Immunology Letters, 31 (1992) 131 - 136
Elsevier IMLET 01724
Monoclonal antibodies against soman: characterization of soman stereoisomers* D a v i d E. L e n z , J e f f r e y J. Y o u r i c k , J. S h a w n D a w s o n a n d Jill Scott United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, U.S.A.
(Received 28 June 1991; accepted 4 September 1991)
1. Summary
2. Introduction
Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman(C+P+,C+P-,C-P+ andC-P-).In all cases the antibodies tested exhibited ICs0 values of 1 0 - 4 - 5 × 1 0 - 6 M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-sOman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers.
Immunologic means of altering the pharmacologic and toxic effects of chemicals have been summarized previously [1]. Recently, Hunter et al. [2] reported on the identification of a monoclonal antibody specific for the toxin anticholinesterase inhibitor pinacolylmethyl phosphonofluoridate (soman, GD), i.e., an anti-soman monoclonal antibody. Brimfield et al. [3] characterized the specificity of monoclonal anti-soman antibodies and Lenz et al. [4] investigated the ability of monoclonal anti-som a n antibodies to confer protection when administered passively to mice. Additional studies on the combining site specificity were carried out by Buenafe and Rittenberg [5] and Buenafe et al. [6] investigated the relationship between fine specificity and particular genes. While these results held the promise of producing a monoclonal antibody which could confer passive protection against soman toxicity in vivo, several technical difficulties remained to be overcome. The antibodies produced by Hunter et al. [2], Brimfield et al. [3] and Lenz et al. [4], while IgG-class antibodies, lacked sufficient affinity to reduce the in vivo concentration of soman to non-toxic levels and they showed only slight stereospecificity. The latter point is of considerable importance since, of the four stereoisomers of soman, two of them, designated C _+P - , are considerably more toxic than the other two, designated C _+P + [7]. I f these two problems could be overcome, then a monoclonal anti-soman antibody might have the requisite properties to confer in vivo protection over an extended ( 1 0 - 20 day) period of time after passive administration.
* The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the "Guide of the Care and Use of Laboratory Animals" of the Institute of Laboratory Animals Resources, National Research Council. Key words: Soman; Antibodies; Stereoisomers; Absolute con-
figuration Correspondence to: D.E. Lenz, United States Army Medical
Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, U.S.A.
0165- 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
131
In the present work, we were able to produce several new monoclonal anti-soman antibodies. We have examined these antibodies for their antibody class and their relative affinities for a racemic soman mixture, each of the soman stereoisomers and a variety of structurally related haptens. We have also been able to produce a monoclonal antiCsPs-soman antibody and have used it to make a preliminary assignment of absolute configuration for the stereoisomers of soman. 3. Materials
and Methods
3.1. Soman-protein conjugates and other chemicals Analogues of soman covalently bound to bovine serum albumin (GD-BSA) or to keyhole limpet hemocyanin (GD-KLH) via a succinamido linking group (Fig. l) at varying weight ratios, e.g., 3 3 - 1 0 0 m g of a particular soman analogue per 160mg of protein, were obtained from Steroids Ltd., Chicago, IL. The material was a lyophilized powder and used as supplied. The analogues included racemic soman as well as the CsPs-, CsPR-, o ,
~ I I
H
The monoclonal anti-soman antibodies were prepared by a modification of the method previously described [2]. Adult female BALB/c mice (Charles River, Boston, MA) were immunized subcutaneously with 100/~g (protein) of soman-BSA for rac~ (CHs)a . ~ C ~
CH= C~
p-amtnophenyl methyl phoephonate
0
3.2. Preparation o f monoclonal antibodies
C~
I I c~
CRPs-, and CRPR-soman stereoisomer analogues. The absolute configuration of the stereoisomer analogues was confirmed by X-ray crystallographic analysis. Soman, isopropylmethyl phosphonofluoridate (sarin) and tabun (Fig. 1) were obtained from the Analytical Chemistry Branch, USAMRICD and were > 95o70 pure as determined by 31p-NMR or gas chromatography. The C-P-, C-P+, C+Pand C + P + soman stereoisomers were obtained from the Prins Maurits Laboratory, TNO, The Netherlands. They were supplied in ethyl acetate at a concentration of 1.5mg/ml and were all greater than 95°7o optical purity and greater than 92°70 chemical purity. They were used as supplied. All other chemicals were the highest purity commercially available and were used as supplied.
