Isolation and characterization of monoclonal antibodies against rat liver epoxide hydrolase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 1, February 15, pp. 79-88, 1983

Isolation CLAUDIA

and Characterization against Rat Liver

of Monoclonal Epoxide Hydrolase

A. TELAKOWSKI-HOPKINS,**’ ANTHONY AND CECIL B. PICKETT*

Antibodies Y. H. LU,t

l Lkpartment

of Biochemical Regulation and tL?epurtment of Animal Lh-ug Metdolism, Merck Sharp & Dohme Research Labomlorim, Rahway, New Jersey 0706.5 Received

July

19, 1982. and in revised

form

September

27, 1982

We have established hybridoma cell lines which secrete monoclonal antibody to rat liver microsomal epoxide hydrolase. All of the monoclonal antibodies formed against epoxide hydrolase are mouse immunoglobulin subclass IgG,. Utilizing double immunodiffusion analysis, we have found that the monoclonal antibodies bind to and precipitate epoxide hydrolase in solubilized rat liver microsomes. Despite the ability of the monoclonal antibodies to precipitate rat liver microsomal epoxide hydrolase, they do not inhibit the catalytic activity of the enzyme but rather stimulate it. The monoclonal antibodies do not precipitate epoxide hydrolase in microsomes isolated from hamster, rabbit, monkey, or human. When rabbit reticulocyte lysates are programmed with rat liver mRNA, the primary translation product of epoxide hydrolase can be immunoprecipitated from the translation system using the monoclonal antibodies. The ability of the antibodies to recognize in vitro synthesized epoxide hydrolase should make them amenable to identify recombinant bacterial clones expressing this protein. The monoclonality of these antibodies and their specificity should provide a useful way of identifying, purifying, and quantitating rat liver epoxide hydrolase as well as examine the expression of the gene(s) encoding epoxide hydrolase in normal rats and in rats exposed to carcinogenic xenobiotics. Recent work from our laboratory has focused on the molecular events which lead to an increase in epoxide hydrolase as a function of induction by various xenobiotits. We have utilized polyclonal antibodies raised against rat liver epoxide hydrolase in immunoelectrophoretic and in vitro translation assays to demonstrate that phenobarbital leads to an increase in the microsomal content of epoxide hydrolase by way of an elevation in functional mRNAs specific for the protein (6,7). Consequently, the molecular events which are responsible for the elevation in epoxide hydrolase appear to be at the transcriptional or post-transcriptional level. Polyclonal antibodies against epoxide hydrolase have been important tools in quantitating this protein in rat liver microsomes and identifying the primary translation product in cell-free protein synthesizing systems. However polyclonal antibodies in general recognize multiple antigenic determinants on proteins and thus are not highly specific if some of these

Cytochrome P-450 and epoxide hydrolase are integral membrane proteins of the endoplasmic reticulum and function in concert in the metabolism of various drugs, mutagens, carcinogens, and other foreign compounds (l-5). The chemically reactive epoxides which are generated by the mixed function oxidase system have various metabolic fates within the cell. The enzymatic conversion of the epoxides to dihydrodiols by epoxide hydrolase represents a major step in the metabolism of these chemically reactive intermediates. Although this reaction is considered a detoxification process, some of the dihydrodiols formed from large polycyclic hydrocarbons can be further metabolized by the cytochrome P-450 system to chemically reactive dihydrodiolepoxides which are rather poor substrates of epoxide hydrolase. These diol-epoxides bind directly to cellular DNA, RNA, and protein. ’ Author dressed.

to whom

all correspondence

should

be ad-

79

ow3-9861/83/030079-10$03.00/0 Copyright All riyhta

e 1981 by Academic Press. Inc. of reproduction in any form reserved

80

TELAKOWSKI-HOPKINS,

determinants are present on other proteins. Consequently assays which are designed to detect proteins present at low concentrations are complicated because of nonspecific binding of the polyclonal antibodies to other antigens. To circumvent these problems, K6hler and Milstein (8) devised a method to construct hybrid myelomas, hybridomas, that make antibody to a single antigenic determinant on an antigen. In this study, we have utilized this technique to prepare monoclonal antibodies against rat liver microsomal epoxide hydrolase. These antibodies are highly specific for rat liver microsomal epoxide hydrolase and are able to bind to and precipitate the antigen in solubilized microsomes as well as in cell-free translation systems programmed with messenger RNA. MATERIALS

