Interaction of monoclonal antibodies with mammalian choline ...

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Aug 9, 1982 - Hybridization and selection in hypoxanthine/aminopterin/ thymidine medium of the fused cell lines were performed as described (10) except ...
Proc. Natl. Acad. Sci. USA Vol. 79, pp. 7031-7035, November 1982 Neurobiology

Interaction of monoclonal antibodies with mammalian choline acetyltransferase (HPLC/neurotransmitter/cholinergic/hybridoma)

G. D. CRAWFORD, L. CORREA, AND P. M. SALVATERRA* Division of Neurosciences, City of Hope Research Institute, Duarte, California 91010

Communicated by Ray D. Owen, August 9, 1982

and functional significance of multiple charge forms of the enzyme (8) and the relationship between the soluble and membrane-bound forms (9). Using experience gained in the production of monoclonal antibodies selective for Drosophila melanogaster choline acetyltransferase (10), we isolated five cell lines secreting different specific monoclonal antibodies directed against the rat brain enzyme. These antibodies were used to determine the relative spatial relationships of their binding domains on the enzyme and were applied as immunohistochemical reagents for localization of cholinergic neurons in rat spinal cord. A recent report of monoclonal antibodies prepared against the bovine enzyme, one of which crossreacts with the rat enzyme, has appeared (11). In addition, two other preliminary reports have described the production of monoclonal antibodies prepared against choline acetyltransferase in species other than rat (12, 13).

ABSTRACT Monoclonal antibodies selective for rat brain choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) were prepared by standard techniques. Five cell lines were isolated from spleen cell-SP/2 hybrids by repetitive cloning with a screening method that used the intrinsic activity of the enzyme. All cell lines secrete immunoglobulin of mouse subclass IgG1, and none inhibit the enzyme activity directly. The size of antibody-enzyme immune complexes formed with different pairs ofthe monoclonal antibodies was determined by gel filtration with HPLC. By comparing the elution position of choline acetyltransferase activity after incubation with paired monoclonal antibodies, the spatial relationship of antibody binding domains relative to each other can be defined and classified as independent, mutually exclusive, or overlapping. Immune complexes in excess of Mr 600,000 were formed by some pairs of antibodies with the antigen, indicating independent binding domains on the enzyme. In one case, the paired antibodies formed an immune complex of only Mr 300,000, indicating that they bound in a mutually exclusive fashion. In most cases, pairs ofantibodies reacted with the enzyme to give simultaneously both higher and lower Mr immune complexes. We conclude that all five antibodies bind to a relatively localized region of the enzyme surface. Antibodies were screened for usefulness as immunohistochemical markers of choline acetyltransferase-containing neurons by using the indirect immunoperoxidase method. One antibody intensely stains cell bodies of motor neurons and processes in a selective manner in the rat spinal cord and brain stem by using aldehyde-fixed tissue; the remaining antibodies do not react with fixed tissue.

MATERIALS AND METHODS Immunization and Hybridization. A young female BALB/c mouse was immunized four times over an 8-wk course with two preparations of rat choline acetyltransferase. Each preparation contained =25 .tg of protein with specific activities of 60 and 72 tkmol/min per mg. The antigen was prepared as described by Dietz and Salvaterra (3) and was delivered subcutaneously, emulsified in Freund's complete adjuvant. The final preparation (5 pug) was injected intravenously 3 days prior to fusion. Hybridization and selection in hypoxanthine/aminopterin/ thymidine medium of the fused cell lines were performed as described (10) except that the myeloma SP/2 line (14) was used in place of the NS-1 line. This procedure yielded growing colonies in 32% of the wells in the four 96-well microtiter plates seeded at the outset. Selection of Hybrids Producing Anti-Choline Acetyltransferase Antibodies. A selection system similar to that used for the production of monoclonal antibodies against Drosophila choline acetyltransferase was used (10). Culture media was screened for its ability to remove enzyme activity from solution. For screening purposes and routine analysis of choline acetyltransferase-directed antibody, 0.8-1.1 milliunits of enzyme activity in the form of a crude rat brain extract was incubated with 100 1A of each culture medium from densely grown wells. After 4-6 hr at 4°C, 15 ,tg of purified mouse IgG and 15 ,ul of goat anti-mouse IgG antiserum was added and incubated overnight at 4°C. After centrifugation, the supernatants were assayed for enzyme activity by the method of Fonnum (15). An arbitrary criterion of 25% decrease in enzyme activity, compared to activity remaining in an equivalent aliquot of parental cell culture medium, was set as the indicator of a positive response for the presence of anti-choline acetyltransferase activity. Hybridoma cultures that met this criterion were expanded to 16 cultures

