Journal of Neuroscience Research 29:77-86 (1991)
Xenopus Oocytes as Immunological Vectors to Produce Monoclonal Antibodies to Rat Brain Antigens C. Matute, G.J. Tigyi, and R. Miledi Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine
A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain-derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA-injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system. Key words: antibodies, mRNA, brain markers, rare antigens INTRODUCTION Monoclonal antibodies (Kohler and Milstein, 1975) are invaluable tools in biology and medicine. Many monoclonal antibodies (mAb) have been produced using neuronal membranes or purified proteins (e.g., Barnstable, 1980; Zipser and McKay, 1981; McKay et al., 1981) to study the nervous system. However, many proteins in nerve cells are rare and difficult to purify. We have developed a new approach to generate mAb’s to neuronal proteins by using Xenopus oocytes as immunological vectors. It is well known that Xenopus oocytes are very efficient translators of heterologus mRNAs (Gurdon et al., 1971) and that oocytes injected 0 1991 Wiley-Liss, Inc.
with brain mRNA express many of the neurotransmitter receptors and voltage-gated ion channels so important to neuronal functions (Gundersen et al., 1983, 1984; Dascal, 1987; Miledi et al., 1989). Moreover, electrophysiological recordings of brain mRNA-injected oocytes show that large currents can be observed in response both to application of trace amounts of neurotransmitter or to their agonists (Miledi et al., 1989). Thus, oocytes can be induced to express large amounts of foreign neuronal proteins, possibly larger amounts than in neuronal membranes, and the oocyte membranes can then be used as antigen to produce mAb to those foreign proteins. We have used this novel approach to generate a set of mAb that bind to rat brain.
MATERIALS AND METHODS Preparation of the Immunogen Rat cerebral cortex poly-(A)+-mRNA was extracted by the phenol-chloroform procedure (Sumikawa et al., 1989) and injected into Xenopus oocytes (50 ng per oocyte). After 3 days, oocytes were treated with collagenase, and the expression of the foreign mRNA was assessed 2 days later by electrophysiologically monitoring the appearance of various neurotransmitter receptors (e.g., ACh, GABA, 5-HT, kainate, glutamate, glycine) as well as voltage-activated ion channels, in the oocyte membrane (Gundersen et al., 1983, 1984; Miledi et al., 1989). Oocyte crude membrane fractions containing endoplasmic reticulum, Golgi apparatus and plasma membranes were obtained by sucrose-density gradient centrifugation (Olhsson et al., I98 1). Homogenates from Received August 3, 1990; accepted October 4, 1990. Address reprint requests to Dr. Ricardo Miledi, Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717. C. Matute is now at Department of Neurosciences, Universidad del Pais Vasco, 48940 Lejona, Spain.
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TABLE I. Quantitative Summary of Representative Fusion Experiments and Patterns of mAbs Reacting With Rat Brain Fusion code Patterns All cells Epend yma Blood vessels Astrocytes Neuropil overall Punctate Cerebellar ml Purkinje cells Cerebellar pinceau White matter Regional
ioo- 1 I1
(00-4 TTTII
too-5 TTlTI
to0-7* TTII
nto**
tiv-2 TTTiv
13 4 0 0 0 0 0 0 0 0 0
2
6 0
8 1 1 1 0 1 1 5 1 3 1
1 3 4 5 14 8 0 0 1 0 0
0 1 0 0 0 3 0 1 0 0 0
1 1
1
1 1 3 1
1
5 11 0 I 0 5 0
0 0 0 0
*Immunized with an N-glycosylated membrane fraction. **nto, neonatally tolerized. Abbreviations: T, treatment with cyclophosphamide; I, immunization with a crude membrane preparation; ml, molecular layer; iv, in vitro immunization; ioo, injected oocyte; too, tolerization followed by immunization with injected oocytes.
TABLE 11. Crass-Reactivities of a Representative Panel of mAb With Different Rat and Frog Tissues Rat brain
Name ~
6G11 1H3 2F8 4E 1 2D3 4E6 5F8 4G8 6F1 I 1B11
~~~
~
Frog brain
Rat muscle
Rat lung
Rat liver
Rat kidney
+ + -
+ +-
+ +-
+ + -
++ -
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
~~
All cells Ependy ma Vessels Astrocytes Neuropil overall Punctate White matter Purkinje cells Cerebellar pinceau Regional
~
-
-
-
~
~
-
~
~
ioo
noo ~~
-
-
-MW of antigen KDa-immunoblot ~
-
+
-
+ + -
+ +
50, 100 (SDS) 28, 43, 96 (SDS) 100 (SDS) 43 (SDS) multiple (SDS) multiple (SDS) 8 5 , 90 (SDS) 500 (NAT) 1,000 (NAT) -a
~, negative immunoreaction; + , positive immunoreaction; noo, native oocyte; ioo, injected oocyte; SDS, immunoblots from SDS gels; NAT, immunoblots from native gels. "No labeled band was found in immunoblots from native and SDS gels.
injected or noninjected oocytes were applied onto a step gradient consisting of lo%, 20%, and 50% sucrose cushions and the membrane fraction at the 20-50 interface was collected after 30-min centrifugation at 15,800g. Membranes were washed with 5 mM HEPES buffer at pH 7.2 and used for immunization or tolerization. The average yield was approximately 50 p g proteinioocyte. This material is referred to as oocyte membranes. For some experiments, the oocyte membrane proteins were solubilized with a mixture of 1% NaDOC, 3% Triton X-100, and 1% Tween 80 detergents in 50 mM Tris-HC1 pH 7.6 in the presence of 1 mM PMSF. The nonsolubilized material was separated by ultracentrifugation at 105g for 1 hr. The supernatant was dialyzed against three changes of 200 vol50 mM Tris-HC1 pH 7.2 buffer overnight at 4°C. The resulting material was batch purified on 1 ml concanavalin A(Con A)-Sepharose 4B lectin affinity gel. The bound glycoproteins were eluted
with 0.5 M methylglucoside in 0.1 M glycine buffer at pH 2.8 and immediately dialyzed against 1,000 vol of 10 mM NaDOC, 50 mM Tris-HC1 pH7.2 and further concentrated in an Amicon pressure cell on PM 10 membranes. This preparation yielded approximately 5 pg protein/oocyte.
Tolerization and Immunization To reduce the immune response to antigens of the native oocyte membrane, 4- to 6-week-old Balb/c mice were immunosuppressed by repeated cyclophosphamide treatment (Matthew and Sandrock, 1987). Briefly, about 0.5- to l-mg membranes from native oocytes was injected intraperitoneally into mice; 10 min, 24 and 48 hr later, animals received 100 mg/kg cyclophosphamide injections. Alternatively, a series of newborn mice were rendered immunotolerant to the native oocyte antigens (Hockfield, 1987). In some experiments, neonatally im-
Antibodies to Brain mRNA-Injected
79
limiting dilutions, expanded, and stored in liquid nitrogen as well as used for ascites production in Pristane primed mice.