AND

METHODS

Pur@xtion of liver microsomd epoxide hydrolase. Rat liver microsomal epoxide hydrolase was purified by the method of Lu and Levin (9) whereas human liver microsomal epoxide hydrolase was purified by the method of Lu et al (10). Immunization of mice and cell fukm. Male BALB/ C mice (Charles River) received three intraperitoneal injections of 120 fig of purified rat liver epoxide hydrolase in complete Freund’s adjuvant (Cappel) over a 5-month period. Three days prior to fusion, the mice received a tail vein injection of 150 pg of epoxide hydrolase. Spleens were removed and perfused with Dulbecco’s modified Eagle’s medium (DMEM) using an l&gauge needle. The spleen cells which were liberated by this procedure were rinsed twice in serumfree DMEM. The parental mouse myeloma cell-line P3-X63-Ag8653 (Human Genetic Mutant Cell Repository GM 3570) was grown on DMEM containing 4.5 g/liter glucose + 10% fetal bovine serum (FBS: GIBCO) + 20 fig/ml 8-azaguanine (Sigma) for 9 days prior to fusion. The medium was changed every 2 days and 12 h prior to fusion. For cell fusion, approximately 10’ spleen cells were pelleted by centrifugation with approximately 2.0 X 10’ myeloma cells. Cell fusion was performed using a slightly modified procedure of Kennett et al. (11). The packed cells were resuspended in 0.4 ml of 30% polyethylene gly~01-1000 (Sigma), pH 7.6, which was maintained at 3’7’C. The cells were centrifuged at 1200 rpm (Beckman) for 5.0 min and the PEG was removed after 8.0 min. Five milliliters of serum-free DMEM was then ’ Abbreviations used: PBS, fetal bovine serum; PEG, polyethylene glycol-1000, ELISA, enzyme-linked immunosorbent assay; HAT, hypoxanthine-aminopterin-thymidine; DMEM, Dulbecco’s modified Eagle’s medium.

LU,

AND

PICKETT

gently added to the pellet and the cells were centrifuged for 2.0 min. The packed cells were diluted with 30.0 ml DMEM containing 10% FBS, 10% RPM1 1640 (GIBCO), 0.1 mM hypoxanthine (Sigma), and 16 PM thymidine (Sigma) and plated out at one drop per well into 96-well microtiter culture plates (Limbro). Cells were grown overnight at 37°C in a humidified 10% CO2 atmosphere and after 24 h one drop of DMEM containing 10% FBS, 10% RPM1 1640, 0.1 mm hypoxanthine, 16 pM thymidine, and 0.4 PM aminopterin (Sigma) was added to each well, thereby completing the HAT medium (the final concentration of aminopterin being 0.2 MM). The cells were maintained at 37°C in a humidified 10% CO2 atmosphere on complete HAT medium for 1 week during which time the cells were fed twice. After 1 week, the HAT medium was gradually replaced by HAT-free medium. Colonies of HAT resistant cells were grown for an additional 8 days with two changes of medium during this period and were scored for colony size. Enzyme-linked immunosorbent assay and cloning by limiting dilution Culture fluids from wells showing positive growth were screened for specific antibody production using an enzyme-linked immunosorbent assay (ELISA) (Bethesda Research Labs). Approximately 10 pg of purified epoxide hydrolase was diluted in 0.1 M sodium carbonate buffer, pH 9.6, and applied to each well of a 96-well polyvinylchloride dish (Dynatech). Ten microliters of 1 mg/ml carbodiimide (Sigma) in carbonate buffer was then added. After overnight incubation at 4”C, the wells were washed with PBS and incubated for 30 min with 0.1 M ammonium chloride. Wells were washed again with PBS and incubated for 30 min with PBS containing 10% BSA (fraction V-Miles) and 0.05% Tween-20 (Sargent Welch). Culture medium, 75 ~1, from the hybridomas or control medium from the parental myeloma cell line was added to the wells and incubated at room temperature for 2.0 h. The plates were washed four times with Buffer A (PBS containing 1.5 mM MgCIZ, 2 mM @-mercaptoethanol, 0.05% Tween-20, and 0.05% sodium azide) and incubated with 50 ~1 of @-galactosidase conjugated to sheep anti-mouse IgG. The enzyme conjugated to sheep anti-mouse IgG was diluted 1:200 with Buffer A. The plates were incubated 2.0 h at room temperature, washed four times with Buffer A and finally incubated for another 1.0 h with 50 ~1 of 1 mg/ml pnitrophenyl+D-galactoside. The reaction was stopped by the addition of 50 ~1 of 0.5 M sodium carbonate and the absorbancies read at 414 nm using a microtiter plate scanner (Titertech, Flow Labs). Colonies which had absorbancies four times greater than background and exhibited significant growth were cloned twice by limiting dilution. Cells were plated out at a density of one cell per well in a 96-well microtiter dish containing an irradiated feeder layer of 3T3 fibroblasts in DMEM. After 3 weeks colonies generated by limiting dilution were again screened by the ELISA. Positive