0
c~- " ~C --0-CH3/
XO . - - P ~ - ~ C - - - - C ~
I I
II
P----F
]
c~ sertn
"hydroxy° eolian 0
H
dtptnecolyl methyl phoephonete
H
I
o
I II I C-.-'-'O--p----.O-...--C~ (Cl'l~ -, I CH= I I H C1%
H
0
F _ P-----~---C----~
I
CHa~ H
dlteopropyl methyl phosphonate H
C~
II I I CmO- - P- ' - " 1 1 - - - ~ " " ~ " " - ~ 2 ~
I
H ...Ckt.
c~
eoIRn
0
II
c..>c - - - ~ - ~ < . ~ "
I I
0
~=',
1 I lmunogen or test antigen
PROTEIN
(CH~}8-N ~
II
N
I tsbun
Fig. 1. Structures of i m m u n o g e n used to elicit antibody response in mice and test haptens.
132
cemic soman or soman-KLH for the soman stereoisomers emulsified in complete Freund's adjuvant. At three succeeding fourteen-day intervals, the mice received a booster immunization in incomplete Freund's adjuvant of 100 #g of soman-BSA or CsP s soman-KLH, 150/~l/animal. After the second and third booster, a sample of blood was obtained by orbital bleeding for testing the antibody response by enzyme immunoassay as described below. If a positive antibody response was obtained after the third booster immunization, spleens of positive responders were removed 4 days after the last booster immunization and splenic lymphocytes were fused with the hypoxanthine-guaninephosphoribosyltransferase-deficient mouse plasmacytoma line P3-X63-AG8.653. Fusion was accomplished with 35°70 polyethylene glycol (M l 1400, American Type Culture Collection, Rockville, MD) and fused cells were plated in 96-well, sterile, flat-bottomed microtiter plates at 5.0 × 105 cells/well. Hybridomas were selected in hypoxanthine-aminopterin-thymidine medium for the first week, with changes of culture medium on alternate days. 3.3. Enzyme immunoassay and cloning Supernatants from 14-day hybridoma cultures were analyzed for anti-soman antibody production by enzyme immunoassay. Wells of flat-bottomed polystyrene microtiter plates (Dyanatech, Alexandria, VA) were incubated with 50 #l of soman-KLH for racemic soman antibodies or CsP s soman-BSA for the stereoisomer immunogens, at a concentration of 50/~g/ml in 0.1M Na2CO 3 (pH9.6) overnight at 4°C. These plates were washed with phosphate-buffered saline (pH7.4) containing 0.5°7o Tween 20 (PBS-T) by five repetitions of filling and emptying each well. Aliquots of hybridoma supernatants (50/A) were added to the somanKLH- or soman-BSA- (see above) coated wells i n duplicate and incubated for 60 min at room temperature. Unbound antibody was washed away as above and 50/zl of rabbit anti-mouse gamma chain alkaline phosphatase-conj ugated antiserum (1:1000 in PBS, Sigma Chemical, St. Louis, MO) was added. Following a 60-min incubation, unbound rabbit antibody was Washed away as above and 50#I aliquots of enzyme substrate (p-nitro-
phenylphosphate; Sigma Chemical Co.), 1 mg/ml in 10o70diethanolamine buffer (pH 9.6), was added. The enzymatic reaction was allowed to proceed and the colored reaction produced was measured spectrophotometrically at 405 nm in a Titertek Multiscan Micro-ELISA Reader (Flow Labs, Vienna, VA). Duplicate determinations > 4 times background were considered to be positive. Positive supernatants were further screened for reactivity with free soman by a modified competitive inhibition enzyme immunoassay (CIEIA) [2]. 4. Results
