Journal of Neuroimmunologv, 27 (1990) 71-78

71

Elsevier JNI 00908

Monoclonal antibody to brain cytoplasmic tetrodotoxin-sensitive protein detects an epitope associated with lymphocytes and involved in lymphocyte activation L.N. Pinchuk and G.V. Pinchuk Department of Neurochemistry, A.A. Bogomoletz Institute of Physiology, Academy of Sciences of the Ukrainian S.S.R., Kiev, Ukraine, U.S.S.R.

(Received 27 April 1989) (Revised, received 6 July 1989 and 5 September 1989) (Accepted 5 September 1989)

Key words: Monoclonal antibody; Cytoplasmic tetrodotoxin-sensitiveprotein; BIP-4 epitope; Lymphocyte; Activation; (Brain)

Summary We have previously derived a monoclonal antibody, BIP-4, which is specific to a mammalian brain protein representing a type of sodium channel. Here we show that this antibody detects an epitope associated with lymphocytes and that it triggers a proliferative response of the cells. BIP-4 epitope can be detected on both human peripheral blood and murine splenic mononuclear cells. Surface immunoglobulin-negative (i.e. resting T) lymphocytes are neither bound by the antibody nor proliferate to it. Proliferative response exerted in 7-day cultures of murine splenic mononuclear cells by recombinant interleukin-2 was blocked by BIP-4 antibody. We conclude that the epitope shared by a type of brain sodium channel protein and lymphocyte surface is involved in some, yet unrecognized, step of immune cell activation.

Introduction Cells of the immune system have membrane-associated ion channels which display several properties similar to those of their excitable cell-associated counterparts. This may be concluded from results obtained in electrophysiologic experiments at the single-cell level, including experiments describing single ion channel behavior (Cahalan et

Address for correspondence: George V. Pinchuk, Department of Neurochemistry, A.A. Bogomoletz Institute of Physiology, 4 BogomoletzSt., Kiev-24, 252601, U.S.S.R.

al., 1987; Choquet and Korn, 1988; Gallin and Sheedy, 1988; McCann et al., 1989). However, the molecules forming the immune cell-associated ion channels are not as yet characterized. Recently, Malysheva et al. (1987) have purified a bovine brain protein capable of incorporating spontaneously into liposomes and conferring a tetrodotoxin-inhibitable sodium permeability on them, thus resembling a type of sodium channel. This protein, termed cytoplasmic tetrodotoxinsensitive protein (CTSP), is water-soluble in contrast with the protein forming the membrane-associated sodium channel. The authors proposed a hypothesis that CTSP is biosynthetically related to

0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

72

the molecule forming the mature sodium channel, possibly a precursor or a degradation product. In line with this hypothesis, BIP-4, a monoclonal antibody derived from the immunization with purified CTSP, has been shown to bind an epitope associated with bovine brain membrane preparations and with cultured murine neuroblastoma cells. BIP-4 binding to neuroblastoma cell monolayers was inhibited by veratrine, a modulator of voltage-gated sodium channel function (Pinchuk et al., 1990). In this paper, we present evidence that the BIP-4 epitope is associated also with cells of immune origin and that it may be related to a molecule involved in lymphocyte activation.

Materials and methods

Sciences of the Ukrainian S.S.R., Kiev). The above antibodies were used as a m m o n i u m sulfate-precipitated Ig fractions of culture supernatants diluted with and dialyzed against either phosphatebuffered saline (PBS), p H 7.4, or culture medium, and stored as aliquots at - 7 0 ° C . Rabbit antimurine Ig (RAM) antiserum was obtained locally. For this purpose, rabbits were hyperimmunized with total murine serum Ig fraction and serum samples having a titer greater than 1:2000 by Ouchterlony testing were collected. Goat antimurine IgG affinity-purified, horseradish peroxidase-conjugated antibody was from Calbiochem, La Jolla, CA, U.S.A. Donkey anti-rabbit lg fluorescein isothiocyanate (FITC)-conjugated antibody was from the G a m a l e y a Institute of Microbiology and Immunology Experimental Plant, Moscow, U.S.S.R. Cells"

