Journal of Autoimmunity (1992) $527-544

Production and Characterization of Human Hybridoma Monoclonal Anti-Sm and Anti-UlRNP Antibodies Spontaneously Produced from Normal Human Lymphocytes

Cynthia J. L. Carruthers and David A. Bell Department

of Medicine,

(Received

Division of Rheumatology, Ontario, Canada 25 November

University Hospital,

1991 and accepted 20 March

London,

1992)

A panel of humamhuman hybridomas secreting monoclonal anti-Sm/ RNP antibodies was established by the fusion of normal human tonsillar lymphocytes to the lymphoblastoid B cell line GM4672. The specificity of these antibodies was studied by direct binding and competitive inhibition in enzyme linked immunosorbent assays (ELISAs), and by immunoblotting. The stable subclones of these hybridoma monoclonal antiSm/RNP antibodies could be classified into four groups according to ELISA. Group I bound Sm/RNP only, group II bound Sm-RNP, Ro/SS-A and La/SS-B, group III bound Sm/RNP and ssDNA, and group IV bound Sm/RNP, Ro/SS-A, La/SS-B and ssDNA. When antibodies from each of the groups were tested by immunoblotting, the following pattern of reactivity emerged. Group II and IV antibodies reacted with UlRNP-A, Sm-B/B, Sm-D and Sm-E proteins, as well as the Ro/SS-A and La/SS-B proteins. In contrast, group I and III antibodies did not bind to any individual protein components of Sm/RNP, Ro/SS-A or La/S%B antigens, but recognized their conformational epitopes. These results, therefore, directly demonstrate for the first time that normal-derived B cells have the genetic potential, revealed here by somatic cell hybridization, to produce anti-Sm/RNP antibody responses which are ordinarily only associated with systemic lupus erythematosus (SLE) and related connective tissue diseases.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of many diverse autoantibodies reactive to a variety of cellular components Correspondence to: Dr David A. Bell, Department of Medicine, London, Ontario, Canada, N6A 5A5.

University Hospital, Box 5339,

527 0896-8411/92/040527+

18 $03.00/O

0 1992 Academic Press Limited

528

Cvnthia T. L. Carruthers and David A. Bell

such as DNA, histones, DNA/histone complex and ribonucleoproteins (Sm, RNP, Ro/SS-A and La/SS-B) [l-4]. While anti-DNA antibodies are the most common and have been studied extensively, antibodies to Sm, which appear to be unique to SLE and to UlRNI?, a serological marker of mixed connective tissue disease, have received much less attention. Relatively little is known about the characteristics of the autoantibody response to Sm/RNP in SLE and about the immunoglobulin (Ig) genes which encode these autoantibodies. Autoantibodies to DNA can be expressed during polyclonal activation of lymphocytes in vitro and have been shown to be encoded by germline Ig genes [5, 61. Anti-Sm antibodies, however, cannot be expressed during polyclonal activation of lymphocytes in vitro, suggesting that these antibodies are not part of the normal B cell repertoire. Further, the spontaneous expression of anti-Sm antibodies in only 10 to 20% of SLE patients and in only some MRL/lpr autoimmune mice has suggested that anti-Sm autoantibodies may result from somatic mutation of germline Ig genes. Moreover, experimental studies in autoimmune mice have suggested that anti-Sm antibodies may have arisen from somatic mutation of Ig genes encoding anti-DNA autoantibodies [7]. Despite this, Sanz et al. [8] have recently reported the existence of an SLE derived hybridoma IgM monoclonal anti-Sm antibody encoded by a variable heavy chain region gene identical to a germline gene segment. This indicates that germline Ig genes exist which can encode anti-Sm autoantibodies in humans. Our studies were designed to determine directly whether the B cells of normal humans have the genetic potential to generate anti-SmjRNP autoantibodies and to examine the relationship of these anti-Sm/RNP autoantibody responses to other anti-RNA protein antigens as well as to anti-DNA autoantibodies and to the polyclonal serum anti-Sm/RNP autoantibody responses of SLE patients. We show here that the lymphocytes of normal individuals can produce IgM anti-Sm/RNP autoantibodies which appear to target the same proteins on immunoblots as the IgG antiSm/RNP autoantibodies of patients with SLE and related connective tissue diseases. This panel of cloned autoantibodies selected for anti-Sm/RNP reactivity reveal some unexpected reactivity to other structurally related autoantigens. Materials