When the supernatant solutions containing antibody derived from mice immunized with the racemic-soman conjugate were screened for monoclonal anti-soman antibodies, fifteen clones tested positive. When these antibodies were tested using an EIA where rabbit anti-mouse-IgG antiserum or rabbit anti-mouse-IgM antiserum was used in place of non-specific rabbit anti-mouse antiserum, a positive response was obtained only with the IgG-specific antiserum, indicating that these antibodies were IgG class antibodies. Of these, six were chosen for further testing and expansion. Each of these six monoclonal antibodies was tested for its relative avidity for soman and several soman analogues by CIEIA. In all cases, the monoclonal antibodies exhibited high specificity for soman and little or no cross reaction with sarin, tabun, pinacolylmethyl phosphonic acid (hydroxy soman), diisopropyl methylphosphonate, dipinacolyl methylphosphonate or p-aminophenyl methylphosphonate (structures of all test haptens given in Fig. 1). Typical results for one monoclonal anti-soman antibody are shown in Table 1 and Fig. 2. The six antibodies were also screened by CIEIA for their ability to bind to t h e C - P - , C - P + , C + P - and C + P + stereoisomers of soman. Each of these antibodies were inhibited equally well by the four soman stereoisomers (data not shown). Supernatants from hybridomas derived from mice immunized with the CsPs-soman-KLH conjugate were screened for monoclonal antibodies against each of the soman stereoisomer-BSA conjugates using an EIA. Over thirty clones tested positive. When tested in an EIA with the three other stereoisomer-BSA conjugates the order of binding 133
TABLE l ICs0 values of haptens b o u n d to monoclonal anti-soman antibody using GDM-KLH coated plates. Compound
IC5o (M)
Hydroxy s o m a n p - A m i n o p h e n y l methylpinacolylphosphonate Diisopropyl methylphosphonate Dipinacolyl methylphosphonate Soman Satin Tabun
- 5 x 10 -3 - 10 - 4 > 10- 3 NS - 8 X 10 - 6 NS NS
two of the soman stereoisomers, ( C - P - )-soman and (C + P + )-soman. Each of these clones was inhibited by the (C - P - )-soman isomer but not the (C + P + )-soman isomer (Fig. 3). 5. Discussion In the current study, we have been able to produce monoclonal anti-soman antibodies that were specific for the fluorophosphonate portion of the soman molecule (Table 1) and also IgG-class antibodies. In previous studies, monoclonal anti-soman IgG-class antibodies were produced [2 - 4] but they exhibited greater specificity with respect to the pinacolyl portion of the soman molecule. That specificity was attributed to the fact that the immunogen used in those experiments was linked to the carrier protein through the position normally occupied by the fluorophosphorus bond. Those monoclonal antibodies, when administered passively in mice, were able to delay the time to death of a lethal dose of soman [4]. In an attempt to make a mono-
NS, not significant. 0.7o -
I
o,6O,, ~
o.~
/
I
•
0.~0. 0,10. O'O~g_8
lg/?
11_6
1E1_5
1El_4
IEL3
HAPTEN CONe. (M)
"~
Fig. 2. Competitive binding assay of hybridoma supernatant solution of monoclonal anti-soman antibody with s o m a n A, tabun o, and hydroxy s o m a n a . All data points are the average of two determinations.
~" ~ "~
preference was CsPR-soman-BSA > CRPs-somanBSA _> CRPR-soman-BSA conjugate (Table2). In each case, the antibodies showed greatest preference for the CsPs-soman-BSA conjugate in an EIA. The six clones which gave the best response in the E I A were further examined using a C I E I A with
1.50 1.40, 1,30' 1.20 1.101 1,00, O.OO' 0.80" 0.70. 0,6O. 0.500.400.300,20 0.10 O.OO
,~-,
,~'-0
,~'-7
,~'-0
I
,~-6
,~'-,
,~'-~
HAPTEN CONC. (M) Fig. 3. Competitive binding assay of hybridoma supernatant solution of monoclonal anti-CsP s s o m a n antibody with (C - P - )s o m a n • and (C + P + )-soman e.