Medium and reagents

RPMI-1640 medium was purchased from Flow Laboratories, Irvine, Scotland, U.K. Fetal calf serum (FCS), bovine serum albumin (BSA) and glutaraldehyde were obtained from Sigma Chemical Corp., St. Louis, MO, U.S.A.; phytohemagglutinin M (PHA-M) was from Calbiochem, La Jolla, CA, U.S.A.; and recombinant human interleukin-2 (rIL-2) was from Biogen, Moscow, U.S.S.R. (kindly provided by Dr. A.L. Pukhalsky, Institute of Medical Genetics, Academy of Medical Sciences of the U.S.S.R., Moscow, U.S.S.R.). A n tibodies

The following murine monoclonal IgG antibodies were used in this study: BIP-4, an antibody prepared in our laboratory, was originally generated against electrophoretically purified CTSP; W 6 / 3 2 , directed against human major histocompatibility complex antigen class 1 (Brodsky et al., 1979); and B2, a species-nonspecific antilymphocyte antibody originally generated against human PHA-stimulated lymphocytes (Volgin et al., 1985). Antibodies W 6 / 3 2 and B2 were generously provided by Dr. A.Yu. Volgin (Institute of Medical Genetics, Moscow). Preimmune murine myeloma antibody was obtained from the MOPC21 cell line (a gift of A.M. Bukhanyevich, A.V. Palladin Institute of Biochemistry, Academy of

Mononuclear cells (MNC) were obtained from samples of venous blood provided by healthy human volunteers, or from teased murine ( C B A / J ) spleens, using the method of BiSyum (1968). Briefly, blood buffy coats or spleen cell suspensions were underlayered with Ficoll-Hypaque mixture, O = 1.077 (Pharmacia, Uppsala, Sweden), and centrifuged. Cells from the interphase were collected, washed 2 - 3 times with PBS and resuspended as indicated below. For some experiments, murine M N C were further depleted of either adherent or surface Ig (sIg)-positive cells. To deplete M N C of adherent cells, they were resuspended in R P M I medium supplemented with L-glutamine and 5% heat-inactivated FCS (hereafter called 'medium'), and the cell concentration adjusted to 2 × 106/ml. The cells were plated onto 35-mm plastic Costar Petri dishes (3 m l / d i s h ) and incubated for 1 h at 3 7 ° C in a humidified atmosphere containing 5% CO 2, after which nonadherent cells were collected, washed twice with PBS and resuspended in medium. This procedure was repeated and then both untreated and adherent cell-depleted M N C were processed for the proliferation assay in the presence of PHA-M, as described below. To deplete M N C of surface Ig (sIg)-positive cells, the ' p a n n i n g ' procedure of Wysocki and Sato (1978), with minor modifications, was used.

73 Briefly, the M N C were resuspended in medium as above and plated onto Costar dishes which had been preincubated with R A M antiserum (diluted 1:4 in PBS) for 4 - 1 6 h at 22°C. Plated cells were incubated as above, after which step the cells which did not attach to the dishes (routinely, about 50% of the spleen cell population) were collected by careful aspiration With a fine Pasteur pipette and washed twice with PBS. Both untreated M N C and cells depleted of sIg-positive cells were processed for immunofluorescent microscopy. In brief, cells from both groups were incubated with R A M antiserum (1 h at 4 ° C ) , washed 4 times and further incubated with donkey anti-rabbit immunoglobulin FITC-conjugated antibody. The cells were washed 4 more, times and inspected with the L U M A M luminescent microscope (Lomo, Leningrad, U.S.S.R.). The percentage of stained cells was determined. Routinely, this was 40-55% in suspensions of untreated cells and less than 5% in suspensions recovered after the ' p a n n i n g ' procedure. Background staining (in suspensions treated with normal rabbit serum instead of RAM) was 2-3%.

