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Monoclonal antibodies to chlorinated dibenzo-p-dioxins
Chemosphere, Vol.15, Nos.9-12, P r i n t e d in G r e a t B r i t a i n
pp 2 O O 7 - 2 0 1 0 ,
1986
0 0 4 5 - 6 5 3 5 / 8 6 $3.00 + .OO P e r g a m o n J o u r n a l s Ltd.
MONOCLONAL ANTIBODIES TO CHLORINATED DIBENZO-p-DIOXINS
S.J. Kennel,
G. Mason and S. Safe
Oak Ridge National Laboratory, Division of Biology, Oak Ridge, TN, 37830 Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX
77843
ABSTRACT
A thyroglobulin conjugate of dioxin (thyroglobulin-2 adipamide, 3,7,8-trichlorodibenzop-dioxin) (TG-TCDD) was used to immunize BALB/c mice. Hybridomas were produced by cell fusion between immune spleen cells and mouse myelomas SP2/O, P3, or NSI. In order to screen the thousands of resultant cultures for production of MoAb, a rapid, solid phase radioimmunoassay for antibody to dioxins was developed. This involved attaching bovine serum albumin coupled with trichlorodibenzo-p-dioxin (BSA-TCDD) to polystyrene plates to be used as a solid phase target antigen for reaction with MoAb. Fourteen hybridomas were identified that produced MoAb reacting with BSA-TCDD but not with BSA alone. Antibodies were tested for binding to BSA-aniline to eliminate those with limited binding specificity. Initial studies indicated that most MoAbs bound BSA-aniline as well as BSA-TCDD. More detailed analyses indicated that while most MoAbs showed some reaction with BSA-aniline, two showed preferential binding to BSA-TCDD of more than 200 fold whereas rabbit antisera demonstrated only a 5-fold discrimination. MoAb 391-IB was purified from mouse ascites fluid and after radioiodination, was tested for direct binding to BSA-TCDD or BSA-aniline. 125I MoAb showed no significant binding to BSA-aniline while demonstrating high binding to BSA-TCDD (Ka : 4.5 x 10~1/mole). Experiments are in progress to use these MoAbs to develop sensitive radioimmunoassays or enzyme linked assays specific for 2,3,7,8-TCDD. INTRODUCTION
The utility of a radioimmunoassay
for 2,3,7,8-TCDD and related
compounds
using
polyclonal antibodies has previously been reported (I). This paper briefly summarize our initial studies on the development of monoclonal antibodies to polychlorinated dibenzo-pdioxins.
MATERIALS AND METHODS
Hybridoma
Production
Female BALB/c mice
were
immunized
with
2-aminc-3,7,8-
trichlorodibenzo-p-dioxin conjugated to thyroglobulin (154 dioxin molecules/thyroglobulin) (TG-TCDD).
Two different protocols were used for different fusions.
Protocol I:
Animals
immunized ip on day -15 and -7 (prior to sacrifice and harvesting of spleen cells on day 0) with 100 ug of T G - T C D D e m u l s i f i e d in 100 ul complete Freund's adjuvant and on days -3, -2, and -I with 400 ug of TG-TCDD emulsified in incomplete Freund's adjuvant (2). Protocol If:
2007
2008
Animals were immunized sc and id with 100 ug of TG-TCDD emulsified in 100 ul complete Freund's adjuvant weekly for four weeks.
For the last 3 days of the fifth week, animals were
boosted each day with 100 ug of TG-dioxin ip in phosphate buffered saline (PBS).
Animals
were sacrificed on the day following the third ip booster. Fusions of imm~me spleen cells and mouse myeloma cells were conducted by the method of Fazekas de St. Groth (3) using parent myelomas P3-X63-Ag8 (P3), SP2/O or NS-I and spleen cells pooled from two immunized animals (either protocol I or II). Hybridomas were screened for antibody production using the solid state radioimmunoassay described below. Antibody Preparations ammonium
Concentrated hybridoma culture fluids (25X) were prepared by
sulfate precipitation and stored at -20oc.
Purification of MoAb 391-IB was
accomplished by a m m o n i u m sulfate precipitation and ion exchange chromatography on DEAE cellulose (DE52; Whatman).
Purified MoAb was radioiodinated with chloramine T and 125I and
freed of aggregates by gel filtration on Ultrogel AcA 34 before use in direct binding experiments (6). Purification and radioiodination of goat antibodies to mouse or rabbit Immtmoglobulin G (IgG) were conducted as described previously (6). Solid Phase Radioimmunoassay
Bovine serum albumin
conjugate of 2-amino-3,7,8-
trichlorodibenzo-p-dioxin (BSA-TCDD) and other protein conjugates were coupled to Immulon II microtest plates (Dynatech) through a bridge to poly-1-1ysine (PLL) (7). Immulon plates were incubated with 50 ul/well of 100 ug PLL per ml of PBS for I h.