TABLE 2 Binding o f C s P s - s o m a n monocional antibody to stereoisomer conjugates in an EIA. Conjugate
Mean A a + S.D. (N = 32)
CsPs-soman
CsPR-soman
CRPs-soman
CRPg-soman
0.882 0.164
0.726 0.153
0.594 0.112
0.596 0.131
aMean of m a x i m u m A405 for each hybridoma supernatant for each test hapten, bData analyzed by one-way A N O V A ; F-ratio = 29.66, F_< 0.00001.
134
clonal antibody with greater specificity for binding to the fluorophosphorus portion of the soman molecule, we chose an immunogen which was coupled to the carrier protein through the pinacolyl portion of the soman molecule. As reported previously [3, 4] the current monoclonal antibodies also demonstrated high specificity for soman in comparison to other, structurally related, haptens (Table 1, Fig. 2). Based on the properties of our current monoclonal anti-soman antibodies we plan to proceed to produce them in sufficient quantity to determine if they afford passive in vivo protection. We also undertook the production of a monoclonal antibody which preferentially bound to the CsPs-soman stereoisomer, using an immunogen of known absolute configuration as determined by Xray crystallographic analysis. The results of an E I A (Table 2) indicate that this antibody exhibited binding specificity for one of the four soman stereoisomers. This was in marked contrast to the antibodies against racemic soman which exhibited only a slight degree of stereospecificity in binding for the four soman stereoisomers [7]. In order to determine if this monoclonal antibody bound one of the toxic soman stereoisomers, either the C - P or C + P - stereoisomer [7], a C I E I A was carried out using pure samples of (C + P + )-soman or its mirror image ( C - P - )-soman (Fig. 3). These results indicate that the monoclonal antibody produced against the CsPs-soman stereoisomer conjugate does bind preferentially to one of the toxic soman stereoisomers, the (C - P - )-soman isomer. Based on these preliminary results we have assigned (C-P-)-soman the absolute configuration of CsP s and (C + P + )-soman the absolute configuration of CRPR. Our results further suggest that the absolute configurations of ( C - P + ) - s o m a n and
(C + P - )-soman are CsP R and CRPs, respectively. As demonstrated in this work, an antibody specific for one of the soman stereoisomers of known absolute stereochemical conformation can prove to be a useful tool in assigning absolute stereochemical conformation to the soman stereoisomers currently identified as C + P + , C + P - , C - P + and C - P - . We are continuing this effort by attempting to produce a monoclonal antibody against the CsPR-soman stereoisomer conjugate which will allow us to confirm our preliminary assignment of absolute configuration. An antibody with stereospecific binding properties toward the toxic isomers and with a sufficiently high binding constant should be more effective in conferring passive in vivo protection against soman toxicity than a monoclonal antibody raised against a racemic immunogen. To that end we intend to produce sufficient quantities in ascites fluid so that the passive protective properties of each of the two antibodies reported here can be compared for their ability to confer protection against soman toxicity in vivo.
References [1] Butler, V.P. (1982) Pharmacol. Rev. 34, 109. [2] Hunter, K.W., Lenz, D.E., Brimfield, A.A. and Naylor, J.A. (1982) FEBS Lett. 149, 147. [3] Brimfield, A.A., Hunter, K.W., Lenz, D.E., Benschop, H.P., Van Dijk, C. and de Jong, L.P.A. 0985) Mol. Pharmacol. 28, 27. [4] Lenz, D.E., Brimfield, A.A., Hunter, K.W., Benschop, H. P., de Jong, L. P.A., Van Dijk, C. and Clow, T. R. (1984) Fund. Appl. Tox. 4, S156. [5] Buenafe, A.C. and Rittenberg, M. B. (1987) Mol. Immunol. 15, 177. [6] Buenafe, A.C., Markowski, F.F. and Rittenberg, M.B. (1989) J. Immunol. 143,539. [7] Benschop, H.P., Konings, C.A.G., van Genderen, J. and de Jong, L. P. A. (1984) Toxicol. Appl. Pharmacol. 72, 61.