Enzyme-linked immunosorbent assay (ELISA) This was based on the methods of Pateraki et al. (1981) and Baumgarten (1986) and was carried out essentially as described (Pinchuk et al., 1988). Briefly, the test cells ( M N C or the cells depleted of either adherent or sIg-positive cells) were seeded into the wells of Terasaki plates (Medical Polymere Plant, Leningrad, U.S.S.R.) at a density of 5 )< 104 cells/well (unless otherwise stated) and fixed by overnight incubation with glutaraldehyde diluted 1 : 2000 in PBS. This allowed the wells to dry and permitted subsequent repeated washings without detectable loss of cells from wells. Subsequently, the cells were washed and incubated with murine monoclonal antibody (diluted as indicated below), and after several washings, with goat anti-murine I g G peroxidase-conjugated antibody (1:2000). Color reaction was developed after the addition of the substrate solution (o-phenylenediamine and H202 in citric buffer, p H 4.7) and terminated by the addition of sulfuric acid. Absorbance at 492 nm was read by the Morphoquant photometer (Carl Zeiss-Jena, Jena, G.D.R.).

Proliferation assays Test cells were washed 3 times, resuspended in medium and plated onto 96-well flat-bottomed tissue culture plates (Dynatech Microtiter, Switzerland) at 105 c e l l s / 2 0 0 / d / w e l l . The appropriate antibody a n d / o r rIL-2 at different concentrations were added to the wells at the start of the culture period. In some experiments, P H A - M was added to the wells at 1 ~ g / m l . The cultures were incubated at 37 ° C in a humidified atmosphere containing 5% CO z, for 72 h, except those with rIL-2 which were incubated for 7 days. 16 h prior to culture termination, 1 /~Ci of [3H]thymidine (Isotop, U.S.S.R.) was added to each well. Subsequently, cultures were harvested onto nitrocellulose filters and isotope uptake measured by liquid scintillation counting using the RackBeta counter (LKB, Stockholm, Sweden).

Results

Binding of BIP-4 antibody to MNC In our previous study (Pinchuk et al., 1990), we found that BIP-4, a monoclonal antibody generated against purified CTSP, detected an epitope associated with bovine brain and murine neuroblastoma cell membrane. In this study, we examined whether or not the BIP-4 epitope was associated with cells of immune origin. To this end, human peripheral blood or rodent spleen M N C were isolated and processed for the ELISA as described above. As shown in Fig. 1, h u m a n M N C were bound by BIP-4 as well as by W 6 / 3 2 and B2 antibodies but not by murine preimmune (myeloma cell-produced) antibody. Similar titration curves were obtained when murine M N C were used as an antigen with the exception that W 6 / 3 2 antibody did not bind these cells when diluted more than 1:200. Rat M N C were also bound by both BIP-4 and B2 antibodies (not shown). We have concluded that BIP-4 detected an epitope associated with both h u m a n and rodent MNC.

Mitogenicity of BIP-4 antibody In both T and B lymphocytes, a limited number of surface epitopes are associated with molecules involved in ligand-induceable proliferation (for re-

74 3 A492

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Fig. 1. Binding of BIP-4 and other murine antibodies to human peripheral blood MNC. The cells were fixed, washed and the wells saturated with 1% BSA. Subsequently, the ELISA was performed as described (Pinchuk et al., 1988; see also Materials and Methods). The antibodies were used as serial dilutions of Ig fractions obtained from culture supernatants by 50% saturated ammonium sulfate precipitation (starting protein concentration, 10 m g / m l , as assessed by the method of Lowry et al., 1951). The curves represent binding of: (1) BIP-4, (2) W6/32, (3) B2, (4) MOPC-21 myeloma preimmune antibody. Binding of the peroxidase-conjugated anti-lgG antibody alone (blank) gave an absorbance of less than 0.150 and was subtracted. Results of a single experiment. Symbols represent means of triplicate determinations. This experiment was repeated 4 times with similar results.