Wells were washed and treated
for 5 min with I% gluteraldehyde in PBS before washing and addition of I ug/well of BSA-TCDD or other conjugates in 50 ul PBS. After incubation on a 60° rotating platform (at 60 rpm) at 4oc for 18 h, blocking solution (100 ul of 25 mg/ml BSA in PBS) was added for a minimum of I h before use in binding tests or storage at -20oc for later use. For indirect binding tests, 50 ul of antibody solution (serum, ascites fluid, hybridoma culture fluid or purified MoAb) diluted in cell growth medium containing 205 fetal bovine serum was added per well and allowed to incubate for 2 h at 4°C before washing and addition of I u g radiolabelled secondary antibody (125I goat antibody to rabbit or mouse IgG) in 50 ul of BSA at 5 mg/ml of PBS.
After incubation of 2 h at 4°C, wells were washed, separated and
analyzed for 125I in a Searle automatic gamma scintillation counter.
A summary of the
binding for several MoAbs is summarized in Figure I. For direct binding assays radioiodinated MoAb 391-IB was diluted in growth medium containing 205 fetal bovine serum and added to wells in 50 ul. the wells were washed and analyzed for 125I.
After Binding for 3 h at 4°C
Values for double reciprocal plots and
determination of binding constants (Ka) were calculated as previously described (8).
RESULTS AND DISCUSSION
The 2-amino-3,7,8-trichlorodibenzo-p-dioxin BSA conjugate has been linked to a solid plate support and used to screen thousands of cultures for the production of MoAb.
Fourteen
hybridomas that produced MoAb reacting with BSA-TCDD but not with BSA were identified; two
2009
A
30-
20
~~O'~BSA_TCD D
10
.o
°BSA amhne f
r
,
c"
0 Z
10-B
•
------.~
0 133 >C3 0 130
81" 6~ 4~
t-Z
2-,~BSA-aniline
>Oc
°
~o5.C"
i
\ "'---..~BSA-TCDD
i o
~]
\BSA-TCDD
0i o
\.
~./ BSA-aniline
64 D 41 -o~:~.~
_] BSA-aniline 10
100
i
i
\----o
1,000
10,000
i
I00,000
ANTI BODY (1/dilution) Fig~e
I
Solid phase radioimmunoassay of rabbit antiserum (Panel A) or ascites fluids containing MoAbs:
Panel B: 391-IB; Panel C:
398-17C; and Panel D: 391-9B.
Each antibody was tested on BSA-TCDD and BSA-aniline.
2010
MoAbs showed >200-fold preferential binding to BSA-TCDD over BSA-aniline whereas rabbit antisera was much less discriminatory.
MoAb 391-IB was purified from ascites fluid,
radiodinated and tested for direct binding to BSA-TCDD and BSA-aniline.
125[-MoAb-391-1B]
demonstrated relatively high binding affinity to BSA-TCDD (Ka = 4.5 x 10-8 i/mol) (Figure 2) and should detect approximately
I ng 2,3,7,8-TCDD per ml of extract.
The specificity and
sensitivity of this MoAb is currently being investigated.
8o- A
v
C~ Z
D 60o 03 .Q
BSA-TCDD
<~ o 40-
/
m
, 20re?
~-----~-- ~ ' - ~
I--t
(',1
~o/./°
~:.-.-- o - -
,
l
1
10
~
,~
125I 591-1B MoAb ADDED Figure 2.
~
~ BSA-aniline I
I
100
1000
(ng)
Direct binding of 1251-1abelled MoAb 391-IB on BSA-TCDD or BSA-aniline. ACKNOWLEDGEMENTS
The financial
assistance of the United States Environmental
Protection
Agency is
gratefully appreciated.
REFERENCES I.
P.W. Albro, M.I. Luster, K. Chae, S.K. Chaudhary, G. Clark, L.D. Lawson, J.T. Corbett and J.D. McKinney, Toxicol. Appl. Pharmacol., 50, 137 (1979).
2.
M. Cianfriglia, D. Armellini, A. Massone and M. Mariani, Hybridoma, 2(4), 451 (1983).
3.
S. Fazekas de St. Groth and M.L. Souan, Science 229, 77(1985).
4.
G. Kohler and C. Milstein, Nature 256, 495 (1975).
5.
G. Kohler and C. Milstein, Eur. J. Immunol. 6, 511 (1976).
6.
S.J. Kennel, L.J. Foote and P.K. Lankford, Cancer Res. 41, 3465 (1981).
7.
S.J. Kennel, J. Immunol. Meth. 55, I (1982).
8.