135
Elsevier IMLET 01724
Monoclonal antibodies against soman: characterization of soman stereoisomers* D a v i d E. L e n z , J e f f r e y J. Y o u r i c k , J. S h a w n D a w s o n a n d Jill Scott United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, U.S.A.
(Received 28 June 1991; accepted 4 September 1991)
1. Summary
2. Introduction
Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman(C+P+,C+P-,C-P+ andC-P-).In all cases the antibodies tested exhibited ICs0 values of 1 0 - 4 - 5 × 1 0 - 6 M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-sOman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers.
Immunologic means of altering the pharmacologic and toxic effects of chemicals have been summarized previously [1]. Recently, Hunter et al. [2] reported on the identification of a monoclonal antibody specific for the toxin anticholinesterase inhibitor pinacolylmethyl phosphonofluoridate (soman, GD), i.e., an anti-soman monoclonal antibody. Brimfield et al. [3] characterized the specificity of monoclonal anti-soman antibodies and Lenz et al. [4] investigated the ability of monoclonal anti-som a n antibodies to confer protection when administered passively to mice. Additional studies on the combining site specificity were carried out by Buenafe and Rittenberg [5] and Buenafe et al. [6] investigated the relationship between fine specificity and particular genes. While these results held the promise of producing a monoclonal antibody which could confer passive protection against soman toxicity in vivo, several technical difficulties remained to be overcome. The antibodies produced by Hunter et al. [2], Brimfield et al. [3] and Lenz et al. [4], while IgG-class antibodies, lacked sufficient affinity to reduce the in vivo concentration of soman to non-toxic levels and they showed only slight stereospecificity. The latter point is of considerable importance since, of the four stereoisomers of soman, two of them, designated C _+P - , are considerably more toxic than the other two, designated C _+P + [7]. I f these two problems could be overcome, then a monoclonal anti-soman antibody might have the requisite properties to confer in vivo protection over an extended ( 1 0 - 20 day) period of time after passive administration.
* The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the "Guide of the Care and Use of Laboratory Animals" of the Institute of Laboratory Animals Resources, National Research Council. Key words: Soman; Antibodies; Stereoisomers; Absolute con-
figuration Correspondence to: D.E. Lenz, United States Army Medical
Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, U.S.A.
0165- 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
131
In the present work, we were able to produce several new monoclonal anti-soman antibodies. We have examined these antibodies for their antibody class and their relative affinities for a racemic soman mixture, each of the soman stereoisomers and a variety of structurally related haptens. We have also been able to produce a monoclonal antiCsPs-soman antibody and have used it to make a preliminary assignment of absolute configuration for the stereoisomers of soman. 3. Materials
and Methods
3.1. Soman-protein conjugates and other chemicals Analogues of soman covalently bound to bovine serum albumin (GD-BSA) or to keyhole limpet hemocyanin (GD-KLH) via a succinamido linking group (Fig. l) at varying weight ratios, e.g., 3 3 - 1 0 0 m g of a particular soman analogue per 160mg of protein, were obtained from Steroids Ltd., Chicago, IL. The material was a lyophilized powder and used as supplied. The analogues included racemic soman as well as the CsPs-, CsPR-, o ,
~ I I
H
The monoclonal anti-soman antibodies were prepared by a modification of the method previously described [2]. Adult female BALB/c mice (Charles River, Boston, MA) were immunized subcutaneously with 100/~g (protein) of soman-BSA for rac~ (CHs)a . ~ C ~
CH= C~
p-amtnophenyl methyl phoephonate
0
3.2. Preparation o f monoclonal antibodies
C~
I I c~
CRPs-, and CRPR-soman stereoisomer analogues. The absolute configuration of the stereoisomer analogues was confirmed by X-ray crystallographic analysis. Soman, isopropylmethyl phosphonofluoridate (sarin) and tabun (Fig. 1) were obtained from the Analytical Chemistry Branch, USAMRICD and were > 95o70 pure as determined by 31p-NMR or gas chromatography. The C-P-, C-P+, C+Pand C + P + soman stereoisomers were obtained from the Prins Maurits Laboratory, TNO, The Netherlands. They were supplied in ethyl acetate at a concentration of 1.5mg/ml and were all greater than 95°7o optical purity and greater than 92°70 chemical purity. They were used as supplied. All other chemicals were the highest purity commercially available and were used as supplied.