views, see Royer and Reinherz, 1987; Valentine et al., 1988). Therefore, we suggested that examination of possible BIP-4 mitogenicity might help understand the nature of the epitope-bearing molecule. In this regard, BIP-4 or control antibodies were added to cultured M N C and proliferation assayed as above. It was found that BIP-4 antibody triggered a moderate but significant proliferative response of human MNC (Fig. 2) and of murine M N C (not shown). This was detectable at an antibody concentration as little as 100 n g / m l and was more prominent at 0.5 ffg/ml. Further increase of BIP-4 concentration caused no increase in the response and concentrations more than 200/~g/ml were not tested because they were found to be cytotoxic as judged by trypan blue exclusion. N o proliferative response was detected in cultures with B2 antibody, consistent with a previous report (Volgin et al., 1985). We concluded that BIP-4 antibody was mitogenic for

M N C but suggested that its mitogenicity was limited to a certain M N C subset because of a rather moderate increase in proliferation in BIP4-containing cultures.

Murine M N C depleted of slg-positive cells do not proliferate in response to BIP-4 antibody To address the question whether the proliferative response triggered by BIP-4 antibody in M N C cultures was a function of a certain M N C subset, we used techniques to deplete the M N C population of either adherent or sIg-positive cells. To test whether or not the procedures were adequate, we examined the cellular response to PHA-M; this was diminished significantly after incubation of the cells in plastic dishes but not after incubation in RAM antiserum-coated dishes (Fig. 3). In addition, the percentage of sIg-bearing cells diminished after 'panning' of murine M N C on the antiserum-coated dishes (see Materials and Methods). Taken together, these data assured us that the procedures used to deplete murine M N C of either adherent or sIg-positive cells indeed led to at least partial depletion of the respective cell type. Therefore, we used these procedures to study the proliferative response of cells, either depleted

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Fig. 2. Effects of B1P-4 and B2 antibodies on proliferation of human peripheral blood MNC. Prior to the experiment, the Ig fractions of the hybridoma culture supernatants were diluted with and extensively dialyzed against the culture medium and aseptically filtered using a 0.22 ~m Millipore filter. Presented are the antibody dose-response proliferation curves in cultures containing BIP-4 (1) and B2 (2). Results of a representative experiment. Symbols represent means_+ SEM of quadruplicate determinations.

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Fig. 3. Incubation of murine MNC in plastic dishes, but not in RAM antiserum-precovered dishes, diminishes the response to PHA-M (see also the text). (1) Untreated MNC, (2) the cells recovered after incubation in plastic dishes (repeated twice), (3) the cells recovered after incubation in the antiserum-coated dishes. Shaded bars: proliferation in the absence of PHA-M; white bars: proliferation in the presence of PHA-M (1/~g/ml).

or not, to BIP-4 antibody. The results (Table 1) indicated that the response was not affected by adherent cell depletion, while it was obviously lacking in cultures depleted of sIg-positive cells. It seemed unlikely that this result was due to some toxic effect of the 'panning' procedure because cells recovered after 'panning' could respond to P H A - M (Fig. 3). We have suggested that M N C

Fig. 4. Binding of BIP-4 antibody (50 / t g / m l ; see also legend to Fig. 1) to murine MNC depleted of either adherent or sIg-positive cells. Curves represent binding of the antibody to: (1) sIg-positive cell-depleted MNC; (2) adherent cell-depleted MNC; (3)untreated cells. Binding of the peroxidase-conjugated antibody alone (blank) did not exceed 0.160 and was subtracted. Symbols are the mean values and SEM of triplicate determinations. The experiment was repeated with similar results.

depleted of sIg-positive cells were unable to proliferate in response to BIP-4 antibody because of one of the following reasons: (i) that these cells had no BIP-4 epitope; or (ii) that they had this epitope but did not enter the cell cycle subsequent to the antibody binding.

