzyme-linked immunosorbent assay). These novel haptens
contain unsaturation between
the halogenated dibenzo-p-dioxin ring system and the protein
to which it is conjugated, presenting
a rigid handle structure. The substitution pattern is identical
with or similar to that of TCDD (i.e.,
2,3,7,8- or 1,2,3,7,8-). Finally, the haptens lack polar groups
for hydrogen bonding. In direct binding
assays using the new polyclonal antibodies there was excellent
recognition of hapten−protein
conjugates, including recognition of those hapten conjugates that were
not used as immunogens
(i.e., assay systems heterologous in hapten structure). These
haptens do elicit selective immune
responses in rabbits. Their evaluation in an ELISA format
demonstrated the usefulness of these
haptens for the detection of dioxins. An IC50 of 0.8
ng/well (16 ng/mL) was observed for an
unoptimized system that used
2,3,7-trichloro-8-methyldibenzo-p-dioxin as an analytical
surrogate
standard.
Keywords: TCDD; dioxin; immunoassay; polyclonal antibodies; hapten
synthesis; polychlorinated
hydrocarbons
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