S.J. Kennel, T. Lankford and K.M. Flynn, Cancer Res 43, 2843 (1983).
pp 2 O O 7 - 2 0 1 0 ,
1986
0 0 4 5 - 6 5 3 5 / 8 6 $3.00 + .OO P e r g a m o n J o u r n a l s Ltd.
MONOCLONAL ANTIBODIES TO CHLORINATED DIBENZO-p-DIOXINS
S.J. Kennel,
G. Mason and S. Safe
Oak Ridge National Laboratory, Division of Biology, Oak Ridge, TN, 37830 Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX
77843
ABSTRACT
A thyroglobulin conjugate of dioxin (thyroglobulin-2 adipamide, 3,7,8-trichlorodibenzop-dioxin) (TG-TCDD) was used to immunize BALB/c mice. Hybridomas were produced by cell fusion between immune spleen cells and mouse myelomas SP2/O, P3, or NSI. In order to screen the thousands of resultant cultures for production of MoAb, a rapid, solid phase radioimmunoassay for antibody to dioxins was developed. This involved attaching bovine serum albumin coupled with trichlorodibenzo-p-dioxin (BSA-TCDD) to polystyrene plates to be used as a solid phase target antigen for reaction with MoAb. Fourteen hybridomas were identified that produced MoAb reacting with BSA-TCDD but not with BSA alone. Antibodies were tested for binding to BSA-aniline to eliminate those with limited binding specificity. Initial studies indicated that most MoAbs bound BSA-aniline as well as BSA-TCDD. More detailed analyses indicated that while most MoAbs showed some reaction with BSA-aniline, two showed preferential binding to BSA-TCDD of more than 200 fold whereas rabbit antisera demonstrated only a 5-fold discrimination. MoAb 391-IB was purified from mouse ascites fluid and after radioiodination, was tested for direct binding to BSA-TCDD or BSA-aniline. 125I MoAb showed no significant binding to BSA-aniline while demonstrating high binding to BSA-TCDD (Ka : 4.5 x 10~1/mole). Experiments are in progress to use these MoAbs to develop sensitive radioimmunoassays or enzyme linked assays specific for 2,3,7,8-TCDD. INTRODUCTION
The utility of a radioimmunoassay
for 2,3,7,8-TCDD and related
compounds
using
polyclonal antibodies has previously been reported (I). This paper briefly summarize our initial studies on the development of monoclonal antibodies to polychlorinated dibenzo-pdioxins.
MATERIALS AND METHODS
Hybridoma
Production
Female BALB/c mice
were
immunized
with
2-aminc-3,7,8-
trichlorodibenzo-p-dioxin conjugated to thyroglobulin (154 dioxin molecules/thyroglobulin) (TG-TCDD).
Two different protocols were used for different fusions.
Protocol I:
Animals
immunized ip on day -15 and -7 (prior to sacrifice and harvesting of spleen cells on day 0) with 100 ug of T G - T C D D e m u l s i f i e d in 100 ul complete Freund's adjuvant and on days -3, -2, and -I with 400 ug of TG-TCDD emulsified in incomplete Freund's adjuvant (2). Protocol If:
2007
2008
Animals were immunized sc and id with 100 ug of TG-TCDD emulsified in 100 ul complete Freund's adjuvant weekly for four weeks.
For the last 3 days of the fifth week, animals were
boosted each day with 100 ug of TG-dioxin ip in phosphate buffered saline (PBS).
Animals
were sacrificed on the day following the third ip booster. Fusions of imm~me spleen cells and mouse myeloma cells were conducted by the method of Fazekas de St. Groth (3) using parent myelomas P3-X63-Ag8 (P3), SP2/O or NS-I and spleen cells pooled from two immunized animals (either protocol I or II). Hybridomas were screened for antibody production using the solid state radioimmunoassay described below. Antibody Preparations ammonium
Concentrated hybridoma culture fluids (25X) were prepared by
sulfate precipitation and stored at -20oc.
Purification of MoAb 391-IB was
accomplished by a m m o n i u m sulfate precipitation and ion exchange chromatography on DEAE cellulose (DE52; Whatman).
Purified MoAb was radioiodinated with chloramine T and 125I and
freed of aggregates by gel filtration on Ultrogel AcA 34 before use in direct binding experiments (6). Purification and radioiodination of goat antibodies to mouse or rabbit Immtmoglobulin G (IgG) were conducted as described previously (6). Solid Phase Radioimmunoassay
Bovine serum albumin
conjugate of 2-amino-3,7,8-
trichlorodibenzo-p-dioxin (BSA-TCDD) and other protein conjugates were coupled to Immulon II microtest plates (Dynatech) through a bridge to poly-1-1ysine (PLL) (7). Immulon plates were incubated with 50 ul/well of 100 ug PLL per ml of PBS for I h.