0
c~- " ~C --0-CH3/
XO . - - P ~ - ~ C - - - - C ~
I I
II
P----F
]
c~ sertn
"hydroxy° eolian 0
H
dtptnecolyl methyl phoephonete
H
I
o
I II I C-.-'-'O--p----.O-...--C~ (Cl'l~ -, I CH= I I H C1%
H
0
F _ P-----~---C----~
I
CHa~ H
dlteopropyl methyl phosphonate H
C~
II I I CmO- - P- ' - " 1 1 - - - ~ " " ~ " " - ~ 2 ~
I
H ...Ckt.
c~
eoIRn
0
II
c..>c - - - ~ - ~ < . ~ "
I I
0
~=',
1 I lmunogen or test antigen
PROTEIN
(CH~}8-N ~
II
N
I tsbun
Fig. 1. Structures of i m m u n o g e n used to elicit antibody response in mice and test haptens.
132
cemic soman or soman-KLH for the soman stereoisomers emulsified in complete Freund's adjuvant. At three succeeding fourteen-day intervals, the mice received a booster immunization in incomplete Freund's adjuvant of 100 #g of soman-BSA or CsP s soman-KLH, 150/~l/animal. After the second and third booster, a sample of blood was obtained by orbital bleeding for testing the antibody response by enzyme immunoassay as described below. If a positive antibody response was obtained after the third booster immunization, spleens of positive responders were removed 4 days after the last booster immunization and splenic lymphocytes were fused with the hypoxanthine-guaninephosphoribosyltransferase-deficient mouse plasmacytoma line P3-X63-AG8.653. Fusion was accomplished with 35°70 polyethylene glycol (M l 1400, American Type Culture Collection, Rockville, MD) and fused cells were plated in 96-well, sterile, flat-bottomed microtiter plates at 5.0 × 105 cells/well. Hybridomas were selected in hypoxanthine-aminopterin-thymidine medium for the first week, with changes of culture medium on alternate days. 3.3. Enzyme immunoassay and cloning Supernatants from 14-day hybridoma cultures were analyzed for anti-soman antibody production by enzyme immunoassay. Wells of flat-bottomed polystyrene microtiter plates (Dyanatech, Alexandria, VA) were incubated with 50 #l of soman-KLH for racemic soman antibodies or CsP s soman-BSA for the stereoisomer immunogens, at a concentration of 50/~g/ml in 0.1M Na2CO 3 (pH9.6) overnight at 4°C. These plates were washed with phosphate-buffered saline (pH7.4) containing 0.5°7o Tween 20 (PBS-T) by five repetitions of filling and emptying each well. Aliquots of hybridoma supernatants (50/A) were added to the somanKLH- or soman-BSA- (see above) coated wells i n duplicate and incubated for 60 min at room temperature. Unbound antibody was washed away as above and 50/zl of rabbit anti-mouse gamma chain alkaline phosphatase-conj ugated antiserum (1:1000 in PBS, Sigma Chemical, St. Louis, MO) was added. Following a 60-min incubation, unbound rabbit antibody was Washed away as above and 50#I aliquots of enzyme substrate (p-nitro-
phenylphosphate; Sigma Chemical Co.), 1 mg/ml in 10o70diethanolamine buffer (pH 9.6), was added. The enzymatic reaction was allowed to proceed and the colored reaction produced was measured spectrophotometrically at 405 nm in a Titertek Multiscan Micro-ELISA Reader (Flow Labs, Vienna, VA). Duplicate determinations > 4 times background were considered to be positive. Positive supernatants were further screened for reactivity with free soman by a modified competitive inhibition enzyme immunoassay (CIEIA) [2]. 4. Results