and methods

Production of normal human:human hybridomas B cells from the tonsil lymphocytes of a normal individual were separated following Ficoll-Hypaque centrifugation by macrophage depletion (plastic adherence) and depletion of T cells with nylon wool columns [9]. The resulting population was 97% B cell enriched (by FACS analysis). These B cells were then fused to the lymphoblastoid cell line, GM4672 (Cell Repository Institute for Medical Research, Camden, NJ, USA) at a 1:l ratio with 0.5 ml of 44.4% polyethylene glycol (PEG 1500, MW 1450; Baker Chemical Co., Phillipsburg, NY, USA) as previously described [ 10,111. The fusion was referred to as the Bud fusion. Hybridomas producing antibodies reactive to Sm/RNP by ELISA were cloned in 96-well Nunclon trays by limiting dilution at 1 cell/well in hybridoma growth media (HGM). Clones producing antibodies reactive to Sm/RNP underwent a second cloning by limiting dilution.

Production and characterization of human antibodies

ELISA

529

IgM quantification

Microtiter well plates (Dynatech Laboratories Inc., Alexandria, VA, USA) were coated overnight at 4°C with 50 ~1 of F(ab’), goat anti-human IgM, Fc specific (10 ug/ml) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) in 15 mM carbonate-35 mM bicarbonate buffer (CB), pH 9.6 or with CB alone which served to monitor the assay for non-specific binding of human IgM. After three washes in Tris-HCl washing buffer (0.1 o/0bovine serum albumin (BSA) in 0.1 M Tris-HCl, pH 7.4 containing 0.05% Tween 20 (TWB) (Fisher Scientific, Fair Lawn, NJ, USA)), wells were blocked for 2 h at room temperature (RT) with 250 ~1 of 2% BSA-O. 1 M Tris-HCl, pH 7.4. After one wash, 50 l.d of pooled human IgM (Cooper Biomedical, West Chester, PA, USA) in dilutions ranging from 0.01 pg/ml to 2 pg/ml or diluted hybridoma supernatants (HS) were added to duplicate wells and incubated overnight at 4°C. Bound human IgM was detected after five washes and the addition of 50 ~1 of alkaline phosphatase-conjugated F(ab’), goat anti-human IgM, Fc specific (Zymed Laboratories Inc., San Francisco, CA, USA) diluted 1: 2,000 in TWB. After 3 h at RT, the plates were washed five times and conjugated antibody was detected using 50 ~1 of p-nitrophenyl phosphate at 1 pg/ml (Sigma Chemical Co., St. Louis, MO, USA) in diethanolamine buffer pH 9.8. After 30 min at 37”C, the reaction was stopped by adding 25 l.tlof 3N NaOH. Optical densities were determined at 405 nm with a Titertek Multiskan (Flow Laboratories Inc., McLean, VA, USA). HS IgM levels were quantified by extrapolation from the dose-response curve of pooled IgM. HGM, GM4672 supernatant and pooled human IgG (2 pg/ml) served as negative controls. ELISA-IgM

light chain expression

This assay was performed as described above for IgM quantification except that the conjugate used here was an F(ab’), goat anti-human lambda (h) or kappa (K) chain-specific antibody (Sigma Chemical Co.) diluted 1:6,000 in 1 %BSA-0.5°/0 BGG-0.1 M Tris-HCl, pH 7.4. Positive controls for h and K light chains consisted of sera from two patients with Waldenstrom’s macroglobulinaemia (IgMh or IgMK). Negative controls consisted of GM4672 supernatant (IgGK), HGM, 1% BSA-0.5% BGG-0.1 M Tris-HCl pH 7.4. ELISA-antibody

reactivity

to DNA and ssDNA

The method used to screen for antibodies to ssDNA and DNA was as previously described [ 111 except that the TWB in this study was 0.1 y0 Tween 20.

ELISA-antibody

reactivity

to SmIRNP,

Ro (SS/A)

and La fSSjl3)

Sm/RNP, Ro/SS-A and La/SS-B afIinity purified antigens from bovine thymus and spleen were purchased from Immunovision, Springdale, AR, USA. Microtiter plate wells were coated overnight at 4°C with antigens (5 l.tg/ml) in CB, or with CB alone. The remaining details of the assay including blocking, washing with TWB and incubation with a conjugate were essentially the same as that described for the IgM

530

Cynthia J. LXarruthers

and David A. Bell

ELISA quantification except that the conjugate for these assays was an F(ab’), goat anti-humam IgM (Jackson Laboratories) used at a 1:2,500 dilution. Positive controls consisted of diluted SLE patient sera with anti-Sm/RNP, anti-Ro/SS-A or antiLa/SS-B antibodies. Negative controls included HGM, 1 y0 BSA-0.5% BGG-0.1 M Tris-HCl pH 7.4 diluent and diluted normal human sera.

