Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.
To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.
Okuyama
Mitsunobu
M
Food & Drug Safety Center, Hatano Research Institute, 729-5, Ochiai, Hadano, Kanagawa 257-8523, Japan.
Kobayashi
Norihiro
N
Takeda
Wakako
W
Anjo
Takako
T
Matsuki
Yasuhiko
Y
Goto
Junichi
J
Kambegawa
Akira
A
Hori
Shinjiro
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001572:Benzofurans / Q000032:analysis / Q000633:toxicity; D002417:Cattle; D004147:Dioxins / Q000032:analysis* / Q000276:immunology*; D004785:Environmental Pollutants / Q000032:analysis* / Q000276:immunology* / Q000633:toxicity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D008895:Milk, Human / Q000737:chemistry*; D000072317:Polychlorinated Dibenzodioxins / Q000031:analogs & derivatives* / Q000032:analysis / Q000633:toxicity
D000911:Antibodies, Monoclonal; D001572:Benzofurans; D004147:Dioxins; D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; C051452:1,2,3,7,8-pentachlorodibenzo-p-dioxin; C023614:dibenzofuran