HATselection
._.
i
assay antibody Fig. 1. Scheme of the experimental design used to raise monoclonal antibodies against membranes from rat cortex mRNAinjected Xenopus oocytes. See text for details.
munotolerized mice were further immunosuppressed against the native membranes with cyclophosphamide when they were 4-6 weeks old. For in vivo immunizations, we used membranes (1 mg protein per mouse) or glycoproteins (90-100 pg per mouse) purified from mRNA-injected oocytes. Several combinations of immunotolerization and immunization schedules were followed (Table I). In all experiments, a 2-week interval between cyclophosphamide treatment and immunization was permitted. Fusions were performed 4 days after the last boost. As a control, a series of mice were immunized with native oocytes. Spleen cells from immunosuppressed or naive mice were immunized in vitro for 5-7 days following the method of Takahashi and colleagues (1987). As immunogen, we used injected oocytes (50-150 per experiment) previously freed of their follicular envelopes by collagenase treatment (Tigyi et al., 1990) and fixed for 30 min with 2% paraformaldehyde in amphibian Ringer’s solution.
Cell Fusion and Cloning After the immunization procedures, spleen cells were fused with Sp2/0-Ag14 myeloma cells using polyethylene glycol (Gefter et al., 1977). Hybrids were seeded in 96- or 24-well plates and raised by standard procedures. Spent hybridoma supernatants were tested initially for immunoreactivity on rat brain sections within 12-25 days after fusion. Positive hybrids were cloned by
Immunohistochemical Procedures Unfixed brains of adult Sprague-Dawley rats were frozen in isopentane at -80°C and stored at this temperature until they were used. Cryostat-cut, 20-ym-thick parasagittal sections were mounted on poly-L-lysine-coated slides and dried on a 37°C slide warmer. Sections were fixed in acetone at -20°C for 10 rnin, air-dried, and rehydrated in phosphate-buffered saline (PBS) (10 mM phosphate buffer, 145 mM NaCl pH 7.4; PBS) before the application of 100- to 2 0 0 - 4 spent hybridoma supernatant. The presence of brain specific antibodies was detected using a mixture of biotinylated antimouse IgM- and IgG-specific antibodies, followed by the biotin-avidin-peroxidase complex (Vectastain, Vector Laboratories, Burlingame, CA). Sections were incubated overnight with supernatants in a humid chamber at room temperature. To detect the immunoperoxidase label, 0.05% diaminobenzidine chromogen was used in the presence of 0.01% hydrogenperoxide substrate. Identical procedures were used to prepare mouse and Xenopus brain sections, or rat liver, muscle, lung, and kidney tissue specimens. Pieces of Xenopus ovaries or injected oocytes were fixed in 0.2% paraformaldehyde, embedded in 8.5% polyacrylamide, frozen, and sectioned as described above. Immunochemical Characterization of the Antigens Brain antigens defined by a representative panel of mAb (Table 11) were characterized using Western-blot techniques as described earlier (Tigyi et al., 1985). Rat brain cytosol and 10 mM CHAPS detergent solubilized membrane fractions were separated either on native 430% polyacrylamide gradient gels, ran in 90 mM TrisHCl, 80 mM boric acid, 2.5 mM EDTA buffer at pH 8.4 or denaturing sodium dodecyl sulfate (SDS) polyacrylamide gels (Nelville, 1971) and electrotransferred to nitrocellulose (Scheiler-Schuell, Keene, NH) with 0. l - y m pore size. Nonspecific binding to the membranes was blocked with 3% bovine serum albumin (BSA) (Sigma, St. Louis, MO) in PBS, followed by 50 mM avidin, and then by 50 mM biotin; 3-mm-wide strips were cut and
cb cx th wm st gr ma
Abbreviations cerebellum cerebral cortex thalamus white matter striatum cerebellar granular layer cerebellar molecular layer
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Fig. 2.
Antibodies to Brain mRNA-Injected
incubated in spent hybridoma supernatant overnight at 4°C. Antibody binding was monitored with the same peroxidase-labeled biotin-avidin system used in immunohistological experiments. Blots were developed with 0.05% diaminobenzidine, 0.01% hydrogen peroxide, 1% cobalt chloride, and 1% nickel chloride. Rabbit antiserum to glial fibrillary acidic protein was obtained from Biogenex Laboratories (Dublin, CA); peroxidaselabeled affinity purified anti-rabbit IgG was purchased from Zymed Laboratories (San Francisco, CA). The scheme of the experimental design is shown in Figure 1.
RESULTS Technique and Effect of Immunization on Number of mAb Obtained Sixteen fusions were done with spleens from mice immunized by different in vivo and in vitro protocols. More than 7,000 supernatants from wells containing growing hybridoma colonies were tested. From all these colony supernatants, 440 were found to contain antibodies reactive with brain sections after repeated testing. Thus, approximately 6% of the wells yielded stable hybrids that produced antibodies to antigens present in rat brain. Table I shows the number of hybrids secreting mAb binding to brain sections together with the types of their staining patterns in representative fusion experiments. From all fusion experiments, immunizations with membrane proteins binding to Con A led to the production of mAb with the greatest variety of immunoreac-
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tivity patterns (see too-7 in Table I), even though the least amount of antigen was injected. Cyclophosphamide-induced immunosuppression was necessary to obtain a more diversified immunoresponse. In immunizations without tolerization, the resulting mAb displayed relatively few immunoreactivity patterns on rat brain sections (see ioo-1 in Table I). In vitro immunizations after immunosuppression induced in vivo yielded neither a larger variety of mAb nor a higher proportion of brain reactive mAb as compared with in vivo immunizations.