ELISA-competitive

inhibition assay

The same ELISA-antibody assay for Sm/RNl? was used here except that HScontaining hybridoma monoclonal antibodies were diluted in HGM to achieve 60% of their reactivity in the Sm/RNP ELISA. To some of the HS, 25 pg, 5 l.tg or 1 ug of inhibitor protein (Sm/U 1RNP, Ro/ S S-A, La/S S-B, ssDNA, RNA) was added. The antigens used in these inhibition experiments were identical to those used in ELISA. HS or HS plus inhibitor, previously incubated in a test tube at 37°C for 2 h with occasional mixing, was added to Sm/RNP coated wells and incubated for 3 h at RT. The wells were washed, the conjugate was added and antibody reactivity was detected as previously described.

ELISA-rheumatoidfactor

activity

Microtiter plate wells were coated overnight at 4°C with 50 ~1 of whole human IgG (Cooper Biomedical), the Fc portion of IgG, or the Fab portions of IgG (Jackson Laboratories) all at a concentration of 10 ug/ml in CB, or CB alone. Wells were washed twice with saline containing 0.05 ‘$” Tween 20 and once with saline. HS were added to duplicate wells and incubated overnight at 4°C. The wells were washed four times with saline containing 0.05% Tween 20 and once with saline. Bound IgM was detected with alkaline phosphatase conjugated F(ab’), goat anti-human IgM, Fc specific, diluted 1:2,500 in 3% BSA-saline-0.05°h Tween 20 and incubated for 3 hat RT. Wells were washed four times with saline-0.05% Tween 20 and once with saline, and developed for 30 min at RT with 50 ~1 of p-nitrophenyl phosphate diluted to 1 pg/ml in diethanolamine buffer. The reaction was stopped by adding 25 ~1 of 3N NaOH and plates were read at 405 nm. Positive controls consisted of diluted sera with known IgM rheumatoid factor activity lacking reactivity to nucleic acid antigens. Negative controls included HGM, GM4672 supernatant, 3% BSA-saline and diluted normal human sera.

Sodium dodecyl sulphate-polyacrylamide

gel electrophoresis

(SDS-PAGE)

Afinity purified Sm/RNP, Ro/SS-A and La/SS-B at 5 pg/lane and high molecular weight markers (BioRad Laboratories, Richmond, CA, USA) were mixed 1:3 with reducing buffer (0.0625 M Tris-HCl, pH 6.8-10% glycerol, 2% SDS, 5% 2-p mercaptoethanol, 0.01% bromophenol blue) and heated for 2 min at 97°C. The samples were centrifuged at 12,500 xg, and added to lanes in an SDSpolyacrylamide gel (4% stacking gel and 12% running gel). The proteins were separated by electrophoresis at 30 mA/gel for 5 h following the method described by Laemmli [12]. High molecular weight markers were run on each gel to identify relative molecular weights of separated antigen proteins. The gels were stained with

Production and characterization of human antibodies

A

C

B

531

D

-8

Figure 1. SDS-PAGE analysisof Sm/RNPRo/SS-A, La/SS-B antigensemployedin ELISA and immunoblotting.10 pgof affinitypurifiedSm/RNP(laneB), Ro/SS-A(laneC) andLa/SS-B(laneD) and high molecularweightmarkers(lane A) were appliedto an SDS-PAGE and stainedwith Coomassie BrilliantBlueas shown.The Sm/RNPproteinsareshown(lane B) at 53 kDa(68 kDa Ul RNP); 27/26kDa (B’/B);22 kDa (C); 12kDa (D); 10kDa (E); 9 kDa (F); and 8 kDa (G). The 60 kDa Ro/SS-A protein (lane C) and the 52 kDa La/SS-B also shown.