MONOCLONAL

ANTIBODIES

TO EPOXIDE

81

HYDROLASE

colonies were transferred to 24-well plates (Costar) containing the irradiated 3T3 cells and were eventually transferred to 25-cm2 flasks (Corning) as the cell population increased. The 25 cm2 flasks did not contain feeder layers and the serum concentration of the medium was raised to 20%. Preparation medium or

of monoclonal

antibodies

from culture

in mouse pedmed aec&eJluid Positive clones were grown on large scale as outlined previously and monoclonal antibody was concentrated from the culture medium by ammonium sulfate precipitation (11). Culture medium was diluted 1:l with saturated ammonium sulfate (pH 7.1) and kept on ice for 1.0 h. The medium was spun at 48,000~ for 30 min at 4°C. The pellet was resuspended in a minimum volume of sterile water and dialyzed against PBS for 48 h. Hybridomas producing antibodies were grown in 75-cm2 flasks containing DMEM plus 20% FBS, eellected by centrifugation, resuspended in PBS, and injected, ip, into pristaned BALB/C male mice (-lo6 cells/injection). After 2-3 weeks the ascites fluids were collected, and the cells removed by centrifugation. Rocket immunoelectrophoresis and do&l& immunodiflwion analysis. Rocket immunoelectrophoresis was performed using a 1% agarose slab gel containing 0.2% Emulgen 911,0.5% sodium eholate, and Trissodium barbital buffer, pH 8.8 (Gelman Instrument Co.). The rocket immunoelectrophoresis procedure has been published in detail (6). The Ouchterlony double-immunodiffusion assay was utilized to determine the class, subclass, and specificity of the monoelonal antibodies. Various antigen or antibody solutions (~-PI samples) were placed in the wells of Ouchterlony dise gels (Hyland Laboratories), incubated at room temperature and observed for precipitin bands which usually occurred within 24 h. Cell-free protein synthesis and immunqnrecipitation using mxmo~kmal antibody against epoxide hydrolase. In vitro synthesized epoxide hydrolase was immu-

noprecipitated from a cell-free translation system using monoclonal antibody against epoxide hydrolase. Liver poly(A+)-RNA was isolated from rats treated acutely with phenobarbital and translated in a rabbit reticulocyte cell-free system using [%S]methionine as the radiolabeled amino acid. Details of the isolation of poly(A+)-RNA and in vitro translation have been published previously (7). After a 90min incubation at 3O”C, the translation mixture was diluted with 1.0 ml 50 mM Tris-HCl, pH 7.5, 6 mM EDTA, 190 mM NaCl, 0.5% sodium cholate, and 0.2% Emulgen 911. The solution was spun for 60 min at 40,000 rpm in a SW-60 rotor (Beckman) to pellet the ribosomes. The resulting supernatant was transferred to 1.5-ml Eppendorf microfuge tubes and 50 ~1 of the monoclonal antibody was added. The antibody had been concentrated from the culture medium by ammonium sulfate precipitation. The sample was incubated overnight at 4°C with end-to-end rotation,