TABLE 1 SURFACE I M M U N O G L O B U L I N - P O S I T I V E CELL DEPLETION ABOLISHES THE P R O L I F E R A T I O N OF M U R I N E MNC TO BIP-4 ANTIBODY Cell type

Expt. No

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0.25

1,00

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7,803 ± 1,433 13,527 _+ 1,501

Untreated MNC

1 2

707 _+ 154 2,171 + 389

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1 2

402 ± 69 4 4 4 ± 33

MNC, depleted of sIg + cells c

1

1,945 + 183 1,203 ± 71

665 ± 960_+ 2,110 + 743 ±

32 148

5,659_+ 7,203 ±

925 814

646 50

1,612 ± 859 ±

853 82

a Expressed as counts per minute (mean _+ SEM of triplicate determinations). b MNC were resuspended in medium and plated onto 35-mm Costar dishes (for details, see text). c MNC were resuspended in medium and plated onto dishes preincubated with RAM antiserum and washed with PBS (for details, see text).

76

Murine M N C depleted of slg-positive cells have few, if any, detectable BIP-4 epitopes To discriminate between the two possible reasons stated above, we examined BIP-4 antibody binding to murine M N C depleted of either adherent or sIg-positive cells using the ELISA technique. In this series of experiments, we used varying concentrations of the antigen (test cells) and a constant dilution of BIP-4 antibody. When either untreated or adherent cell-depleted M N C were used as the test antigen, the absorbance grew commensurate with the increase of the cell concentration. Conversely, when the sIg-positive celldepleted population was used as the antigen, no significant growth of the absorbance was detected (Fig. 4). Therefore, it seemed likely that M N C depleted of sIg-positive cells (T lymphocytes and accessory cells) had no BIP-4 epitopes or the quantity of these epitopes was too small to be detected in the ELISA.

BIP-4 antibody blocks rlL-2-induced proliferation The results described in the previous sections allowed us to suggest that BIP-4-induced proliferation was a function of sIg-positive (B) lymphocytes or a subset because murine sIg-positive celldepleted M N C (a population comprising T lymphocytes and accessory cells) had few, if any, BIP-4 epitopes and did not proliferate in response to the antibody. Recent studies have shown that interleukin-2, a lymphokine with a broad spectrum of immunomodulating activities, can enhance the proliferation of preactivated B lymphocytes (Mingari et al., 1984). Moreover, rIL-2 is able to bind to, and to enhance immunoglobulin secretion of, resting B cells (Bich-Tuy and Fauci, 1985). Therefore, we assumed that it would be interesting to examine whether or not the proliferative response induced by BIP-4 antibody would be affected by rIL-2. Preliminary experiments failed to reveal a significant modulation of the above response if rIL-2 was added to standard 3-day cultures. Under these conditions, rIL-2 did not cause a proliferative response on its own. However, the lymphokine did induce a dose-dependent proliferative response in prolonged (7-day) cultures of murine MNC. One representative experiment is shown in Fig. 5. As shown, the response peaked at an rIL-2 concentra-

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Fig. 5. The proliferative response to IL-2 is blocked by BIP-4 antibody. Human rlL-2 (Biogen, Moscow) was tested in the standard cytotoxic lymphoid line (CTLL) assay (Gillis et al., 1978), as indicated by the manufacturer. Prior to the experiment, the gamma-irradiated preparation was diluted in medium and added to MNC cultures to give final concentrations indicated on the abscissa. The cells were cultured as described in Materials and Methods. Presented are the rIL-2 dose-response proliferation curves in cultures containing the following antibody (100 ng/ml): (1) BIP-4, (2) B2, (3) medium only. Symbols are means_+SEM of quadruplicate determinations (data from an experiment which was repeated twice with similar results).

tion as low as 0.5 U / m l , which is consistent with the observations of Lakhanpal et al. (1987) who examined the response of human peripheral blood T cells to rIL-2. Lymphokine concentrations greater than 10 U / m l caused no significant proliferative response (reproducibility of this result has not yet been explained, possibly attributable to the particular rIL-2 preparation used). Interestingly, the proliferative response triggered by rIL-2 was completely abolished when BIP-4 antibody (100 n g / m l ) was present in the cultures. Thus, although rIL-2 seems to have no effect on the proliferative response triggered by BIP-4 antibody, the latter is able to block the proliferative response caused by rIL-2 within the population of murine MNC.