Characterization of Antigens A selected group of the typical histological binding patterns observed is shown in Figures 2 and 3; mAb were arbitrarily classified into the following subgroups: 1. Non-brain-specific mAb recognizing rat antigens a. mAb cross-reacting with Xenopus tissues, e.g., 6G 1 1 (Fig. 2A) b. mAb non-cross-reacting with Xenopus tissues, e.g., 2F8 (Fig. 2B) 2. Brain-specific mAb a. Overall reactivity with higher regional intensity, e.g., 4E6 (Fig. 2C,E); l B l l (Fig. 2D,F) b. Region-specific antibodies, e.g., 5F8 (Fig. 3A,C) c. Cell type-specific markers, e.g., 1H3 (Fig. 3B); 4G8 (Fig. 3D); 4E1 (Fig. 3F) d. Nerve-ending specific antibodies, e.g., 6F11 (Fig. 3E)
Cross-reactivities of mAb with Xenopus brain, native and injected oocytes, as well as different rat tissues, together with their immunoblotting patterns are summarized in Table 11. Antibody 6GI I , that belongs to group 1.a in the above classification, reacted with an epitope Fig. 2. Patterns of immunoreactivity in parasagittal frozen present in nuclei of neural and non-neural cells in both sections from rat brain obtained with monoclonal antibodies rat and Xenopus tissues (Fig. 2A). Many mAb had the raised against rat cortex mRNA-injected oocytes. A: Labeling same reactivity, and their incidence was the highest, in in cerebral cortex with mAb 6Gll-stained nuclei in both neufusions made with nontolerized mice. This antibody recronal and non-neuronal cells. B: Immunoreactivity of mAb ognized 50- and 100-kDa molecular-weight proteins also 2F8 was restricted to blood vessels, as shown in this microphotograph taken from the cerebral cortex. C: Immunolabeling present in the solubilized membrane fraction. mAb 2F8 with rnAb 4E6 was particularly intense in the granular layer of recognized an epitope restricted to blood vessels (Fig. the cerebellum, in the deeper layers of the cerebral cortex, and 2B). Unlike 6Gl1, mAb 2F8 did not cross-react with in the thalamus. Note the absence of immunoreactivity in the frog brain. 4 white matter. D: Antibody l B l l strongly bound to the cereThe remaining 7 mAb (whose immunoreactivity bellar granular layer, deeper layers of the cerebral cortex, most pattern is shown in Figs. 2 and 3 ) were specific for rat subcortical areas, and white matter. Note that the labeling in brain tissue. Figure 2C illustrates a panoramic view of striatum is restricted to fiber bundles. E: Higher magnification the staining pattern of mAb 4E6, which bound to dotlike of cortical layer VI. from section shown in C. Note the dense, puncta-like immunoreactivity in the neuropil and its associa- structures (Fig. 2E). Labeling of mab 4E6 was intense in tion with cell bodies (arrows). F: Staining in cerebellar cortex layer V1 of cerebral cortex (Fig. 2E), in hippocampal with 1 B 1 1 . Immunoreactivity is localised to the granular layer area CA3, in the thalamus, in some area of the brainand white matter. Higher-power magnification of photograph stem, and in the granular layer of cerebellar cortex (Fig. shown in D. Calibration bars: C,D: 2.5 mm; A: 140 pm; 2C). No binding of mAb 4E6 was observed in the white matter. B,E,F: 80 p m .
Fig. 3 .
Antibodies to Brain mRNA-Injected
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Another antibody, 1B11, displayed intense binding contacts made between Purkinje cells and basket cells. to many areas of the brain including the white matter Partial characterization of the antigen recognized by this (Fig. 2D,F). In the cerebral cortex, the distribution of antibody was previously published (Tigyi et al., 1990). staining was similar to that observed with 4E6, except Two other mAb families showing faint and diffuse for the presence of a narrow, weakly labeled band local- staining in rat brain sections were obtained. The first ized in some portions of layer I11 of visual and soma- group was termed overall immunoreactive mAb (e.g., tosensory cortex. mAb 1B11 showed a particularly in- 2D3 in Table 11). This hard-to-characterize group of anteresting pattern of immunoreactivity in the cerebellar tibodies was especially numerous when neonatally tolercortex, where no labeling was found in the molecular ized mice were used for fusion (Table I). The second layer, in contrast to the intense staining observed in the group of mAb was found to label selectively the molecgranular layer (Fig. 2F). In the striatum, l B l l exclu- ular layer of the cerebellum. sively bound to fiber tracts (Fig. 2D), while 4E6 reacted All antibodies listed in this panel cross-reacted with exclusively with the neuropil. mouse brain, displaying the same pattern of immunoreAntibody 5F8 is a marker for the white matter of activity as in rat brain. mAb 6G 1 1, 2D3,4E6, 1B 11 , and the rat central nervous system (CNS) (Fig. 3A,C). This 1H3 reacted with sections of Xenopus brains. ImmunomAb did not cross-react with the white matter of the frog histochemical experiments were carried out to test the brain (Table 11). In Western blots, 5F8 recognized a dou- presence of these antigens in native oocytes and in blet of proteins with 85- and 90-kDa molecular weights mRNA-injected oocytes. None of the antibodies shown in both the cytosolic and the membrane protein fractions. in Table I1 reacted with sections prepared from 0.2% mAb lH3 bound to the ependymal cells outlining paraformaldehyde-fixed native oocytes. However, sevthe ventricles (Fig. 3B). It also labeled some component eral mAb in Table I1 showed detectable binding to idenof the neuropil abundant in the granular layer of cerebel- tically fixed sections of mRNA-injected oocytes. lar cortex. mAb 4E1, a marker for astrocytes (Fig. 3F), produced a staining pattern very similar to that observed with specific antiserum to glial fibrillary acidic protein (GFAP). Because of this similarity, 4E1 was tested on DISCUSSION Producing mAb to complex antigenic mixtures immunoblots and compared with an anti-GFAP antiserum (Fig. 4). Both mAb 4E1 and the antiserum recog- from various tissues has several advantages and disadnized a band at 43-kDa molecular weight, suggesting vantages as discussed by several groups of investigators that 4El was indeed specific for an epitope present in the (Barnstable, 1980; Fujita et al., 1982; Hawkes et a]., 1982; Sternberger et al., 1982; Reichardt and Matthew, GFAP molecule. Two groups of mAbs reacted exclusively with ele- 1982; De Blas et al., 1984; Hockfield, 1987). The major ments of the cerebellar cortex (Fig. 3D,E). Antibody drawback of such an approach is the unpredictability of 4G8 intensely stained Purkinje cell bodies and, to a cer- the types of antigens yielding mAb and the relative abuntain degree, their dendritic arborizations as well as some dance of mAb recognizing immunodominant epitopes processes in the granular layer (Fig. 3D). mAb 6 F l l (e.g., see Matthew and Sandrock, 1987). Although serecognized an epitope present in the pinceau synapse lective immunosuppression methods have recently been formed between basket cell axons and the initial segment developed and applied with some success to eliminate of the Purkinje cell axons. Thus, antibody 6 F l l exclu- the effect of immunodominant epitopes (Golumbeski and sively marked one of the four different types of synaptic Dimond, 1986; Matthew and Sandrock, 1987; Hockfield, 1987), techniques directing the immunoresponse to a restricted variety of minor antigens have not been available for practical use. Here, we have explored the posFig. 3 . Patterns of immunoreactivity in rat brain parasagittal sibility of using Xenopus oocytes as immunological vecfrozen sections obtained with mAb raised against rat cortex tors to produce mAb to brain antigens. mRNA-injected oocytes. A: Antibody 5F8 is a marker for CNS In the present study, cryostat-cut sections from rat white matter. B: Ependymal cells outlining the lateral ventricle brain were used to select hybridomas that were produced stained with antibody 1H3.C: Higher magnification of A taken following immunizations with membranes of oocytes infrom a cerebral cortex showing intense labeling of white matjected with mRNA extracted from rat cerebral cortex. ter. D: Labeling of cerebellar Purkinje cells by 4G8. Note the presence of some visible reactivity in granular layer. E: Anti- This highly sensitive procedure preserves most of the body 6 F l l bound to pinceau terminals between basket and natural conformation of the CNS constituents and perPurkinje cells. F: Astrocytes (arrows) in cerebellar cortex la- mits the anatomical localization of epitopes recognized beled with mAb 4E1. Calibration bars: A: 2.5 mm; B,D,E,F: by mAb (Fujita et al., 1982). Antibodies such as 6F11, which marks the cerebellar pinceau terminals, might 140 km; C: 200 pm.
