Anal. Chem. 1980, 52, 1497-1500
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Radioimmunoassay for Quantitation of 2,3,7,8-Tetrachlorodibenzof uran Michael I. Luster,” Phillip W. Albro, Kun Chae, Lela D. Lawson, Jean T. Corbett, and James
(4.
McKinney
Laboratory of Environmental Chemistry, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
A radioimmunoassay is described for the determination of 2,3,7,8-tetrachiorodibenrofuran (TCDF) in commercial preparations of polychlorinated biphenyls (PCBs) as well as environmental samples including animal tlswes. A feature of the assay method is the use of nonionic detergent to solubilize the hydrophobic dibenzofurans in a manner permitting their binding to antibodies. The detection limits of the assay range between 20 pg and 4.0 ng of 2,3,7,8-TCDF. Extensive cross-reactivity studies indicated that the antiserum was fairly specific for TCDF although some cross-reactivity was found to structurally similar compounds. Values for TCDF in various Arociors and animal tissues by radioimmunoassay correlated with those obtained by gas chromatography-mass spectrometry analysis. The radioimmunoassayis applicable to screening samples in order to minimize the demand for mass spectrometric screening and should be simple enough to be performed in most clinical laboratories.
Polychlorinated dibenzofurans (PCDFs) are a class of highly toxic, widespread environmental pollutants belonging to the polyhalogenated aromatic hydrocarbon series. PCDFs have been identified in various commercial preparations of polychlorinated biphenyls (PCB) ( 1 , 2), pentachlorophenol ( 3 ) , herbicide orange ( 4 ) as well as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)(5). PCDFs have also been detected in PCBcontaminated rice oil that was responsible for the human intoxication referred to as “Yusho” and was subsequently identified in tissues from individuals with Yusho disease (6). T h e primary PCDF isomer found as a contaminant in PCB as well as Yusho rice oil is 2,3,7,8-tetrachlorodibenzofuran (TCDF; 7), probably the most toxic member of this series with a n acute oral LD50of 5-10 Mg/kg body weight in the adult guinea pig (8). Because of the low levels of TCDF that one can expect to find in environmental samples, its high toxicity, and its likelihood to be found with structurally similar compounds, very sensitive and specific analytical methodology is required for monitoring levels in environmental samples. The present method of choice for P C D F analysis involves column chromatography cleanup followed by high-resolution gas chromatography (GC) and quantitation by mass spectrometry (MS) (9). Comparison of t h e retention times using highresolution GC with authentic standard has been used for qualitative analysis; however, the number of synthetic standards is limited in comparison to the number of potential isomers (10). Because of the limited number of laboratories capable of performing PCDF analysis with GC-MS and lack of a confirmatory method not based on these procedures, we have developed a radioimmunoassay (RIA) for the determination of T C D F in environmental samples.