In order to study the biochemical properties and regulation of cholinergic neural transmission, it will be important to have reagents that can be used as probes for the important biosynthetic enzyme, choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Numerous attempts have been made to isolate the enzyme in homogenous form (1-6), and some recent reports claim to have produced small amounts of protein from mammalian tissue that may approach this goal (5, 6). Unfortunately, the disappointingly low yields of these protocols virtually preclude a rigorous biochemical assessment of purity or a molecular characterization of the protein and, therefore, make monospecific antibody production problematic (7). Choline acetyltransferase has been purified to homogeneity from Drosophila, and a limited characterization of the protein has been reported (6). However, the mammalian enzyme remains largely uncharacterized. Production of specific monoclonal antibodies to choline acetyltransferase not only would eliminate the need for a rigorously purified antigen but also would supply a useful structural probe of the enzyme surface. Antibodies with appropriate specificity could be used to answer questions about the molecular nature The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. § 1734 solely to indicate this fact.

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To whom reprint requests should be addressed.

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to stabilize the population. A positive culture from these was then cloned repetitively by a limiting dilution method. Purification and Analysis of Monoclonal Anti-Choline Acetyltransferase Antibodies. A clone of each positive cell line was grown in fetal calf serum-supplemented medium, and the secreted antibody was concentrated by ammonium sulfate precipitation at 34%. The subclass of each antibody was then determined by Ouchterlony analysis ofthis material with subclassspecific antisera. Monoclonal antibodies were prepared on a large scale by protein A-Sepharose adsorption chromatography from ascites fluids grown from the same clone (16). All cell lines were established as ascites tumors in retired breeders after being cloned at least twice. Three of the cell lines produced ascites that had very low amounts ofantibody. Carrier IgG used for the immunoprecipitation assays was produced in a similar manner from pooled whole-mouse sera. Analysis of Immune Complexes. The molecular size of immune complexes formed in the presence of crude choline acetyltransferase preparation and one or more monoclonal antibodies was determined by gel filtration with HPLC. In contrast to our results with the monoclonal antibodies against Drosophila choline acetyltransferase (10) none of the antibodies against mammalian choline acetyltransferase were found to significantly inhibit the enzyme activity. Therefore, the elution position of immune complexes from the HPLC chromatography could be monitored by measuring enzyme activity. The enzyme present in brain extracts was concentrated 10-fold by preparing a 40-60% ammonium sulfate fraction of the extract and subsequently dialyzing it against several changes of 0.1 M sodium phosphate (pH 7.4). It was necessary to clarify these samples by using the Beckman air-driven centrifuge (30 psi, 1 psi = 6,895 Pa; 25 cm) before HPLC chromatography to avoid obstructing the column. Immune complexes were formed by incubating (40C for 3 hr) 100 ul of the enzyme sample with one or more of the desired protein A-Sepharose-purified antibodies at 50 ,ug/ml for each antibody. A portion (50 jul) of this mixture was then chromatographed on a BioRad TSK250 column developed with 0.1 M sodium phosphate (pH 7.4) at a flow rate of 0.3 ml/min. The effluent was fractionated into 250-1.l aliquots, and each fraction was immediately assessed for enzyme activity (13). In some cases the antibodies were omitted, and the entire elution profile was monitored for enzyme activity. Peak positions were compared relative to A280 of internal and external standards. The A280 profile was monitored as a measure of the reproducibility of the enzyme elution profile, and the elution position was found to vary by ± 250 1A. Molecular weight standards included: thyroglobulin, Mr 670,000; IgG, Mr 155,000; ovalbumin, Mr 45,000; myoglobin, Mr 18,500; and cyanocoalbumin, Mr 4,500 (17). Protein and Enzyme Activity Estimation. Protein levels were estimated by the Lowry technique (18), and choline acetyltransferase activity was determined by the method of Fonnum (15) with a substrate mixture containing 1.85 X 104 cpm of [1-14C]acetyl-CoA per nmol. NaDodSO4 Electrophoresis. Ascites fluids and protein ASepharose-purified antibodies were analyzed by electrophoresis on a Laemmli-type NaDodSO4/polyacrylamide gel system (19). The resolution gel matrix contained 10% acrylamide and 0. 1% N,N'-methylenebisacrylamide. Immunocytochemistry. Adult Sprague-Dawley rats were perfused through the vascular system with a solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.12 M phosphate buffer (pH 7.3). Sections from the medulla and cervical spinal cord were processed for light microscopic immunocytochemistry by the peroxidase-antiperoxidase method (20) as modified by Wainer and Levey (21). Sections were incubated

Proc. Natl. Acad. Sci. USA 79 (1982)

in solutions of each of the monoclonal antibodies at a concentration of 2.5 ,4g of antibody per ml of buffer for 18 hr at 40C. Control sections were incubated in an identically prepared monoclonal antibody against an irrelevant antigen not present in the nervous system. A detailed report describing the immunocytochemical procedures will appear elsewhere.