When the supernatant solutions containing antibody derived from mice immunized with the racemic-soman conjugate were screened for monoclonal anti-soman antibodies, fifteen clones tested positive. When these antibodies were tested using an EIA where rabbit anti-mouse-IgG antiserum or rabbit anti-mouse-IgM antiserum was used in place of non-specific rabbit anti-mouse antiserum, a positive response was obtained only with the IgG-specific antiserum, indicating that these antibodies were IgG class antibodies. Of these, six were chosen for further testing and expansion. Each of these six monoclonal antibodies was tested for its relative avidity for soman and several soman analogues by CIEIA. In all cases, the monoclonal antibodies exhibited high specificity for soman and little or no cross reaction with sarin, tabun, pinacolylmethyl phosphonic acid (hydroxy soman), diisopropyl methylphosphonate, dipinacolyl methylphosphonate or p-aminophenyl methylphosphonate (structures of all test haptens given in Fig. 1). Typical results for one monoclonal anti-soman antibody are shown in Table 1 and Fig. 2. The six antibodies were also screened by CIEIA for their ability to bind to t h e C - P - , C - P + , C + P - and C + P + stereoisomers of soman. Each of these antibodies were inhibited equally well by the four soman stereoisomers (data not shown). Supernatants from hybridomas derived from mice immunized with the CsPs-soman-KLH conjugate were screened for monoclonal antibodies against each of the soman stereoisomer-BSA conjugates using an EIA. Over thirty clones tested positive. When tested in an EIA with the three other stereoisomer-BSA conjugates the order of binding 133
TABLE l ICs0 values of haptens b o u n d to monoclonal anti-soman antibody using GDM-KLH coated plates. Compound
IC5o (M)
Hydroxy s o m a n p - A m i n o p h e n y l methylpinacolylphosphonate Diisopropyl methylphosphonate Dipinacolyl methylphosphonate Soman Satin Tabun
- 5 x 10 -3 - 10 - 4 > 10- 3 NS - 8 X 10 - 6 NS NS
two of the soman stereoisomers, ( C - P - )-soman and (C + P + )-soman. Each of these clones was inhibited by the (C - P - )-soman isomer but not the (C + P + )-soman isomer (Fig. 3). 5. Discussion In the current study, we have been able to produce monoclonal anti-soman antibodies that were specific for the fluorophosphonate portion of the soman molecule (Table 1) and also IgG-class antibodies. In previous studies, monoclonal anti-soman IgG-class antibodies were produced [2 - 4] but they exhibited greater specificity with respect to the pinacolyl portion of the soman molecule. That specificity was attributed to the fact that the immunogen used in those experiments was linked to the carrier protein through the position normally occupied by the fluorophosphorus bond. Those monoclonal antibodies, when administered passively in mice, were able to delay the time to death of a lethal dose of soman [4]. In an attempt to make a mono-
NS, not significant. 0.7o -
I
o,6O,, ~
o.~
/
I
•
0.~0. 0,10. O'O~g_8
lg/?
11_6
1E1_5
1El_4
IEL3
HAPTEN CONe. (M)
"~
Fig. 2. Competitive binding assay of hybridoma supernatant solution of monoclonal anti-soman antibody with s o m a n A, tabun o, and hydroxy s o m a n a . All data points are the average of two determinations.
~" ~ "~
preference was CsPR-soman-BSA > CRPs-somanBSA _> CRPR-soman-BSA conjugate (Table2). In each case, the antibodies showed greatest preference for the CsPs-soman-BSA conjugate in an EIA. The six clones which gave the best response in the E I A were further examined using a C I E I A with
1.50 1.40, 1,30' 1.20 1.101 1,00, O.OO' 0.80" 0.70. 0,6O. 0.500.400.300,20 0.10 O.OO
,~-,
,~'-0
,~'-7
,~'-0
I
,~-6
,~'-,
,~'-~
HAPTEN CONC. (M) Fig. 3. Competitive binding assay of hybridoma supernatant solution of monoclonal anti-CsP s s o m a n antibody with (C - P - )s o m a n • and (C + P + )-soman e.