A

6

FIG. 1. (A) Immunological detection of mouse antibody titer against purified rat liver epoxide hydrolase. Before fusing isolated mouse spleen cells with the parental myeloma cell, P3-X63-Ag8653, serum was collected from mice which had received injections of purified rat liver epoxide hydrolase and examined for the presence of antibodies against epoxide hydrolase by rocket immunoelectrophoresis. Seven microliters of purified rat liver epoxide hydrolase (36 pg/ml) was placed in the well and electrophoresed into 1% agarose gel containing mouse serum. The rocket-shaped preeipitin band was detected by staining the dried gel with Coomassie Blue R-250. (B) Ouchterlony immunodiffusion analysis of the class of antibodies being secreted by hybridoma (16-15). Hybridoma 16-15 was examined for the production of antibodies by double immunodiffusion analysis. The clone was grown in large cultures and the antibodies were concentrated from the culture medium by ammonium sulfate precipitation. The concentrated monoclonal antibodies were reacted against various rabbit anti-mouse class and subclass antibodies. The center well contained seven microliters of the concentrated monoclonal antibodies. Well 1, rabbit anti-mouse IgGzb; well 2, rabbit anti-mouse IgG*; well 3, rabbit anti-mouse IgM; well 4, rabbit anti-mouse IgGi; well 5, rabbit antimouse IgG%. The precipitin bands were detected by staining with Coomassie Blue G-250.

82

TELAKOWSKI-HOPKINS,

LU,

FIG. 2. (Upper) Control P3-X63-Ag8653 cells on HAT ridomas on HAT selective medium 8 days postfusion. Twenty r-five microliters (Miles) was then added tinued for an additional

of rabbit anti-mouse IgGl and the incubation was con2.0 h. Finally, 100 pl of a

AND

selective

PICKETT

medium

for 5 days.

(Lower)

Hy-

protein A-Sepharose CL-4B slurry (Pharmacia) 7was added and the incubation was continued for anot .her 2.0 h. The protein A-Sepharose beads containing the

MONOCLONAL

ANTIBODIES

antigen-monoclonal antibody-IgG, complex were spun down and washed three times with 1 ml 50 mM TrisHCl, pH 7.5,6 mM EDTA, 190 mM NaCI, 0.5% sodium cholate, and 0.2% Emulgen 911. To elute the antigen, the sample was boiled for 5 min in 100 ~15 mM, TrisHCI, pH 7.5, 5% SDS, 20% glycerol, 10 mM DTT, and 2% fi-mercaptoethanol. The eluted antigen was run on a 7.5% SDS-polyacrylamide gel (12). In order to visualize the radiolabeled epoxide hydrolase, the gel was subjected to fluorography using PHIENHANCE (New England Nuclear Corp.). kfeasurement of catal@ic activity c&3- binding of mono&ma1 antibody to epoxide hydrolase. Monoclonal antibodies which had been concentrated by ammonium sulfate precipitation from culture medium were preincubated at room temperature with purified epoxide hydrolase (0.21 rg/ml). Following preincubation epoxide hydrolase activity was determined by measuring the conversion of [‘7-3H]styrene oxide to [7-3H]styrene glycol (13). RESULTS

Production of mouse serum antibodies and mono&ma1 antibodies against rat liver epoxide hydrolase. Male BALB/C mice which were immunized over a 5-month period with highly purified rat liver epoxide hydrolase produced serum antibodies which were detected by their ability to precipitate the purified protein in a rocket immunoelectrophoresis assay (Fig. 1A). Spleen cells (~10~) were isolated from these animals and fused with 2 X 10’ myeloma cells. The fusion resulted in the growth of hybrid cells in 99 of the 496 wells which contained selective HAT medium (Fig. 2). Hybrid cells (30 wells) which exhibited significant growth on the selective medium were screened for the production of epoxide hydrolase antibody by ELISA as described under Materials and Methods. The assay utilizes &galactosidase conjugated to sheep anti-mouse IgG and p-nitrophenyl-P-D-galactoside as substrate. As can be seen from Fig. 3, approximately 19 wells exhibiting growth produced antibodies against epoxide hydrolase as indicated by an absorbancy of at least two times over background in the ELISA. One well, No. 16, was selected for subcloning since growth in this well was very vigorous. Subcloning was achieved by limiting dilution at a cell density of one cell per well in a 96-well microtiter dish. We found that it was necessary to plate the cells out onto an irradiated layer of 3T3 fibroblasts in order to insure growth of the hybrid cells at the low plating den-