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946740-
c
Fig. 4. Western blots with mAb 4E1 (lane B), and a commercial antiserum against GFAP (lane C). mAb 4E1 and antiGFAP antiserum recognized a 43-kDa band (arrow) in rat cerebellum separated by SDS-PAGE. Lane A was stained with India ink. Numbers indicate migration distances and corresponding molecular masses for marker proteins.
have been omitted in enzyme-linked immunosorbent assay (ELISA) or radio immunoassay (RIA)-based tests. mAb derived from mice directly immunized with crude membranes from brain mRNA-injected oocytes recognized only a few different epitopes, as seen from the results of the ioo-l fusion in Table I. Therefore, we have applied neonatally acquired (Hockfield, 1987) and cyclophosphamide-induced immunotolerization methods (Matthew and Sandrock, 1987), or a combination of both, in an attempt to enhance the immunoresponse against brain proteins transplanted into oocyte membranes. Both methods appeared to be useful in generating mAb to rare antigens. However, it is surprising that,
after 16 fusion experiments, we obtained mAb with only 1 1 different histological binding patterns. This fact might be due in part to the presence of immunodominant epitopes among the expression products of the mRNA injected into oocytes. Several lines of evidence suggest that most antigens defined by this panel of mAb are not shared with native oocytes. First, binding of all these mAb to native oocytes was not detected in immunocytochemical experiments. Second, out of a family of mAb raised against native oocytes, only three cross-reacted with rat brain sections and displayed binding to ependymal cells or a general staining. However, not all mAb in the panel shown in Table I1 reacted with mRNA-injected oocytes, suggesting that the amount of antigen might be beyond detection limits or, alternatively, that the antigen was chemically modified during fixation of the oocytes with paraformaldehyde. Several panels of mAb showing restricted patterns of immunoreactivity in brain have been described after immunizations with rat cerebral cortex (De Blas et al., 1984) or with rat cerebellum synaptosomal plasma membranes (Hawkes et al., 1982). Interestingly, some of the antibodies obtained by these procedures appear to have immunohistochemical characteristics identical to those of some mAb described here, reinforcing the idea that antibodies in our panel were generated in response to brain-derived proteins contained in the oocyte membrane preparation used as immunogen. However, using Xenopus oocytes as an immunological vector, we were able to raise mAb detecting new rare antigens. In evaluating the potential of the oocyte as an immunological expression vector for rare neuronal antigens, one has to consider the possibility that some antibodies obtained in this study might be produced by fusions between myelomas and lymphocytes secreting natural autoantibodies. The existence of natural autoantibodies has been reported (Sotelo et al., 1980; Dighiero et al., 1982); lymphocytes producing such antibodies could be immortalized by the hybridoma technique from nonimmunized mice (Dighiero et al., 1983), as well as from mitogen-stimulated spleen cells (Guilbert et al., 1985; Freitas et al., 1986). Although it is possible that this might be the case for some of the mAb described here, it seems unlikely because most mAb in the panel did not show a wide range of cross-reactivities with multiple organs (see Table II), as generally occurs with autoantibodies (Garcelli et al., 1984; Notkins and Prabkahar, 1986; Hartmann et al., 1989). In addition, most natural autoantibodies recognize cytoskeletal self-antigens, including tubulin, actin, myosin, and spectrin (Stefansson et al., 1985; Freitas et al., 1986; Notkins and Prabkahar, 1986), a property not shared by mAb described in this report.
Antibodies to Brain mRNA-Injected
mAb 4E1 was the only antibody in the panel that bound to an identified antigen, GFAP, according to immunohistochemical and immunoblotting techniques. Table I1 includes several mab that recognize novel brainspecific antigens whose detailed characterization needs further study. From these, 6F11 stands out as a synaptic marker for one of the four types of synapses between basket and Purkinje cells, as recently reported (Tigyi et al., 1990). Thus, 6Fll is a marker for a subset of synaptic contacts formed by one class of cells onto another, the cerebellar pinceau terminals. Other terminals formed by the same class of basket cells onto the same class of Purkinje cells were not stained. These data suggest that nerve endings originating from a single neuron might differ in their immuno-chemical composition. To explore the full potential that the oocyte system may offer as an immunological vector for production of mAb to brain antigens will require further experiments. In such experiments, it should be feasible to raise useful mAb to oocytes that have been injected with a single mRNA, synthesized in vitro from cDNA coding for a neurotransmitter receptor, or to oocytes injected with highly enriched size fractionated mRNA coding for one receptor or channel protein, In vitro immunizations with such mRNA-injected oocytes might also be an alternative to raise valuable mAb to the “active external sites” of desired transmembrane proteins “transplanted” from the brain into oocytes.
ACKNOWLEDGMENTS The skillful technical assistance of Cindy Asselin, Irma Juarez, Lilly Greco, and David Scharberg is greatly appreciated. We are thankful to A.L. De Blas, S.H. Hendry, H. Killackey, and I. Parker for critical reading of the manuscript. This work was supported by a grant to R.M. from the Klingenstein Foundation and partly by PHS grant NS23284 from the National Institutes of Health.