Wells were washed and treated
for 5 min with I% gluteraldehyde in PBS before washing and addition of I ug/well of BSA-TCDD or other conjugates in 50 ul PBS. After incubation on a 60° rotating platform (at 60 rpm) at 4oc for 18 h, blocking solution (100 ul of 25 mg/ml BSA in PBS) was added for a minimum of I h before use in binding tests or storage at -20oc for later use. For indirect binding tests, 50 ul of antibody solution (serum, ascites fluid, hybridoma culture fluid or purified MoAb) diluted in cell growth medium containing 205 fetal bovine serum was added per well and allowed to incubate for 2 h at 4°C before washing and addition of I u g radiolabelled secondary antibody (125I goat antibody to rabbit or mouse IgG) in 50 ul of BSA at 5 mg/ml of PBS.
After incubation of 2 h at 4°C, wells were washed, separated and
analyzed for 125I in a Searle automatic gamma scintillation counter.
A summary of the
binding for several MoAbs is summarized in Figure I. For direct binding assays radioiodinated MoAb 391-IB was diluted in growth medium containing 205 fetal bovine serum and added to wells in 50 ul. the wells were washed and analyzed for 125I.
After Binding for 3 h at 4°C
Values for double reciprocal plots and
determination of binding constants (Ka) were calculated as previously described (8).
RESULTS AND DISCUSSION
The 2-amino-3,7,8-trichlorodibenzo-p-dioxin BSA conjugate has been linked to a solid plate support and used to screen thousands of cultures for the production of MoAb.
Fourteen
hybridomas that produced MoAb reacting with BSA-TCDD but not with BSA were identified; two
2009
A
30-
20
~~O'~BSA_TCD D
10
.o
°BSA amhne f
r
,
c"
0 Z
10-B
•
------.~
0 133 >C3 0 130
81" 6~ 4~
t-Z
2-,~BSA-aniline
>Oc
°
~o5.C"
i
\ "'---..~BSA-TCDD
i o
~]
\BSA-TCDD
0i o
\.
~./ BSA-aniline
64 D 41 -o~:~.~
_] BSA-aniline 10
100
i
i
\----o
1,000
10,000
i
I00,000
ANTI BODY (1/dilution) Fig~e
I
Solid phase radioimmunoassay of rabbit antiserum (Panel A) or ascites fluids containing MoAbs:
Panel B: 391-IB; Panel C:
398-17C; and Panel D: 391-9B.
Each antibody was tested on BSA-TCDD and BSA-aniline.
2010
MoAbs showed >200-fold preferential binding to BSA-TCDD over BSA-aniline whereas rabbit antisera was much less discriminatory.
MoAb 391-IB was purified from ascites fluid,
radiodinated and tested for direct binding to BSA-TCDD and BSA-aniline.
125[-MoAb-391-1B]
demonstrated relatively high binding affinity to BSA-TCDD (Ka = 4.5 x 10-8 i/mol) (Figure 2) and should detect approximately
I ng 2,3,7,8-TCDD per ml of extract.
The specificity and
sensitivity of this MoAb is currently being investigated.
8o- A
v
C~ Z
D 60o 03 .Q
BSA-TCDD
<~ o 40-
/
m
, 20re?
~-----~-- ~ ' - ~
I--t
(',1
~o/./°
~:.-.-- o - -
,
l
1
10
~
,~
125I 591-1B MoAb ADDED Figure 2.
~
~ BSA-aniline I
I
100
1000
(ng)
Direct binding of 1251-1abelled MoAb 391-IB on BSA-TCDD or BSA-aniline. ACKNOWLEDGEMENTS
The financial
assistance of the United States Environmental
Protection
Agency is
gratefully appreciated.
REFERENCES I.
P.W. Albro, M.I. Luster, K. Chae, S.K. Chaudhary, G. Clark, L.D. Lawson, J.T. Corbett and J.D. McKinney, Toxicol. Appl. Pharmacol., 50, 137 (1979).
2.
M. Cianfriglia, D. Armellini, A. Massone and M. Mariani, Hybridoma, 2(4), 451 (1983).
3.
S. Fazekas de St. Groth and M.L. Souan, Science 229, 77(1985).
4.
G. Kohler and C. Milstein, Nature 256, 495 (1975).
5.
G. Kohler and C. Milstein, Eur. J. Immunol. 6, 511 (1976).
6.
S.J. Kennel, L.J. Foote and P.K. Lankford, Cancer Res. 41, 3465 (1981).
7.
S.J. Kennel, J. Immunol. Meth. 55, I (1982).
8.
S.J. Kennel, T. Lankford and K.M. Flynn, Cancer Res 43, 2843 (1983).