RESULTS Selection of Cell Lines. The fusion mixture was plated into four 96-well microtiter plates; after 2 wk of selection in hypoxanthine/aminopterin/thymidine medium, 32% of the wells contained growing cultures. Nineteen wells initially screened positive for anti-choline acetyltransferase activity, and each was expanded into 16 wells. The purpose of this strategy was to enhance the population ratio of antibody-producing cells over nonproducers in some of the wells and, thus, to strengthen the cultures that remained positive. Many of these subeloned lines reverted to nonproducers in all wells or were overgrown by cells producing antibodies that were not of interest, but eventually five cell lines were selected for cloning by limiting dilution. For two cell lines (1E6 and 4G5), all growing wells were positive for anti-choline acetyltransferase activity when tested by the enzyme-depletion method after two cloning operations. In contrast, 600,000. This suggests that both antibodies can bind independently to choline acetyltransferase. An example of two antibodies that mutually excluded each other's binding is shown in Fig. 1E. The elution profile after incubation with the antibodies 3F12 paired with 4D7 indicated a complex of Mr 300,000 and was identical to the elution position of either antibody alone. In many cases, the elution profile indicated the formation of both the higher and lower molecular weight type of immune complex. An example of this, shown in Fig. 1F, is the combination ofantibodies 4D7 and 4G5, perhaps indicating partially overlapping binding domains. The immune complexes formed upon incubation of choline acetyltransferase with each permutation of antibody pairs is summarized in Table 1. Only a single pair of antibody binding domains were mutually exclusive. Four pairs showed independent binding, whereas the remaining five showed partial overlap. The data on spatial organization of antibody binding domains is summarized in the form of a Venn diagram in Fig. 2. Each

DISCUSSION A number of attempts have been made in the past to generate specific antibodies reactive with mammalian choline acetyltransferase (22-26). Several of these reports have been criticized because the antigen was inadequately characterized (7). Use of the hybridoma technology for specific antibody production presents a solution to this problem by obviating the requirement for pure antigen. In addition, a panel of antibodies reactive with single determinants on the enzyme can be generated and used for a number of biochemical studies of the surface features of choline acetyltransferase. The critical feature in applying the hybridoma technology to select a specific antibody to an impure antigen is the screening system used. A system based on enzyme-linked immunosorbent assay that uses one of the choline acetyltransferase preparations now available as the adsorbed target suffers from the same criticisms applicable to polyclonal antisera-i.e, an in-

2F11

FIG. 2. Venn diagram showing the relationship between antibody binding domains inferred from HPLC of immune complexes. Each domain is represented as an oval. Independent domains do not intersect, interacting domains are shown with a slight overlap, and mutually exclusive domains are represented by major overlap.

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FIG. 3. Immunocytochemistry of choline acetyltransferase with antibody 3F12. (A) A section through the medulla shows choline acetyltransferasepositive motor neurons in the hypoglossal (H) and ambiguus (A) nuclei as well as in the dorsal motor nucleus of the vagus nerve (V). The axons of these neurons also are stained intensely in the emerging vagal and hypoglossal cranial nerves. (B) A section of cervical spinal cord shows choline acetyltransferase-positive motor neurons within the anterior horn. (C) A photomicrograph of a section from the cervical spinal cord incubated in a control monoclonal antibody solution shows no specific staining. In both experimental and control tissue, erythrocytes are stained due to endogenous peroxidase-like activity. (Bar = 300 ,Am.)

completely characterized antigen such as choline acetyltransferase may be grossly contaminated with other proteins. A recent preparation suggests that enzyme-specific activities of 150 pumol/min are obtainable in small amounts (12), and, in the rat, this suggests that choline acetyltransferase comprises no more than 1 part in 100,000 of the total protein. We have chosen to select hybridomas that will remove choline acetyltransferase activity from solutions. An implicit assumption of this method is that the enzyme exists in solution unassociated with other molecules. A large body of evidence supports this contention (8). By using a second antibody precipitation system to remove immune complexes from solution, five cell lines were established that produce antibodies reactive with choline acetyltransferase. All cell lines produce mouse subclass IgG1, enabling the antibody to be purified out of ascites tumors by protein A adsorption chromatography. Given the large variation in the antibody content of the ascites fluids, this step was necessary for a direct comparison of the effect of the antibodies at the same concentration on enzyme activity. In contrast to antibodies produced against Drosophila choline acetyltransferase (10), these antibodies against rat choline acetyltransferase were not found to inhibit the enzyme activity at concentrations up to 100 /g/ ml. This indicates that the antibodies react at a position(s) away from the active site of the enzyme. Because the immune complexes were enzymologically active, it was possible to directly monitor antibody-enzyme interaction by observing the change in gel filtration elution position of enzyme activity after reaction with antibodies. In all cases, the enzyme activity was found completely within the peaks corresponding to immune complexes. The cytoplasmic form of rat brain choline acetyltransferase has been shown to be a globular protein of M, 68,000 (8). By using an approximation of 40 nm as the diameter of a molecule this size, it can be calculated that choline acetyltransferase has a surface area of approximately 6,400 nm2. Although the actual antibody-binding determinant is usually the size of 6 or 7 amino acids (27) or perhaps 2 nm2, the area swept out by the binding region of one antibody arm has been estimated as 35 nm2 (27). It is reasonable to expect that if the determinants recognized by the five monoclonal antibodies were distributed randomly