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ducing hybridomas by in vitro stimulation of murine spleen cells. J Immunol Methods 96:247-253. Tigyi G , Juntti N , Johansson KE (1985): Screening for monoclonal antibodies against a mycoplasmal membrane protein by protein electroblotting. Proteins Biol Fluids 33:575-578. Tigyi G , Matute C, Miledi R (1990): Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA injected Xenopus oocytes. Proc Natl Acad Sci USA 87:528-532. Zipser B, McKay R (1981): Monoclonal antibodies distinguish identifiable neurons in the leech. Nature 289:549-554.
Xenopus Oocytes as Immunological Vectors to Produce Monoclonal Antibodies to Rat Brain Antigens C. Matute, G.J. Tigyi, and R. Miledi Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine
A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain-derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA-injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system. Key words: antibodies, mRNA, brain markers, rare antigens INTRODUCTION Monoclonal antibodies (Kohler and Milstein, 1975) are invaluable tools in biology and medicine. Many monoclonal antibodies (mAb) have been produced using neuronal membranes or purified proteins (e.g., Barnstable, 1980; Zipser and McKay, 1981; McKay et al., 1981) to study the nervous system. However, many proteins in nerve cells are rare and difficult to purify. We have developed a new approach to generate mAb’s to neuronal proteins by using Xenopus oocytes as immunological vectors. It is well known that Xenopus oocytes are very efficient translators of heterologus mRNAs (Gurdon et al., 1971) and that oocytes injected 0 1991 Wiley-Liss, Inc.
with brain mRNA express many of the neurotransmitter receptors and voltage-gated ion channels so important to neuronal functions (Gundersen et al., 1983, 1984; Dascal, 1987; Miledi et al., 1989). Moreover, electrophysiological recordings of brain mRNA-injected oocytes show that large currents can be observed in response both to application of trace amounts of neurotransmitter or to their agonists (Miledi et al., 1989). Thus, oocytes can be induced to express large amounts of foreign neuronal proteins, possibly larger amounts than in neuronal membranes, and the oocyte membranes can then be used as antigen to produce mAb to those foreign proteins. We have used this novel approach to generate a set of mAb that bind to rat brain.
MATERIALS AND METHODS Preparation of the Immunogen Rat cerebral cortex poly-(A)+-mRNA was extracted by the phenol-chloroform procedure (Sumikawa et al., 1989) and injected into Xenopus oocytes (50 ng per oocyte). After 3 days, oocytes were treated with collagenase, and the expression of the foreign mRNA was assessed 2 days later by electrophysiologically monitoring the appearance of various neurotransmitter receptors (e.g., ACh, GABA, 5-HT, kainate, glutamate, glycine) as well as voltage-activated ion channels, in the oocyte membrane (Gundersen et al., 1983, 1984; Miledi et al., 1989). Oocyte crude membrane fractions containing endoplasmic reticulum, Golgi apparatus and plasma membranes were obtained by sucrose-density gradient centrifugation (Olhsson et al., I98 1). Homogenates from Received August 3, 1990; accepted October 4, 1990. Address reprint requests to Dr. Ricardo Miledi, Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717. C. Matute is now at Department of Neurosciences, Universidad del Pais Vasco, 48940 Lejona, Spain.
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TABLE I. Quantitative Summary of Representative Fusion Experiments and Patterns of mAbs Reacting With Rat Brain Fusion code Patterns All cells Epend yma Blood vessels Astrocytes Neuropil overall Punctate Cerebellar ml Purkinje cells Cerebellar pinceau White matter Regional
ioo- 1 I1
(00-4 TTTII
too-5 TTlTI
to0-7* TTII
nto**
tiv-2 TTTiv
13 4 0 0 0 0 0 0 0 0 0
2
6 0
8 1 1 1 0 1 1 5 1 3 1
1 3 4 5 14 8 0 0 1 0 0
0 1 0 0 0 3 0 1 0 0 0
1 1
1
1 1 3 1
1
5 11 0 I 0 5 0
0 0 0 0
*Immunized with an N-glycosylated membrane fraction. **nto, neonatally tolerized. Abbreviations: T, treatment with cyclophosphamide; I, immunization with a crude membrane preparation; ml, molecular layer; iv, in vitro immunization; ioo, injected oocyte; too, tolerization followed by immunization with injected oocytes.
TABLE 11. Crass-Reactivities of a Representative Panel of mAb With Different Rat and Frog Tissues Rat brain
Name ~
6G11 1H3 2F8 4E 1 2D3 4E6 5F8 4G8 6F1 I 1B11
~~~
~
Frog brain
Rat muscle
Rat lung
Rat liver
Rat kidney
+ + -
+ +-
+ +-
+ + -
++ -
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
~~
All cells Ependy ma Vessels Astrocytes Neuropil overall Punctate White matter Purkinje cells Cerebellar pinceau Regional
~
-
-
-
~
~
-
~
~
ioo
noo ~~
-
-
-MW of antigen KDa-immunoblot ~
-
+
-
+ + -
+ +
50, 100 (SDS) 28, 43, 96 (SDS) 100 (SDS) 43 (SDS) multiple (SDS) multiple (SDS) 8 5 , 90 (SDS) 500 (NAT) 1,000 (NAT) -a
~, negative immunoreaction; + , positive immunoreaction; noo, native oocyte; ioo, injected oocyte; SDS, immunoblots from SDS gels; NAT, immunoblots from native gels. "No labeled band was found in immunoblots from native and SDS gels.
injected or noninjected oocytes were applied onto a step gradient consisting of lo%, 20%, and 50% sucrose cushions and the membrane fraction at the 20-50 interface was collected after 30-min centrifugation at 15,800g. Membranes were washed with 5 mM HEPES buffer at pH 7.2 and used for immunization or tolerization. The average yield was approximately 50 p g proteinioocyte. This material is referred to as oocyte membranes. For some experiments, the oocyte membrane proteins were solubilized with a mixture of 1% NaDOC, 3% Triton X-100, and 1% Tween 80 detergents in 50 mM Tris-HC1 pH 7.6 in the presence of 1 mM PMSF. The nonsolubilized material was separated by ultracentrifugation at 105g for 1 hr. The supernatant was dialyzed against three changes of 200 vol50 mM Tris-HC1 pH 7.2 buffer overnight at 4°C. The resulting material was batch purified on 1 ml concanavalin A(Con A)-Sepharose 4B lectin affinity gel. The bound glycoproteins were eluted
with 0.5 M methylglucoside in 0.1 M glycine buffer at pH 2.8 and immediately dialyzed against 1,000 vol of 10 mM NaDOC, 50 mM Tris-HC1 pH7.2 and further concentrated in an Amicon pressure cell on PM 10 membranes. This preparation yielded approximately 5 pg protein/oocyte.