Discussion

The results presented in this paper can be summarized as follows. BIP-4, a monoclonal antibody originally derived from a cytoplasmic protein with

77 some of the properties of a b r a i n cell s o d i u m channel, detected a n epitope associated with the cells of the i m m u n e system. This a n t i b o d y was mitogenic to i m m u n e cells though the m o d e of its m i t o g e n i c i t y allowed us to suggest that a proliferative response triggered b y this a n t i b o d y was a f u n c t i o n of some i m m u n e cell subset. M u r i n e splenic M N C depleted of slg-positive cells had few, if any, BIP-4 epitopes a n d did n o t proliferate in response to the a n t i b o d y . Therefore, BIP-4 antib o d y b o u n d to a n d triggered the mitogenesis of slg-positive (B) lymphocytes or of their subset. I n p r o l o n g e d cultures c o n t a i n i n g r l L - 2 a n d n o other d e f i n e d stimulant, BIP-4 a n t i b o d y blocked the l y m p h o k i n e - i n d u c e d proliferative response. M a n y questions, of course, arise c o n c e r n i n g the n a t u r e of the i m m u n e cell-associated BIP-4 epitope. First, what molecule(s) bears the BIP-4 epitope o n the surface of b o t h excitable (brain, n e u r o b l a s t o m a ) a n d i m m u n e cells? F u r t h e r studies m u s t be u n d e r t a k e n to address this q u e s t i o n a n d they might be of critical i m p o r t a n c e for u n d e r s t a n d i n g whether or not the BIP-4-carrying molecule is related to the family of ion channels. Second, what is the true origin of the cells within the i m m u n e cell p o p u l a t i o n which proliferate in the response to BIP-4 a n d what physiological significance this response might have? T o answer this question, it will be necessary to study the effects of BIP-4 o n extensively purified l y m p h o c y t e subsets. Finally, it is i n t r i g u i n g to elucidate the mecha n i s m b y which the a n t i b o d y blocks r l L - 2 - i n d u c e d proliferation. It is t e m p t i n g to speculate that the p h e n o m e n o n is based o n some h o m o l o g y between the putative BIP-4 epitope-carrying molecule a n d a structure c o m p r i s i n g part of the IL-2 receptor (possibly, one of the receptor s u b u n i t s - - see Smith (1988) for a review of IL-2 receptor structure). Therefore, future studies at the molecular level c o n c e r n i n g the n a t u r e of BIP-4 epitope-carrying molecule might also be useful for u n d e r s t a n d i n g the i n t i m a t e m e c h a n i s m s of the present immunologic phenomena.

Acknowledgements W e would like to t h a n k Prof. P.G. K o s t y u k a n d Dr. M.K. Malysheva for their helpful criticism,

Drs. A.L. Pukhalsky for the generous gift of rlL-2 a n d useful advice, A.Yu. Volgin for antibodies, a n d A.M. B u k h a n y e v i c h for the m y e l o m a cell line. The expert assistance of Dr. P.G. K l e r i n g in operating with the M o r p h o q u a n t p h o t o m e t e r a n d V.N. G o l o b o r o d ' k o with the L U M A M microscope is gratefully acknowledged.