on the surface of the enzyme, then all five could bind simultaneously. This has been observed for myoglobin, a globular protein of Mr 14,500, to which four out of five monoclonal antibodies were able to bind simultaneously (28). Clearly, however, this is not the case for the antibodies described here. No antibody binds independently of more than two other antibodies, and each antibody binding domain overlaps with two other domains. The binding domains and, therefore, the antibody determinants appear to be clustered in a localized region of the enzyme. This localized region may be analogous to the main immunogenic region inferred by Lindstrom et al. (29) for binding of monoclonal antibodies to Torpedo and Electrophorous acetylcholine receptor proteins. They found that many of their antibodies recognized a localized region comprising an area made up of two subunits and that the binding of many of these antibodies were conformation dependent. Many of their antibodies crossreacted with the nicotinic receptor present on mammalian skeletal muscle as well. The conservation of this striking surface feature has led Lindstrom to propose a physiological relevance, as yet undefined, to the region marked by the determinant cluster. The presence of a similar determinant cluster on choline acetyltransferase may reflect a region of the molecule involved in noncatalytic functions of the enzyme such as attachment to membranes, transmitter packaging, transport signalling, or regulation of activity. In addition to revealing an interesting and new feature of the enzyme, the use of the monoclonal technology has produced a very specific immunohistochemical reagent for cholinergic tissue. Only one of the antibodies, 3F12, is able to recognize its determinant after fixation. Even though the binding domain of antibody 4D7 overlaps extensively with 3F12, neither its domain nor the nearby domains of 2F11 nor 1E6 appear conserved after fixation. Thus, it is apparent that the domains recognized by antibodies 4D7 and 3F12 are significantly different. In fact, each antibody-binding determinant is unique because it interacts with different combinations in the HPLC experiments. The mechanism by which the four determinants are destroyed by fixation is unknown but could include either direct covalent modification of lysine residues or shifts in the conformation of the determinants.

Neurobiology: Crawford et aL The authors gratefully acknowledge the preliminary anatomical work done by Drs. Houser and Vaughn and Mr. Barber of this division. In addition, Dr. Wainer and Mr. Levey, Department of Pediatrics, University of Chicago, kindly provided a sample of their monoclonal antibody against bovine choline acetyltransferase. Antibody against chicken erythrocyte was provided as a control by Dr. Miller, Department of Cytogenetics, City of Hope. The support of National Institutes of Health Grant NS-18858 is gratefully acknowledged. 1. Rossier, J. (1976) J. Neurochem. 26, 543-548. 2. Ryan, R. L. & McClure, W. 0. (1979) Biochemistry 18, 53575365. 3. Dietz, G. W. & Salvaterra, P. M. (1980)J. BioL Chem. 255, 1061210617. 4. Chao, L.-P. (1980) J. Neurosci. Res. 5, 85-115. 5. Eckenstein, F., Barde, Y.-A. & Thoenen, H. (1981) Neuroscience 49, 45-58. 6. Slemmon, J. R., Crawford, G. C., Salvaterra, P. M. & Roberts, E. (1982) J. BioL Chem. 257, 3847-3852. 7. Rossier, J. (1975) Brain Res. 98, 619-622. 8. Malthe-Sorrenson, D. (1979) Prog. Brain Res. 49, 45-58. 9. Smith, C. P. & Carroll, P. T. (1980) Brain Res. 185, 363-371. 10. Crawford, G., Slemmon, J. R. & Salvaterra, P. M. (1982)J. BioL Chem. 257, 3853-3856. 11. Levey, A. I., Aoki, M., Fitch, F. W. & Wainer, B. (1981) Brain Res. 218, 383-387. 12. Eckenstein, F., Schwab, M. & Thoenen, H. (1981) Trans. Soc. Neurosci. 103.1 (abstr.). 13. Ross, M. E., Park, D. H., Teitelman, G. N., Reis, D. J. & Joh, T. H. (1981) Trans. Soc. Neurosci. 40.8 (abstr.).

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