TO

EPOXIDE

HYDROLASE

83

1.5 0 -2

1.0 .5

B I 2 3 5 6 1 6 9 10111213151611W19202122W2525212~2930 FIG. 3. Enzyme-linked immunosorbent assay of hybridomas generated by cell fusion. Approximately 2 weeks after fusion of mouse spleen cells with the parental myeloma cells, hybridomas exhibiting growth were screened for the production of antibody against rat liver epoxide hydrolase. One well, 16, which had an absorbance greater than three times over background in the ELISA, was chosen for subcloning as described under Materials and Methods.

sity. After 3 weeks in culture, clones generated by limiting dilutions were rescreened by ELISA. Approximately 73% of the individual clones derived from well 16 were positive for epoxide hydrolase antibody production, Positive clones were further subcloned by limiting dilution and rescreened by ELISA. The positive clones were transferred to 24-well plates and eventually to 25-cm2 flasks as the cell population increased. The hybridomas producing epoxide hydrolase antibodies were grown further in ‘75-cm2 flasks and the culture fluids were concentrated by ammonium sulfate precipitation. The antibody contents of the concentrates were examined by Ouchterlony double-immunodiffusion analysis. Ouchterlony double-immunodflusion analysis. The hybridoma clone No. 16-15, resulting from the fusion of spleen cells of epoxide hydrolase immunized mice with the P3-X63-Ag8653 myeloma cells produced monoclonal antibodies which formed a clear precipitin band between purified rat liver epoxide hydrolase or with solubilized microsomes isolated from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and Aroclor 1254treated rats (Fig. 4). In addition the monoclonal antibody also formed a precipitin band when reacted against antimouse IgG, (Fig. 1B). We observed no reaction with anti-mouse IgGza, IgGeb, IgG,, IgA, or IgM. Thus all of the hybrid clones producing antibodies against rat liver epoxide hydrolase were of the subclass IgGl _

TELAKOWSKI-HOPKINS,

LU,

AND

PICKETT

1

2 3 i

k

I

FIG. 4. Ouchterlony double immunodiffusion analysis of monoclonal antibody to epoxide hydrolase. Culture media from hybridoma 16-15 and the parental myeloma cell line, P3-X63-Ag3653, were concentrated by ammonium sulfate precipitation and applied to the center wells. In a-k, the center wells contained concentrated culture media from hybridoma 16-15 whereas in 1 the center well contained concentrated culture media from myeloma cells P3-X63-Ag3653. All microsomal samples were solubilized in 0.25 M sucrose, 0.01 M Tris-HCl, pH 7.4,0.5% sodium cholate, and 0.2% Emulgen 911. The Ouchterlony gels were comprised of agar and did not contain any detergents. (a) Wells 1, 3, and 5 contained solubilized liver microsomes isolated from untreated rabbits (1.3 mg/ml). Wells 2 and 4 contained purified rat liver epoxide hydrolase (0.15 mg/ml). (b) Wells 1, 3, and 5 contained solubilized liver microsomes isolated from phenobarbital-treated rabbits (2.9 mg/ ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (c) Wells 1, 3, and 5 contain solubilized liver microsomes isolated from 6-naphthoflavone-treated rabbits (1.5 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (d) Wells 1,3, and 5 contain