EXPERIMENTAL Materials. RIA grade bovine serum albumin (BSA), bovine gamma globulin (BGG), bovine thyroglobulin (Type I), and
Sephadex G-100 were purchased from Sigma Chemical Co. (St. Louis, Mo). Rabbit y-globulin was from h‘Iiles Lab., Inc. (Elkhart, Ind.). Silica gel, Ninhydrin, thionyl chloride, and Cutscum detergent were obtained from Fisher Scientific Co. (Raleigh, N.C.). Freund’s adjuvants were obtained from Difco (Detroit, Mich.). Organic solvents were from Burdick and Jackson Lab., Inc. (Muskegon, Mich.). Goat anti-rabbit y-globulin was from Calbiochem (San Diego, Calif.). Sodium [lz5I]iodide (17 Ci/mg; “low pH”) was obtained from New England Nuclear (Boston, Mass.). The 3,4,3’,4’-tetrachloroazobenzenewas purchased from RFR (Hope, R.I.). All other biphenyls, dioxins, and furans were provided by John A. Moore of this institute. Aroclors 1242, lot No. KA 491, and 1016 lot No. KA 706, were obtained from Monosanto in 1970. Instrumentation. The following instrumentation was used: Hewlett-Packard Model 5750 gas chromatograph with @N-electron capture detector; VG-Micromass ZAB-SF high resolution mass spectrometer with Varian 1400 gas chromatograph and Finnigan INCOS 2300 data system; Packard Model 5210 Auto Gamma scintillation spectrometer; IEC Model PR-2 centrifuge; Heat System Ultrasonics, Inc., sonic bath; Cary Model 17 UV-Visible spectrophotometer; and Gilford Model 300-N microsample spectrophotometer. Preparation of Hapten- Protein Conjugates a n d Hadiohapten was tracer. The 4-amino-2,7,8-trichlorodibenzofuran synthesized as described previously (11). The hapten was converted into adipamide derivative which was then reacted with thyroglobulin or BSA via the mixed anhydride method (12). Detailed procedures for the preparation of adipamide derivative, conjugation with proteins, and subsequent characterizations were described previously (13,14). The conjugates, thyroglobulin- and BSA-[4-amino-2,7,8-trichlorodibenzofuran], contained 94 and 12 mol of hapten per mol of protein, respectively. The lZ5I-labeledderivative was prepared by converting unlawith beled 4-N-(5-iodovaleramido)-2,7,8-trichlorodibenzofuran carrier free NalZ5I. Identical labeling procedures were followed as described for labeling of dioxin (13) and biphenyl ( 1 4 ) derivatives. The final product 4-N-(5-’251-iodovaleramido)-2,7,8-trichlorodibenzofuran had a specific activity of 70 Ci/mmol and greater than 95% of the product was precipitable with specific antiserum. Immunization. New Zealand rabbits were injected intradermally in the back and footpad with 0.5 mg of thyroglobulin4-amino-2,7,8-trichlorodibenzofuran (TriCDF). The antigen was dissolved in 0.4 mL of Tris buffer and emulsified in 0.6 mL of complete Freund’s adjuvant. Intramuscular booster injections were given biweekly using similar volumes in incomplete Freund’s adjuvant. Antibody production was initially screened by immunodiffusion analysis in agar gel against a heterologous protein and later by RIA. Usually carrier (BSA-4-amino-2,7,8-TriCDF) 3 or 4 injections were required to obtain antiserum that showed strong precipitin bands in immunodiffusion analysis against the TCDF derivative conjugated to the heterologous protein. General Principle. A dilution of antiserum to TCDF capable of binding 40% of the lZ5I-labeleddibenzofuran derivative was preincubated with the detergent emulsion of sample extract followed by incubation with the radiotracer. Following equilibrium, goat antibodies against rabbit y-globulin were added to precipitate the antibody-hapten complex and the amount of lZ5I in the precipitate was determined. The extent to which preincubation with the test material decreased the amount of lZ5I precipitated relative to the control value was a measure of the amount of TCDF in the test sample.