References Baumgarten, A. (1986) Cell ELISA for quantitation of leucocyte antigens: requirements for calibration. J. Immunol. Methods 94, 91-98. Bich-Tuy, L.-T. and Fauci, A.S. (1985) Direct effect of interleukin 2 on the differentiation of human B cells which have not been preactivated in vitro. Eur. J. lmmunol. 15, 1075-1082. BiSyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 99, 77-82. Brodsky, F.M., Parham, P., Barnstable, C.J., Crumpton, M.J. and Bodmer, W.F. (1979) Monoclonal antibodies for analysis of the HLA system, lmmunol. Rev. 47, 32-62. Cahalan, M.D., Chandy, K.G., DeCoursey, T.E., Gupta, S., Lewis, R.S. and Sutro, J.B. (1987) Ion channels in T lymphocytes. Adv. Exp. Biol. Med. 213, 85-101. Choquet, D. and Korn, H. (1988) Modulation of voltage-dependent potassium channels in B lymphocytes. Biochem. Pharmacol. 37, 3797-3802. Gallin, E.K. and Sheedy, P.A. (1988) Leucocyte ion channels and their functional implications. In: I. Goldstein, R. Synderman and E.K. Gallin (Eds.), Inflammation, Raven Press, New York, pp. 651-664. Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A. (1978) T cell growth factor: parameters of production and a quantitative microassay for activity. J. lmmunol. 120, 2027-2035. Lakhanpak S., Gonchoroff, N.J. and Handwerger, B.S. (1987) Interleukin-2 induces proliferation of normal 'resting' human T cells in the absence of other known external stimulation. Cell. Immunol. 106, 62-75. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol method. J. Biol. Chem. 193, 265-267. Malysheva, M.K., Lishko, V.K., Zhukareva, V.A., Lysenko, V.V. and Tretyakov, L.A. (1987) Purification of cytoplasmic tetrodotoxin-sensitive protein from bovine brain tissue. Neurophysiol. (Kiev) 19, 202-209. McCann, F.V., McCarthy, D.C., Keller, T.M. and Noelle, R.J. (1989) Characterization of a large-conductance non-selective anion channel in B lymphocytes. Cell. Signal. 1, 31-44. Mingari, M.C., Gerosa, F., Carra, G., Accolla, R.C., Moretta, A., Zubler, R.H., Waldmann, T.A. and Moretta, L. (1984) Human interleukin-2 promotes proliferation of activated B cell via surface receptors similar to those of activated T cells. Nature 312, 641-645.

78 Pateraki, E., Guesdon, J.L., Serie, C. and Avrameas, S. (1981) TerELISA: the ELISA test performed in Terasaki plates. J. Immunol. Methods 46, 361 368. Pinchuk, G.V., Pinchuk, L.N. and Gerasymenko, O.V. (1988) Murine neuroblastoma cells absorb anti-cytoplasmic tetrodotoxin-sensitive protein antibodies. Neurophysiol. (Kiev) 20, 98-105. Pinchuk, G.V., Malysheva, M.K., Pinchuk, L.N., Gerasymenko, O.V. and Zhukareva, V.A. (1990) Possible relationship of brain cytoplasmic tetrodotoxin-sensitive protein to voltage-gated sodium channel shown by monoclonal antibody. J. Neuroimmunol. 26, 91-96. Royer, H.D. and Reinherz, E.L. (1987) T lymphocytes: ontogeny, function and relevance to clinical disorders. New Engl. J. Med. 318, 1136-1142.

Smith, K.A. (1988) Interleukin-2: inception, impact, and implications. Science 240, 1169 1176. Valentine, M.A., Clark, E.A., Shu, G.L., Norris, N.A. and Ledbetter, J.A. (1988) Antibody to a novel 95 kDa surface glycoprotein on human B cells induces calcium mobilization and B cell activation. J. Immunol. 140, 4071-4078. Volgin, A.Yu., Pinchuk, G.V., Chernikov, V.G., Korogodin, D.V., Kasimirskaya, V.V. and Pevnitzky, L.A. (1985) Strategy for establishing hybridomas producing antibodies to cell surface antigens. In: A. Novokhatsky et al. (Eds.), Monoclonal Antibodies in Microbiology and Virology, Meditzina, Moscow, pp. 150-161 (in Russian). Wysocki, J.L. and Sato, V.L. (1978) Panning for lymphocytes: a method for cell selection. Proc. Natl. Acad. Sci. U.S.A. 75, 2844 2846.

Monoclonal antibody to brain cytoplasmic tetrodotoxin-sensitive protein detects an epitope associated with lymphocytes and involved in lymphocyte activation.

We have previously derived a monoclonal antibody, BIP-4, which is specific to a mammalian brain protein representing a type of sodium channel. Here we...
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