MONOCLONAL

ANTIBODIES

When the monoclonal antibodies from 1615 were examined for their ability to precipitate epoxide hydrolase in microsomes from other species, we observed no precipitin band formation (Fig. 4). In contrast, polyclonal antibodies raised in rabbits against epoxide hydrolase cross-reacted with microsomes isolated from hamster, monkey, and human (data not shown). Similarly, we found that the monoclonal antibodies of clone 16-15 did not form a precipitin band when reacted against purified human liver epoxide hydrolase whereas the conventional polyclonal rabbit antiserum against rat liver epoxide hydrolase formed a strong crossreaction with the human liver epoxide hydrolase showing a line of partial identity (Fig. 5). Cells from clones 16-15 and 16-19 were also grown as ascites tumors in pristanetreated BALB/C mice. The cell-free ascites supernatant was reacted against purified rat liver epoxide hydrolase. The results indicated a significantly higher epoxide hydrolase antibody titer when the cells were grown as an ascites tumor (data not shown). We have also subjected hybridoma cell line 16-15 and 16-19 to a second subcloning (hybridoma cell lines 1615-44, 16-19-10, and 16-19-15). Using the monoclonal antibodies produced by these hybridoma cell lines, we have obtained identical double immunodiffusion data as presented in Figs. 4 and 5 (data not shown). Cell-free translation and immunoprecip itatim of epoxide hydrolme. In order to de-

TO

EPOXIDE

HYDROLASE

85

termine the utility of the monoclonal antibodies against epoxide hydrolase, we examined the ability of the antibody to recognize and bind to the primary translation product of epoxide hydrolase. As can be seen from Fig. 6, the monoclonal antibody can be used to immunoprecipitate %radiolabeled epoxide hydrolase from an in vitro translation system directed by poly(A+)-RNA isolated from phenobarbital-treated rats. Since protein A does not bind mouse IgGi with high affinity, we utilized an intermediate antibody, rabbit anti-mouse IgGl, to bind the monoclonal antibodies. We then recovered the monoclonal antibody-anti-mouse IgG, complex using protein A. Although it is feasible to eliminate the protein A from this assay and utilize only the rabbit anti-mouse IgGl to recover the monoclonal antibody, we found that the protein A-Sepharose beads can be handled very easily and washed properly to remove any contaminating background. Finally, if the monoclonal antibody is not added, we observe no precipitation of radiolabeled epoxide hydrolase from the translation system (Fig. 6). Effect of wumoclonal antibody binding on the catalytic activity of pm$ied epoxide hydrolase. In order to ascertain whether the binding of the monoclonal antibodies to purified epoxide hydrolase inhibited catalytic activity, three hybridoma clones (1615-44, 16-19-10, and 16-19-15) producing monoclonal antibodies against epoxide hydrolase were preincubated with purified epoxide hydrolase and the activity was

solubilized liver microsomes isolated from untreated rats (3.1 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (e) Wells 1,3, and 5 contain solubilized liver microsomes from phenobarbital-treated rats (3.1 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrase (0.15 mg/ml). (f) Wells 1, 3, and 5 contain solubilized liver microsomes from a-methylcholanthrene-treated rats (3.4 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (g) Wells 1, 3, and 5 contain solubilized liver microsomes from Aroclor-treated rats (2.8 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (h) Wells 1, 3, and 5 contain solubilized liver microsomes isolated from untreated hamsters (2.0 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (i) Wells 1, 3, and 5 contain solubilized liver microsomes from 3-methylcholanthrene-treated hamsters (3.7 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (j) Wells 1, 3, and 5 contain solubilized liver microsomes from an untreated monkey (2.4 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ml). (k) Wells 1,3, and 5 contain solubilized liver microsomes from a human (10.0 mg/ml). Wells 2 and 4 contain purified rat liver epoxide hydrolase (0.15 mg/ ml). (1) Wells 1, 2, and 3 contain purified rat liver epoxide hydrolase (0.15 mg/ml). Wells 4 and 5 contain solubilized liver microsomes from phenobarbital-treated rats (3.1 mg/ml). Although we have not presented the data in this figure, the microsomal protein concentrations were varied betwen 9 and 1 mg/ml and at no concentration did we detect a precipitin reaction between the monoclonal antibody and the liver microsomes isolated from rabbit, hamster, monkey, or human.

