Chemosphere, Vol.16, Nos.8/9, pp 1635-1639, 1987 0045-6535/87 $3.00 + .OO Printed in Great Britain Pergamon Journals Ltd.

DEVELOPMENT OF AN IMMUNOASSAY FOR CHLORINATED DIOXINS BASED ON A MONOCLONAL ANTIBODY

AND AN ENZYME LINKED IMMUNOSORBENT ASSAY (EUSA)

Larry Stanker, Bruce Watk,ns, Martin Vandedaan and Wdham L. Budde 1

Biomedical Sciences Division, Lawrence Livermore National Laboratory, P.O. Box 5507, Livermore, CA 94550

and U.S. Environmental Protection Agency 1 , Office of Research and Development, USA.

ABSTRACT

A set of monoclonal antibodies to dioxin have been developed. These form the bases for a competition enzyme-linked immunosorbent assay capable of detecting 0.5 ng of 2,3,7,8-tetrachlorodibenzodioxin.

INTRODUCTION

Effective environmental monitoring of toxic chemicals requires the analysis of a large number of samples. The cost and

time required to analyze for some chemicals by traditional methods often severely limits the scope and thoroughness of a

sampling effort. Since most of the samples tested are usually negative, a rapid screening assay is useful to separate the

samples which are of no concem from those which require further investigation. Immunoassays are an example of this

type of assay. They are physical assays which depend on the laws of mess action and should not be confused with

biological assays which depend on a biological response. Immunoassays have been widely accepted in the clinical

laboratories but have yet to be exploited for environmental monitoring. These assays are rapid, inexpensive, and can be

quantitative with detection limits in the low picogram level. Albro, Luster and their co-workers developed a series of of

immunoassays for polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated

biphenyls (PCDs) based on rabbit polyclonal sera and 125Iodine-labeled haptens [1, 2, 3]. These assays suffered from

nonspecificity and the need to frequently synthesize high specific-activity 125Iodine-labeled 2,3,7,8-TCDD. A set of

monoclonal antibodies (mAbs) to dioxin have recently been developed by Kennel et al. [4]. However, these recognized

dioxin only when it was conjugated to a protein and thus are not suitable for an immunoassay to detect free dioxin in

environmental samples. In contrast, we have isolated 5 mAbs that recognize free 2,3,7,8-TCDD in solution. These mAbs

are referred to as DD-1, -3, -4, -5, and -6, and from the basis of a competition enzyme-linked immunosorbent assay

(ELISA) that is able to detect PCDDs and PCDFs at the ng.

METHODS

1-amino-3,7,8-triCDD (amino-triCDD), and 1-nitro-3,7,8-triCDD were synthesized in our laboratodes by published methods

[5], 1-N-(adipamino)-3,7,8-tdCDD (triCDD-A) was synthesized to provide a basis for linking the dioxin hapten to proteins [1]

(both bovine serum albumin, triCDD-A-BSA and rabbit serum albumin triCDD-A-RSA) for immunization of BALB/cBkl mice

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(Bantin and Kingman, Fremont, CA), Biozzi mice [6] and ELISA development. Mouse spleen cells were fused with SP2/0

myeloma cells [7] using cell fusion and growth conditions as described by Stanker et al. [8]. Hybridomas were screened for

anti-dioxin activity using an ELISA [8] that incorporated triCDD-A-RSA as antigen. A competition ELISA was developed as

follows. Microtiter wells were coated with 0.5 pg/well of IriCDD-A-RSA conjugate (100 pl/well of antigen in carbonate buffer

for 18h at 4°C [8]). Competitor, dissolved in hexane, was added to a glass vial and dried under a stream of nitrogen. 500 ILl

of a 1 rng/ml solution of BSA in 0.01M phosphate buffer, 0.15M NaCI, pH 72 (PBS-7) was added, the vial was tightly

capped and sonicated for 2h in a Bransonio 220 ultrasonic cleaner (Branson, Shelton, CT). 100 p.I of this sonicated

solution containing the competitor was then added to wells of an antigen coated, ovalbumin blocked microtiterplate. A 2-

fold dilution series in PBS-7 was then made in the microtiter plate and 100 td of hybridoma antibody (1 pg antibody/well in

PBS-7) was added immediately to each well. The plate was then incubated for I hr at room temperature, washed and

processed as for a standard ELISA [8] using 50 p J/well of peroxidase conjugated goat-anti mouse immunoglobulin (United

States Biochemioals, Cleveland OH) and 2,2 azino-di-3-ethylbenzthiazoline as substrate.

RESULTS

Five anti-dioxin rnAbs, referred to as DD-1, DD-3, DD-4, DD-5, and DD-6, were isolated, each from an independent fusion.

These antibodies recognized 2,3,7-triCDD-A-BSA and 2,3,7-triCDD-A-RSA but did not recognize BSA or RSA (data not

shown). Each antibody was able to detect free 2,3,7,8-TCDD in a competition ELISA. Shown in Figure 1 are data from 15

independent competition ELISAs using DD-3 and 2,3,7,8-TCDD as competitor. These assays were run over a 2 month

period using different lots of microtiter plates and IriCDD-A-RSA as coating antigen. The concentration of 2,3,7,8-TCDD

inhibiting 50% of the activity (150) varied from 0.5 to 3.0 ng.