Tolerization and Immunization To reduce the immune response to antigens of the native oocyte membrane, 4- to 6-week-old Balb/c mice were immunosuppressed by repeated cyclophosphamide treatment (Matthew and Sandrock, 1987). Briefly, about 0.5- to l-mg membranes from native oocytes was injected intraperitoneally into mice; 10 min, 24 and 48 hr later, animals received 100 mg/kg cyclophosphamide injections. Alternatively, a series of newborn mice were rendered immunotolerant to the native oocyte antigens (Hockfield, 1987). In some experiments, neonatally im-
Antibodies to Brain mRNA-Injected
79
limiting dilutions, expanded, and stored in liquid nitrogen as well as used for ascites production in Pristane primed mice.
HATselection
._.
i
assay antibody Fig. 1. Scheme of the experimental design used to raise monoclonal antibodies against membranes from rat cortex mRNAinjected Xenopus oocytes. See text for details.
munotolerized mice were further immunosuppressed against the native membranes with cyclophosphamide when they were 4-6 weeks old. For in vivo immunizations, we used membranes (1 mg protein per mouse) or glycoproteins (90-100 pg per mouse) purified from mRNA-injected oocytes. Several combinations of immunotolerization and immunization schedules were followed (Table I). In all experiments, a 2-week interval between cyclophosphamide treatment and immunization was permitted. Fusions were performed 4 days after the last boost. As a control, a series of mice were immunized with native oocytes. Spleen cells from immunosuppressed or naive mice were immunized in vitro for 5-7 days following the method of Takahashi and colleagues (1987). As immunogen, we used injected oocytes (50-150 per experiment) previously freed of their follicular envelopes by collagenase treatment (Tigyi et al., 1990) and fixed for 30 min with 2% paraformaldehyde in amphibian Ringer’s solution.
Cell Fusion and Cloning After the immunization procedures, spleen cells were fused with Sp2/0-Ag14 myeloma cells using polyethylene glycol (Gefter et al., 1977). Hybrids were seeded in 96- or 24-well plates and raised by standard procedures. Spent hybridoma supernatants were tested initially for immunoreactivity on rat brain sections within 12-25 days after fusion. Positive hybrids were cloned by
Immunohistochemical Procedures Unfixed brains of adult Sprague-Dawley rats were frozen in isopentane at -80°C and stored at this temperature until they were used. Cryostat-cut, 20-ym-thick parasagittal sections were mounted on poly-L-lysine-coated slides and dried on a 37°C slide warmer. Sections were fixed in acetone at -20°C for 10 rnin, air-dried, and rehydrated in phosphate-buffered saline (PBS) (10 mM phosphate buffer, 145 mM NaCl pH 7.4; PBS) before the application of 100- to 2 0 0 - 4 spent hybridoma supernatant. The presence of brain specific antibodies was detected using a mixture of biotinylated antimouse IgM- and IgG-specific antibodies, followed by the biotin-avidin-peroxidase complex (Vectastain, Vector Laboratories, Burlingame, CA). Sections were incubated overnight with supernatants in a humid chamber at room temperature. To detect the immunoperoxidase label, 0.05% diaminobenzidine chromogen was used in the presence of 0.01% hydrogenperoxide substrate. Identical procedures were used to prepare mouse and Xenopus brain sections, or rat liver, muscle, lung, and kidney tissue specimens. Pieces of Xenopus ovaries or injected oocytes were fixed in 0.2% paraformaldehyde, embedded in 8.5% polyacrylamide, frozen, and sectioned as described above. Immunochemical Characterization of the Antigens Brain antigens defined by a representative panel of mAb (Table 11) were characterized using Western-blot techniques as described earlier (Tigyi et al., 1985). Rat brain cytosol and 10 mM CHAPS detergent solubilized membrane fractions were separated either on native 430% polyacrylamide gradient gels, ran in 90 mM TrisHCl, 80 mM boric acid, 2.5 mM EDTA buffer at pH 8.4 or denaturing sodium dodecyl sulfate (SDS) polyacrylamide gels (Nelville, 1971) and electrotransferred to nitrocellulose (Scheiler-Schuell, Keene, NH) with 0. l - y m pore size. Nonspecific binding to the membranes was blocked with 3% bovine serum albumin (BSA) (Sigma, St. Louis, MO) in PBS, followed by 50 mM avidin, and then by 50 mM biotin; 3-mm-wide strips were cut and
cb cx th wm st gr ma
Abbreviations cerebellum cerebral cortex thalamus white matter striatum cerebellar granular layer cerebellar molecular layer
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Fig. 2.
Antibodies to Brain mRNA-Injected
incubated in spent hybridoma supernatant overnight at 4°C. Antibody binding was monitored with the same peroxidase-labeled biotin-avidin system used in immunohistological experiments. Blots were developed with 0.05% diaminobenzidine, 0.01% hydrogen peroxide, 1% cobalt chloride, and 1% nickel chloride. Rabbit antiserum to glial fibrillary acidic protein was obtained from Biogenex Laboratories (Dublin, CA); peroxidaselabeled affinity purified anti-rabbit IgG was purchased from Zymed Laboratories (San Francisco, CA). The scheme of the experimental design is shown in Figure 1.
RESULTS Technique and Effect of Immunization on Number of mAb Obtained Sixteen fusions were done with spleens from mice immunized by different in vivo and in vitro protocols. More than 7,000 supernatants from wells containing growing hybridoma colonies were tested. From all these colony supernatants, 440 were found to contain antibodies reactive with brain sections after repeated testing. Thus, approximately 6% of the wells yielded stable hybrids that produced antibodies to antigens present in rat brain. Table I shows the number of hybrids secreting mAb binding to brain sections together with the types of their staining patterns in representative fusion experiments. From all fusion experiments, immunizations with membrane proteins binding to Con A led to the production of mAb with the greatest variety of immunoreac-
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tivity patterns (see too-7 in Table I), even though the least amount of antigen was injected. Cyclophosphamide-induced immunosuppression was necessary to obtain a more diversified immunoresponse. In immunizations without tolerization, the resulting mAb displayed relatively few immunoreactivity patterns on rat brain sections (see ioo-1 in Table I). In vitro immunizations after immunosuppression induced in vivo yielded neither a larger variety of mAb nor a higher proportion of brain reactive mAb as compared with in vivo immunizations.