86

TELAKOWSKI-HOPKINS,

LU,

AND

PICKETI

FIG. 5. Ouchterlony double immunodiffusion analysis of monoclonal and polyclonal antibody versus human liver epoxide hydrolase. (A) Monoclonal antibodies against purified rat liver epoxide hydrolase were examined for cross-reactivity with human liver epoxide hydrolase by Ouchterlony double immunodiffusion. Center well: ammonium sulfate concentrated monoclonal antibody, 16-15. Wells 1 and 3 contain 7 ~1 of purified rat liver epoxide hydrolase (0.15 mg/ml). Wells 2 and 4 contain 7 ~1 of purified human liver epoxide hydrolase (0.12 mg/ml). (B) Rabbit polyclonal antibodies against rat liver epoxide hydrolase were examined for their cross-reactivity with purified human liver epoxide hydrolase. Center well contains 7 pl of rabbit polyclonal antibodies against rat liver epoxide hydrolase. Wells 1 and 3 contain 7 pl of purified rat liver epoxide hydrolase (0.15 mg/ml). Well 2 contains 7 ~1 of purified human liver epoxide hydrolase (0.12 mg/ml).

measured. The three clones represent subclones of 16-15 and 16-19. We found that all three monoclonal antibodies stimulated rather than inhibited the catalytic activity of the purified enzyme (Table I). In contrast, polyclonal antibodies against rat epoxide hydrolase had no effect on the catalytic activity. DISCUSSION

In this investigation we describe the production and characterization of monoclonal antibodies against rat liver microsomal epoxide hydrolase. The monoclonal

antibodies bind to and precipitate the purified epoxide hydrolase in solution as well as the protein in solubilized microsomes isolated from control, phenobarbital, 3methylcholanthrene, and Aroclor-treated rats. Furthermore the monoclonal antibodies recognize and bind to the primary translation product of epoxide hydrolase which is synthesized in in vitro protein synthesizing systems. Finally when the purified enzyme is incubated with the monoclonal antibodies, a slight stimulation rather than an inhibition of catalytic activity was observed.

TABLE EFFECT

OF MONOCLONAL

ANTIBODIES

Antibody (A)

None Myeloma Hybridoma Hybridoma Hybridoma

P3-X63-Ag8653 16-15-14 16-19-10 16-19-15

(B) None Control serum* Polyclonal antibody ‘Catalytic monoclonal ’ Control

AGAINST

EPOXIDE

I HYDROLASE

Epoxide hydrolase (nmol styrene formed/min/mg

ON STYRENE

activity” glycol protein)

OXIDE

HYDRATION

Percentage control

460 443 549 554 503

100 97 119 120 109

438 430 434

100 98 100

activity was determined as described under Materials or polyclonal antibodies with purified rat liver epoxide serum and polyclonal antibody against rat liver epoxide

and Methods after preincubating hydrolase for 30 min. hydrolase were raised in rabbits.

the

MONOCLONAL

ANTIBODIES

MW x 1O-3

94 68 45-r

-

A

I3

FIG. 6. Immunoprecipitation of in vitro synthesized rat liver epoxide hydrolase by monoclonal antibody. NaDodSOJpolyacrylamide gel electrophoresis of FSlmethionine-labeled rat liver epoxide hydrolase obtained from in vitro translation mixtures programmed with poly(A+)-RNA isolated from phenobarbital-treated rats. Lane A represents a fluorogram of in vitro synthesized epoxide hydrolase immunoprecipitated from the cell-free protein synthesis system by monoclonal antibody from hybridoma 1615. Lane B represents a control experiment where monoclonal antibody against rat liver epoxide hydrolase was not present. The arrow corresponds to the position of purified epoxide hydrolase in the Coomassie brilliant blue-stained gel from which the fluorogram was obtained.