This article not subject to U.S. Copyright. Published 1980 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980
Table I. Cross-Reactivity in PCDF RIAa so-
8- 60x
m0 \
m
40-
20 -
I 0 01
005
01
05 IO ng /tube 2.3.7,sTCDF
I 50
Figure 1. Standard curve for 2,3,7,8-TCDFwith antiserum against 4-amino-2,7,8-TriCDFin the presence of Cutscum detergent
Tissue Extraction a n d Cleanup. Aroclors, liver, and adipose tissue were processed as described by Albro and Corbett (15). The final residues in 2.0 mL of benzene were placed in airtight vials and frozen at -20 "C until needed. Assay Buffer. The assay buffer was phosphate-buffered saline (PBS),20 mmol/L potassium phosphate, 140 mmol/L NaC1, and 200 mg/L sodium azide, adjusted to pH 7.3. The antiserum was diluted with this buffer to which was added, per liter, 1.0 g of bovine y-globulin and 200 mg of rabbit y-globulin as a carrier. Assay Procedure. A nonionic detergent (Cutscum) was added to the buffer system in the assays to solubilize these rather hydrophobic compounds in a manner permitting them to bind to antibodies. The solubility of polychlorinated aromatic hydrocarbons in physiological buffer containing detergent systems has been discussed in detail previously (13). Cutscum itself, at this concentration, causes a 15% inhibition of the antibody-antigen reaction throughout the entire curve. Samples (standards or unknowns) were diluted in benzene to the desired concentration, added in duplicate to 12 X 75 mm glass tubes and evaporated to dryness under nitrogen. Methanol (0.2 mL) containing 1 % Cutscum (v/v) was added to each tube and the methanol was likewise evaporated. PBS (0.2 mL) was added and the tubes were placed in an Ultramet I1 sonic cleaner for 30 min. After cooling, 0.2 mL of antiserum diluted in PBS with carrier proteins (capable of binding 40% of the radiotracer) was added to the tubes, which were then incubated for 30 min at 37 "C. 1251-labeledradioligand was then added in PBS with 1 % Cutscum in a volume of 25 WL (- 14 000 dpm) followed by an additional 30-min incubation at 37 "C, then 65 h at 4 "C. Each tube then received 0.2 mL of cold goat anti-rabbit y-globulin appropriately diluted with PBS containing 0.05 mM EDTA. Following incubation for an additional 6 h at 4 "C all tubes were centrifuged (4 "C, 500 g, 30 min) and the radioactivity in the precipitate w8s measured in a y counter. Control tubes consisted of blanks (no antiserum), radiolabeled antigen only, and tracer tubes (no unlabeled inhibitor). Standard curves were obtained using various concentrations of TCDF ranging from 20 pg to 4.0 ng analyzed in triplicate. Calibration curves were constructed by plotting (BIB,) X 100 vs. ng TCDF/tube added on semi-log paper where B represents blank substracted cpm in tubes containing unlabeled affector and Eo represents blank subtracted cpm in tubes lacking affector (tracer tubes). Details of these procedures have been described elsewhere (13, 14, 16).
RESULTS A N D D I S C U S S I O N Antisera avidity was compared in sera from rabbits immunized with thyroglobulin conjugated 4-amino-2,7,8-triCDF by a series of antibody titration curves as described by Hunter ( 1 7 ) . Avidity is related to the binding affinity of the antibody population and is not necessarily related to titer. Since one of the antisera demonstrated relatively high avidity, although a low titer, compared to the others, it was used in subsequent experiments. A dilution of this antiserum ( - 1:2000) that bound about 40% of the radiotracer dose in the absence of unlabeled T C D F was used. A typical standand curve for inhibition by TCDF of radioactive tracer binding to the antiserum in the presence of Cutscum is shown in Figure 1. The range of this RIA for TCDF is 20 pg to 4.0 ng if no interference
compound dibenzofurans unchlorinated 2,8-C1 3,6-C1 2,3,8-C13 2,3,6,8-C14 2,3,7,8-Br 2,3,7,8-CI4 1,3,4,7,8-C1, 2,3,4,6,7,a-ci6 1,2,3,4,6,7,8,9-C18 biphenyls 2,4,2' ,4'-C1, 2,6,2',6'-C1, 3,4,3',4'-Cl, 3,4,3',4'-(CH,), 3,5, 3',5'-C1, 3,4,5,3' ,4', 5' -C1, others 3,4,3',4'-C14bipheny1 ether 3,4,3' ,4' C1, biphenylene 2,3,7,8-tetrachlorodi benzo-p-dioxin 3,4,3' ,4'-tetrachloroazobenzene RIA working proteinsC
ng required for 50% crossinhibition of curve reactivity >100.0 >100.0 17.0 0.45 1.25 0.64 0.25 >100.0 12.0 >100.0 9.9
> 00.0