TABLE 2 Binding o f C s P s - s o m a n monocional antibody to stereoisomer conjugates in an EIA. Conjugate
Mean A a + S.D. (N = 32)
CsPs-soman
CsPR-soman
CRPs-soman
CRPg-soman
0.882 0.164
0.726 0.153
0.594 0.112
0.596 0.131
aMean of m a x i m u m A405 for each hybridoma supernatant for each test hapten, bData analyzed by one-way A N O V A ; F-ratio = 29.66, F_< 0.00001.
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clonal antibody with greater specificity for binding to the fluorophosphorus portion of the soman molecule, we chose an immunogen which was coupled to the carrier protein through the pinacolyl portion of the soman molecule. As reported previously [3, 4] the current monoclonal antibodies also demonstrated high specificity for soman in comparison to other, structurally related, haptens (Table 1, Fig. 2). Based on the properties of our current monoclonal anti-soman antibodies we plan to proceed to produce them in sufficient quantity to determine if they afford passive in vivo protection. We also undertook the production of a monoclonal antibody which preferentially bound to the CsPs-soman stereoisomer, using an immunogen of known absolute configuration as determined by Xray crystallographic analysis. The results of an E I A (Table 2) indicate that this antibody exhibited binding specificity for one of the four soman stereoisomers. This was in marked contrast to the antibodies against racemic soman which exhibited only a slight degree of stereospecificity in binding for the four soman stereoisomers [7]. In order to determine if this monoclonal antibody bound one of the toxic soman stereoisomers, either the C - P or C + P - stereoisomer [7], a C I E I A was carried out using pure samples of (C + P + )-soman or its mirror image ( C - P - )-soman (Fig. 3). These results indicate that the monoclonal antibody produced against the CsPs-soman stereoisomer conjugate does bind preferentially to one of the toxic soman stereoisomers, the (C - P - )-soman isomer. Based on these preliminary results we have assigned (C-P-)-soman the absolute configuration of CsP s and (C + P + )-soman the absolute configuration of CRPR. Our results further suggest that the absolute configurations of ( C - P + ) - s o m a n and
(C + P - )-soman are CsP R and CRPs, respectively. As demonstrated in this work, an antibody specific for one of the soman stereoisomers of known absolute stereochemical conformation can prove to be a useful tool in assigning absolute stereochemical conformation to the soman stereoisomers currently identified as C + P + , C + P - , C - P + and C - P - . We are continuing this effort by attempting to produce a monoclonal antibody against the CsPR-soman stereoisomer conjugate which will allow us to confirm our preliminary assignment of absolute configuration. An antibody with stereospecific binding properties toward the toxic isomers and with a sufficiently high binding constant should be more effective in conferring passive in vivo protection against soman toxicity than a monoclonal antibody raised against a racemic immunogen. To that end we intend to produce sufficient quantities in ascites fluid so that the passive protective properties of each of the two antibodies reported here can be compared for their ability to confer protection against soman toxicity in vivo.
References [1] Butler, V.P. (1982) Pharmacol. Rev. 34, 109. [2] Hunter, K.W., Lenz, D.E., Brimfield, A.A. and Naylor, J.A. (1982) FEBS Lett. 149, 147. [3] Brimfield, A.A., Hunter, K.W., Lenz, D.E., Benschop, H.P., Van Dijk, C. and de Jong, L.P.A. 0985) Mol. Pharmacol. 28, 27. [4] Lenz, D.E., Brimfield, A.A., Hunter, K.W., Benschop, H. P., de Jong, L. P.A., Van Dijk, C. and Clow, T. R. (1984) Fund. Appl. Tox. 4, S156. [5] Buenafe, A.C. and Rittenberg, M. B. (1987) Mol. Immunol. 15, 177. [6] Buenafe, A.C., Markowski, F.F. and Rittenberg, M.B. (1989) J. Immunol. 143,539. [7] Benschop, H.P., Konings, C.A.G., van Genderen, J. and de Jong, L. P. A. (1984) Toxicol. Appl. Pharmacol. 72, 61.
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