Characterization of Antigens A selected group of the typical histological binding patterns observed is shown in Figures 2 and 3; mAb were arbitrarily classified into the following subgroups: 1. Non-brain-specific mAb recognizing rat antigens a. mAb cross-reacting with Xenopus tissues, e.g., 6G 1 1 (Fig. 2A) b. mAb non-cross-reacting with Xenopus tissues, e.g., 2F8 (Fig. 2B) 2. Brain-specific mAb a. Overall reactivity with higher regional intensity, e.g., 4E6 (Fig. 2C,E); l B l l (Fig. 2D,F) b. Region-specific antibodies, e.g., 5F8 (Fig. 3A,C) c. Cell type-specific markers, e.g., 1H3 (Fig. 3B); 4G8 (Fig. 3D); 4E1 (Fig. 3F) d. Nerve-ending specific antibodies, e.g., 6F11 (Fig. 3E)
Cross-reactivities of mAb with Xenopus brain, native and injected oocytes, as well as different rat tissues, together with their immunoblotting patterns are summarized in Table 11. Antibody 6GI I , that belongs to group 1.a in the above classification, reacted with an epitope Fig. 2. Patterns of immunoreactivity in parasagittal frozen present in nuclei of neural and non-neural cells in both sections from rat brain obtained with monoclonal antibodies rat and Xenopus tissues (Fig. 2A). Many mAb had the raised against rat cortex mRNA-injected oocytes. A: Labeling same reactivity, and their incidence was the highest, in in cerebral cortex with mAb 6Gll-stained nuclei in both neufusions made with nontolerized mice. This antibody recronal and non-neuronal cells. B: Immunoreactivity of mAb ognized 50- and 100-kDa molecular-weight proteins also 2F8 was restricted to blood vessels, as shown in this microphotograph taken from the cerebral cortex. C: Immunolabeling present in the solubilized membrane fraction. mAb 2F8 with rnAb 4E6 was particularly intense in the granular layer of recognized an epitope restricted to blood vessels (Fig. the cerebellum, in the deeper layers of the cerebral cortex, and 2B). Unlike 6Gl1, mAb 2F8 did not cross-react with in the thalamus. Note the absence of immunoreactivity in the frog brain. 4 white matter. D: Antibody l B l l strongly bound to the cereThe remaining 7 mAb (whose immunoreactivity bellar granular layer, deeper layers of the cerebral cortex, most pattern is shown in Figs. 2 and 3 ) were specific for rat subcortical areas, and white matter. Note that the labeling in brain tissue. Figure 2C illustrates a panoramic view of striatum is restricted to fiber bundles. E: Higher magnification the staining pattern of mAb 4E6, which bound to dotlike of cortical layer VI. from section shown in C. Note the dense, puncta-like immunoreactivity in the neuropil and its associa- structures (Fig. 2E). Labeling of mab 4E6 was intense in tion with cell bodies (arrows). F: Staining in cerebellar cortex layer V1 of cerebral cortex (Fig. 2E), in hippocampal with 1 B 1 1 . Immunoreactivity is localised to the granular layer area CA3, in the thalamus, in some area of the brainand white matter. Higher-power magnification of photograph stem, and in the granular layer of cerebellar cortex (Fig. shown in D. Calibration bars: C,D: 2.5 mm; A: 140 pm; 2C). No binding of mAb 4E6 was observed in the white matter. B,E,F: 80 p m .
Fig. 3 .
Antibodies to Brain mRNA-Injected
83
Another antibody, 1B11, displayed intense binding contacts made between Purkinje cells and basket cells. to many areas of the brain including the white matter Partial characterization of the antigen recognized by this (Fig. 2D,F). In the cerebral cortex, the distribution of antibody was previously published (Tigyi et al., 1990). staining was similar to that observed with 4E6, except Two other mAb families showing faint and diffuse for the presence of a narrow, weakly labeled band local- staining in rat brain sections were obtained. The first ized in some portions of layer I11 of visual and soma- group was termed overall immunoreactive mAb (e.g., tosensory cortex. mAb 1B11 showed a particularly in- 2D3 in Table 11). This hard-to-characterize group of anteresting pattern of immunoreactivity in the cerebellar tibodies was especially numerous when neonatally tolercortex, where no labeling was found in the molecular ized mice were used for fusion (Table I). The second layer, in contrast to the intense staining observed in the group of mAb was found to label selectively the molecgranular layer (Fig. 2F). In the striatum, l B l l exclu- ular layer of the cerebellum. sively bound to fiber tracts (Fig. 2D), while 4E6 reacted All antibodies listed in this panel cross-reacted with exclusively with the neuropil. mouse brain, displaying the same pattern of immunoreAntibody 5F8 is a marker for the white matter of activity as in rat brain. mAb 6G 1 1, 2D3,4E6, 1B 11 , and the rat central nervous system (CNS) (Fig. 3A,C). This 1H3 reacted with sections of Xenopus brains. ImmunomAb did not cross-react with the white matter of the frog histochemical experiments were carried out to test the brain (Table 11). In Western blots, 5F8 recognized a dou- presence of these antigens in native oocytes and in blet of proteins with 85- and 90-kDa molecular weights mRNA-injected oocytes. None of the antibodies shown in both the cytosolic and the membrane protein fractions. in Table I1 reacted with sections prepared from 0.2% mAb lH3 bound to the ependymal cells outlining paraformaldehyde-fixed native oocytes. However, sevthe ventricles (Fig. 3B). It also labeled some component eral mAb in Table I1 showed detectable binding to idenof the neuropil abundant in the granular layer of cerebel- tically fixed sections of mRNA-injected oocytes. lar cortex. mAb 4E1, a marker for astrocytes (Fig. 3F), produced a staining pattern very similar to that observed with specific antiserum to glial fibrillary acidic protein (GFAP). Because of this similarity, 4E1 was tested on DISCUSSION Producing mAb to complex antigenic mixtures immunoblots and compared with an anti-GFAP antiserum (Fig. 4). Both mAb 4E1 and the antiserum recog- from various tissues has several advantages and disadnized a band at 43-kDa molecular weight, suggesting vantages as discussed by several groups of investigators that 4El was indeed specific for an epitope present in the (Barnstable, 1980; Fujita et al., 1982; Hawkes et a]., 1982; Sternberger et al., 1982; Reichardt and Matthew, GFAP molecule. Two groups of mAbs reacted exclusively with ele- 1982; De Blas et al., 1984; Hockfield, 1987). The major ments of the cerebellar cortex (Fig. 3D,E). Antibody drawback of such an approach is the unpredictability of 4G8 intensely stained Purkinje cell bodies and, to a cer- the types of antigens yielding mAb and the relative abuntain degree, their dendritic arborizations as well as some dance of mAb recognizing immunodominant epitopes processes in the granular layer (Fig. 3D). mAb 6 F l l (e.g., see Matthew and Sandrock, 1987). Although serecognized an epitope present in the pinceau synapse lective immunosuppression methods have recently been formed between basket cell axons and the initial segment developed and applied with some success to eliminate of the Purkinje cell axons. Thus, antibody 6 F l l exclu- the effect of immunodominant epitopes (Golumbeski and sively marked one of the four different types of synaptic Dimond, 1986; Matthew and Sandrock, 1987; Hockfield, 1987), techniques directing the immunoresponse to a restricted variety of minor antigens have not been available for practical use. Here, we have explored the posFig. 3 . Patterns of immunoreactivity in rat brain parasagittal sibility of using Xenopus oocytes as immunological vecfrozen sections obtained with mAb raised against rat cortex tors to produce mAb to brain antigens. mRNA-injected oocytes. A: Antibody 5F8 is a marker for CNS In the present study, cryostat-cut sections from rat white matter. B: Ependymal cells outlining the lateral ventricle brain were used to select hybridomas that were produced stained with antibody 1H3.C: Higher magnification of A taken following immunizations with membranes of oocytes infrom a cerebral cortex showing intense labeling of white matjected with mRNA extracted from rat cerebral cortex. ter. D: Labeling of cerebellar Purkinje cells by 4G8. Note the presence of some visible reactivity in granular layer. E: Anti- This highly sensitive procedure preserves most of the body 6 F l l bound to pinceau terminals between basket and natural conformation of the CNS constituents and perPurkinje cells. F: Astrocytes (arrows) in cerebellar cortex la- mits the anatomical localization of epitopes recognized beled with mAb 4E1. Calibration bars: A: 2.5 mm; B,D,E,F: by mAb (Fujita et al., 1982). Antibodies such as 6F11, which marks the cerebellar pinceau terminals, might 140 km; C: 200 pm.