Two hybridoma cell lines generated from the fusion, 16-15 and 16-19, are highly specific for rat liver epoxide hydrolase and are not able to precipitate the enzyme from microsomes isolated from rabbit liver, hamster liver, monkey liver, or human liver. Similarly the monoclonal antibodies are not able to precipitate purified human liver epoxide hydrolase. Unlike the monoclonal antibodies, rabbit polyclonal antibodies produced a strong cross-reaction with the purified human liver epoxide hydrolase and a weak to strong cross-reaction with the microsomes isolated from the other species. All of the precipitin reactions with the polyclonal antibodies against the various microsomal samples or

TO

EPOXIDE

HYDROLASE

8'7

the purified human liver epoxide hydrolase display lines of partial identity with rat liver epoxide hydrolase. Recently DuBois et al. (14) have reported that the human and rat liver epoxide hydrolase share a great deal of structural homology. Therefore it is not surprising that the polyclonal antibodies against rat liver epoxide hydrolase cross-react strongly with purified human liver epoxide hydrolase. The inability of the monoclonal antibody to precipitate human liver epoxide hydrolase suggests that the monoclonal antibody is directed against an antigenic determinant in the rat epoxide hydrolase which is not present in the human liver epoxide hydrolase. The possibility cannot be ruled out that the monoclonal antibodies bind weakly to the epoxide hydrolase in the various microsomal samples since the immunodiffusion assay measures only the ability of the antibody to precipitate the antigen. Since monoclonal antibodies are directed to a single antigenic site they generally do not precipitate the protein antigens they bind. However, rat liver microsomal epoxide hydrolase is very hydrophobic and tends to aggregate. Consequently, the precipitation reaction which we observe between the monoclonal antibodies and epoxide hydrolase may be due to the tendency of the enzyme to aggregate. Similar findings have been reported recently with monoclonal antibodies against rabbit liver cytochrome P-450 LM2 and the 3-methylcholanthrene inducible rat liver cytochrome P-450 (15, 16). Finally, the fusion of the precipitin bands of the purified rat liver microsomal epoxide hydrolase and the solubilized microsomes from rats treated with various xenobiotics indicates that the epoxide hydrolases in these various microsomes are immunologically identical. At no time did we observe more than a single precipitin band or precipitin lines of partial identity between the purified rat liver epoxide hydrolase and the various rat liver microsomes. Taken together, these data suggest the presence of a single form of the enzyme. The monoclonal antibodies which have been produced in this study can recognize and bind to epoxide hydrolase synthesized

88

TELAKOWSKI-HOPKINS,

in cell-free systems programmed with rat liver poly(A+)-RNA isolated from phenobarbital-treated rats. These data indicate that the .antibody can be utilized as a screening reagent to identify recombinant DNA clones which are expressing epoxide hydrolase. The advantage of using a monoclonal antibody over the conventional polyclonal antibodies would be a lower background which is due to nonspecific binding of the antibody to various proteins or to a cross-reaction between the antibody and some other antigen. Recently Kemp and Cowan (17) reported on a direct immunoassay for detecting colonies containing polypeptides encoded by cloned DNA segments. This procedure should be amenable to the use of monoclonal antibodies. Although the monoclonal antibodies bind and precipitate epoxide hydrolase, we found that the antibody did not inhibit the catalytic activity of the purified protein. Therefore, the antigenic determinant for these antibodies does not appear to be necessary for the catalytic activity. The slight stimulation in catalytic activity observed with the monoclonal antibody has also been found previously with polyclonal antibody against epoxide hydrolase (18). However in the present study polyclonal antibody against rat liver epoxide hydrolase had no effect on the catalytic activity. The increase in catalytic activity suggests that binding of the antibody to the enzyme alters the configuration of the active site to facilitate catalysis. Our studies suggest that monoclonal antibodies against epoxide hydrolase will be a very useful tool not only to study the structure and function of the enzyme but also to identify epoxide hydrolase in cellfree systems and in bacterial cells expressing the protein. These findings should add significantly to our understanding of the regulation and expression of epoxide hydrolase as well as its role as a preneoplastic antigen during hepatocarcinogenesis (19, 20). ACKNOWLEDGMENTS We would like to acknowledge Dr. C. von Bahr of Karolinska Institutet for providing the human liver

LU.

AND

PICKETT

microsomes and Dr. J. Y. C. Chiang of Northeastern Ohio University for rabbit, hamster, and monkey liver microsomes. We would also like to thank Joan Kiliyanski for her assistance in the preparation of this manuscript. REFERENCES

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