protein

together

with degradation

products

at 28,20

and 10 kDa (lane D) are

Coomassie Brilliant Blue (CBB) for 1 h at RT and destained in 40% methanol/lO% acetic acid for 3 h with several changes of the destain solution. The Sm/RNP preparations contained proteins of 53 kDa as a possible degradation product of the 68 kDa protein, 27/26 kDa doublet (B’/B), 22 kDa (C), 12 kDa (D), 10 kDa (E) and 9 kDa (F) and 8 kDa (G) in size. Protein A (approximately 32 kDa in size) could not be detected by CBB staining. The Ro/SS-A antigen preparation contained a 60 kDa protein and the La/SS-B antigen preparation contained proteins of 53 kDa and possible degradation products at 37, 28, 20 and 10 kDa (Figure 1). The La/SS-B protein has been reported to be approximately 50 kDa, but as shown here and by others, this protein may undergo rapid degradation into lower molecular weight immunoreactive products [ 131. Immunoblotting The above SDS-PAGE antigens, separated as described, were electrophoretically transferred to nitrocellulose (BioRad), as described by Tobin [ 141, in transfer buffer (25 mM Tris base, 192 mu glycine, 20% methanol, pH 8.3) at 75 V for 3 h. The nitrocellulose was then blocked overnight at 4°C with Tris buffered saline (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4) (TBS) containing 5% skim milk powder (Carnation Inc., Toronto, Ontario, Canada). Nitrocellulose strips were cut, washed in TBS and incubated for 3 h at RT with HS, diluted normal or SLE patient sera.

532

Cynthia J. L. Carruthers and David A. Bell

After three washes in TBS-0.05% Tween 20, the strips were incubated for 3 hat RT with alkaline phosphatase conjugated F(ab’), goat anti-human IgM, Fc specific diluted 1:2,500 in 1% BSA, 2% BGG-TBS. The strips were washed three times, incubated with substrate buffer (0.1 M Tris-HCl, 0.1 M NaCl, 50 mM MgCl,, pH 9.5) for 20 min and in 5-bromo-4-chloro-3-indolylphosphate p-toluidine (BCIP)/ Nitroblue Tetrazolium Chloride (NBT) chromogenic substrate (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg, MD, USA) for 20 min. Molecular weights of the antibody-bound substrate-stained antigen protein bands were approximated using prestained molecular weight markers as a reference. Dot blot analysis

Nitrocellulose was soaked for 1 h in TBS and placed in the Bio-dot microfiltration apparatus (BioRad). To each well was added 1 pg of Sm/RNP diluted in 100 ~1 of TBS, or TBS alone, to monitor iron-specific binding of human IgM. This was allowed to pass through the filter by gravity flow. Some preparations of Sm/RNP were heated for 2 min at 97°C or treated with 2% SDS or 5% 2P-mercaptoethanol before addition to the wells. To ensure maximum binding of the antigen to the nitrocellulose, 100 ~1 of TBS was added to each well and filtered by gravity flow. The nitrocellulose was blocked by adding 200 l.tl of 1% BSA-TBS. Two hundred yl of TBS containing 0.05% Tween (TTBS) was added to each well, filtered under vacuum and the procedure repeated a second time to wash the wells. One hundred ~1 of HS, diluted SLE patient sera, or negative controls were added and filtered by gravity flow. Three TTBS washes were performed to remove unbound antibody. One hundred ~1 of alkaline phosphatase conjugated F(ab’), goat anti-human IgM, Fc specific diluted 1:2,500 in TBS was added, filtered by gravity flow and unbound second antibody washed off with two TTBS washes. Bound antibody was detected by placing the nitrocellulose in BCIP/ NBT diluted in TBS for 20 min. To stop the reaction, the nitrocellulose was placed in H,O and air dried on filter paper. Immunoprecipitation

Immunoprecipitation experiments using HeLa cells were performed according to Forman et al. [ 151. Representative HS from each group of anti-Sm/RNP clones were concentrated by Amicon to approximately 100 pg IgM and bound to protein A coupled to a rabbit anti-human IgM antibody (Jackson Laboratories). Following extraction and precipitation, the RNA from the HeLa cell immunoprecipitations were fractioned on a denaturing polyacrylamide gel (10% polyacrylamide-10 N urea1 gels) and silver stained. Positive controls for this assay included SLE serum with known IgG anti-Sm/RNP, anti-Ro/SS-A or anti-La/SS-B antibodies. Results Fusion and cloning strategy

One hundred and fifteen of 210 wells (2 x lo5 cells/well) seeded showed growth by 6 weeks. These growth characteristics were similar to those reported from our

Production and characterization of human antibodies

533

laboratory with other humanhuman hybridomas using the same fusion partner [ 111. Hybridomas growing in 84/l 15 wells produced IgM, while hybridomas in the remaining 31 wells produced either no detectable Ig or IgG with no identified specificity. Fifteen of the 84 IgM producers exhibited anti-Sm/RNP reactivity by ELISA, but none of the remaining IgG-positive or IgM-negative hybridomas showed binding to Sm/RNP. The 15 IgM anti-Sm/RNP-positive hybridomas were selected for cloning twice by limiting dilution at 1 cell/well. The percentage growth ranged from 3.8 to 50% for the first and 0.8 to 17% for the second cloning. Employing these data, a probability for monoclonality of 98.7% for the subclones was derived, according to the method of Collar and Collar [ 161. All subclones were stable for longer than 6 to 12 months.