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946740-
c
Fig. 4. Western blots with mAb 4E1 (lane B), and a commercial antiserum against GFAP (lane C). mAb 4E1 and antiGFAP antiserum recognized a 43-kDa band (arrow) in rat cerebellum separated by SDS-PAGE. Lane A was stained with India ink. Numbers indicate migration distances and corresponding molecular masses for marker proteins.
have been omitted in enzyme-linked immunosorbent assay (ELISA) or radio immunoassay (RIA)-based tests. mAb derived from mice directly immunized with crude membranes from brain mRNA-injected oocytes recognized only a few different epitopes, as seen from the results of the ioo-l fusion in Table I. Therefore, we have applied neonatally acquired (Hockfield, 1987) and cyclophosphamide-induced immunotolerization methods (Matthew and Sandrock, 1987), or a combination of both, in an attempt to enhance the immunoresponse against brain proteins transplanted into oocyte membranes. Both methods appeared to be useful in generating mAb to rare antigens. However, it is surprising that,
after 16 fusion experiments, we obtained mAb with only 1 1 different histological binding patterns. This fact might be due in part to the presence of immunodominant epitopes among the expression products of the mRNA injected into oocytes. Several lines of evidence suggest that most antigens defined by this panel of mAb are not shared with native oocytes. First, binding of all these mAb to native oocytes was not detected in immunocytochemical experiments. Second, out of a family of mAb raised against native oocytes, only three cross-reacted with rat brain sections and displayed binding to ependymal cells or a general staining. However, not all mAb in the panel shown in Table I1 reacted with mRNA-injected oocytes, suggesting that the amount of antigen might be beyond detection limits or, alternatively, that the antigen was chemically modified during fixation of the oocytes with paraformaldehyde. Several panels of mAb showing restricted patterns of immunoreactivity in brain have been described after immunizations with rat cerebral cortex (De Blas et al., 1984) or with rat cerebellum synaptosomal plasma membranes (Hawkes et al., 1982). Interestingly, some of the antibodies obtained by these procedures appear to have immunohistochemical characteristics identical to those of some mAb described here, reinforcing the idea that antibodies in our panel were generated in response to brain-derived proteins contained in the oocyte membrane preparation used as immunogen. However, using Xenopus oocytes as an immunological vector, we were able to raise mAb detecting new rare antigens. In evaluating the potential of the oocyte as an immunological expression vector for rare neuronal antigens, one has to consider the possibility that some antibodies obtained in this study might be produced by fusions between myelomas and lymphocytes secreting natural autoantibodies. The existence of natural autoantibodies has been reported (Sotelo et al., 1980; Dighiero et al., 1982); lymphocytes producing such antibodies could be immortalized by the hybridoma technique from nonimmunized mice (Dighiero et al., 1983), as well as from mitogen-stimulated spleen cells (Guilbert et al., 1985; Freitas et al., 1986). Although it is possible that this might be the case for some of the mAb described here, it seems unlikely because most mAb in the panel did not show a wide range of cross-reactivities with multiple organs (see Table II), as generally occurs with autoantibodies (Garcelli et al., 1984; Notkins and Prabkahar, 1986; Hartmann et al., 1989). In addition, most natural autoantibodies recognize cytoskeletal self-antigens, including tubulin, actin, myosin, and spectrin (Stefansson et al., 1985; Freitas et al., 1986; Notkins and Prabkahar, 1986), a property not shared by mAb described in this report.
Antibodies to Brain mRNA-Injected
mAb 4E1 was the only antibody in the panel that bound to an identified antigen, GFAP, according to immunohistochemical and immunoblotting techniques. Table I1 includes several mab that recognize novel brainspecific antigens whose detailed characterization needs further study. From these, 6F11 stands out as a synaptic marker for one of the four types of synapses between basket and Purkinje cells, as recently reported (Tigyi et al., 1990). Thus, 6Fll is a marker for a subset of synaptic contacts formed by one class of cells onto another, the cerebellar pinceau terminals. Other terminals formed by the same class of basket cells onto the same class of Purkinje cells were not stained. These data suggest that nerve endings originating from a single neuron might differ in their immuno-chemical composition. To explore the full potential that the oocyte system may offer as an immunological vector for production of mAb to brain antigens will require further experiments. In such experiments, it should be feasible to raise useful mAb to oocytes that have been injected with a single mRNA, synthesized in vitro from cDNA coding for a neurotransmitter receptor, or to oocytes injected with highly enriched size fractionated mRNA coding for one receptor or channel protein, In vitro immunizations with such mRNA-injected oocytes might also be an alternative to raise valuable mAb to the “active external sites” of desired transmembrane proteins “transplanted” from the brain into oocytes.
ACKNOWLEDGMENTS The skillful technical assistance of Cindy Asselin, Irma Juarez, Lilly Greco, and David Scharberg is greatly appreciated. We are thankful to A.L. De Blas, S.H. Hendry, H. Killackey, and I. Parker for critical reading of the manuscript. This work was supported by a grant to R.M. from the Klingenstein Foundation and partly by PHS grant NS23284 from the National Institutes of Health.
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