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1637

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FIGURE 2: Reactivity of individual mAbs to different congeners of PCDD, PCDF and PCB. The 150 values for each congener was determined and the activity normalized to the 150 for 2,3,7,8-TCDD and assigned a value of 100. Panels A- B, mAb DD-1; C-D, mAb DD-3; E-F, mAb DD-4; G-H, mAb DD-5; I-J, rnAb DD-6. Key to congeners: 1, dibenzodioxin (DD); 2, 1-CDD; 3, 2,7-diCDD; 4, 1,2,4-tdCDD; 5, 1,2,3,4-TCDD; 6, 2,3,7,8-TCDD; 7, 1,3,7,8-TCDD; 8, mixed isomers 1,2,3,7/1,2,38-TCDD; 9, mixed isomers 1,3,6,8/1,3,7,9-TCDD; 10, 1,2,3,4,7-pentaCDD; 11, 1,2,3,7,8-pentaCDD; 12, mixed isomers 1,2,3,6,7,8/1,2,3,7,8,9-hexaCDD; 13, OCDD; 14, dibenzofuran (DF); 15, 2,8-diCDF; 16, 2,3,7,8-TCDF; 17, 2,3,4,7,8-pentaCDF; 18, 1,2,3,4,8,9,-hexaCDF; 19, OCDF; 20 2,2',4,6-tetrachlorobiphenyl (2,2',4,7-TCBP); 21,

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3,3',4,4'-TCBP; 22, 2,2'3,4,5-pentaCBP; 23, 2,2',3,4,4',5-hexa CBP; 24, 2,2',3,4,5,5',6-heptaCBP; 25, 2,2',3,3',4,4',6-heptaCBP; 26, 2,2',3,3',4,5,6,6'-octaCBP; 27, 2,2',3,3',4,4',5,5'-octaCBP. PCDDs and PCDFs were analytic standards purchased from Cambridge Isotope Laboratories (Wobum, MA, USA).

Using the above competition ELISA we determined the 150 values for numerous PCDDs, PCDFs, and PCBs. These data

were then normalized to the 150 for 2,3,7,8-TCDD and are shown in Figure 2a-j. The 5 antibodies recognized similar but

not identical PCDD and PCDF congeners. All of the mAbs recognized 2,7-diCDD, 2,3,7,8-TCDD, the mixed isomers

1,2,3,7/1,2,3,8-TCDD, 1,3,7,8-TCDD, the mixed isomers 1,3,6,8/1,3,7,9~TCDD, and 1,2,3,7,8-pentaCDD. More poorly

recognized were 1,2,3,4,7-pentaCDD and the mixed isomers 1,2,6,7/1,2,8,9-TCDD. The hexachloro isomers tested

were differentially recognized by the 5 rnAbs_ The sample containing the mixed isomers 1,2,3,6,7,8/1,2,37,8,9-

hexaCDD was recognized only by DD-1, while 1,2,3,4,8,9-hexaCDF was not recognized by any of the mAbs. A number of

isomers were not recognized including nonchiorinated and monochlorinated dioxin and OCDD. Also shown in Figure 2 is

the reactivity of the 5 mAbs to different PCDFs and PCBs. The reactivity pattern of the PCDFs were similar to that seen for

the PCDDs. Generally, the PCBs tested were not recognized by the antibodies. However, weak reactivity to the coplanar

3,3',4,4'-TCBP was observed with mAbs DD-1, DD-6, and to some extent with DD-3.

DISCUSSION

Monoclonal antibodies DD-1, DD-3, DD-4, DD-5, and DD-6 are able to recognizing 2,3,7,8-TCDD. However, it is clear that

not all dioxin congeners are recognized equally by each mAb. In general the antibodies appear to preferentially recognize

tetra and pentachloro dioxins and dibenzofurans with lateral chlorines. However, it appears that antibody binding is

enhanced if the number one position has a chlorine, not surprising since the immunogen had an amino substitution at this

position. The specific congener recognition pattern, although, is distinct for each antibody, (e.g., the differential binding

to the hexachloro isomers). The only PCB recognized was the coplanar 3,3',4,4'-TCBP, and onty significantly so by DD-I.

MAbs DD-4, and DD-5 did not recognize any of the PCBs tested even when present at 2000 times the concentration of

2,3,7,8-TCDD.

The results presented here show that monoclonal antibodies can be generated to small organic haptens such as

dibenzodioxin. Furthermore, such antibodies are suitable reagents for development of immunoassays capable of

detecting environmentally significant levels of dioxin. The competition ELISA assay described here is able, in the case of

DD-3, to easily detect 0.5 ng of 2,3,7,8-TCDD and lower levels of other congeners. Such an assay should be useful as a

preliminary screen to determine which samples need fudher characterization using standard analytical chemical

procedures.

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The mAbs described here preferentially recognize the tetrachloro isomers of dibenzodioxin and dibenzofuran. This

undoubtedly is a result of the manner in which the antibodies were generated (i.e., using 3,7,8,-triCDD-A-BSA as

immunogen). It should be possible to develop antibodies specific for octa or hoptachloro dibenzodloxins, and develop a

series of immunoassays that would integrate over the entire spectrum of dioxin congeners. Such an assay scheme may

provide quantitative data regarding the levels of different congeners present in a sample

ACKNOWI.EDGMENTS

We wish to thank Ms. Nina Rogers, Ms Cynthia Thomas, and Mr. Rodrigo Devivar for their excellent technical assistance. Work was performed under the auspices of the U.S. Department of Energy by the Lawrence Liverrnore National Laboratory under contract No. W-7405-ENG-48, and supported in pad by the U.S. Envimnrnental Protection Agency through interagency agreement No. DW89931433-01-0.

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