ELISA

antibody binding spec$cities

The subclones selected for anti-Sm/RNP reactivity by ELISA were also tested by ELISA for reactivity with other relevant autoantigens (DNA; ssDNA; Ro/SS-A; La/SS-B). The antibody reactivities of subclones could be classified into four groups according to their ELISA antibody binding characteristics (Figure 2). Group I (Bud 46) antibodies showed reactivity to Sm/RNP only; group II (Bud 94,91) cloned antibodies showed reactivity to Sm/RNP, Ro/SS-A and La/SS-B; group III (Bud 1, 45, 47) antibodies showed reactivity to Sm/RNP and ssDNA; and group IV (Bud 114) antibodies showed reactivity to Sm/RNP, Ro/SS-A, La/SS-B and ssDNA. None of these antibodies bound to DNA or cardiolipin, but some bound to IgG (see below). The IgM levels of the HS from these clones ranged from 0.2 pg/ml to 8.5 pg/ml. There was no consistent correlation between IgM concentrations and ELISA reactivity to any of the specific antigens tested here. The IgM concentrations here are consistent with the IgM concentrations in HS from other human lymphocyte fusions performed in this laboratory [ll]. Group I, III and IV antibodies all expressed K light chains, while group II antibodies expressed h light chains (Figure 2).

ELISA

rheumatoidfactor activity

The cloned hybridoma antibodies to anti-Sm/RNP which also bound Ro/SS-A (groups II and IV) showed variable binding reactivity to human IgG. Both group II and group IV antibodies showed binding to whole human IgG and IgG Fc while group II antibodies also bound to IgG Fab.

Specificity of binding of cloned hybridoma antibodies Inhibition of antibody binding to Sm/RNP coated to ELISA wells by homologous or other antigens in solution was employed to confirm antibody binding specificities. Representative inhibition results for clones from each of the four different groups are shown in Figure 3. Group I (Bud 46.15.1) antibodies bound to Sm/RNP only by ELISA. Five pg of homologous Sm/RNP produced greater than 50% inhibition, while 25 pg of

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Production and characterization of human antibodies % Bindmg

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Figure 3. Inhibition of anti-Sm/RNP ELISA reactivity for representative subcloned antibodies from each group. Various concentrations of Sm/RNP ( n ), Ro/SS-A (A), La/SS-B (A) and ssDNA (0) were preincubated with representative cloned hybridoma antibodies from each of the groups prior to determining their anti-Sm/RNP reactivity in ELISA. Figure 3a (group I; Bud 46.15.1); Figure 3b (group II; Bud 94.9.2); Figure 3c (group III; Bud 47.8.1); Figure 3d (group IV; Bud 114.4.2).

Ro/SS-A, La/SS-B and ssDNA, did not significantly inhibit anti-Sm/RNP binding (Figure 3a). Group II (Bud 94.9.2) antibodies bound Sm/RNP, Ro/SS-A and La/SS-B by ELISA. Greater than 50% inhibition was observed with 25 ug of Sm/RNP but, like group I antibodies, little inhibition (less than 20%) was achieved with 25 ltg of Ro/SS-A or La/SS-B and no inhibition was observed with ssDNA (Figure 3b).

536

Cynthia J. L. Carruthers and David A. Bell

-32 -28

-13

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DEFGHIJ

KL

MNOPQ

RSTUV

Figure 4. Immunoblotting of cloned hybridoma monoclonal antibodies to Sm/RNP. Ten ug of affinity purified Sm/RNP protein separated on SDS-PAGE was electrophoretically transferred to nitrocellulose and incubated with diluted (l/100) normal human sera (lane A), GM4672 supernatant (lane B), HGM (lane C), diluted (l/100) anti-Sm/RNP SLE sera (lanes D to J) and cloned hybridoma supernatants (lanes KtoV).Bud91.3.1,91.3.2,91.3.7(lanesK,LandM)andBud94.9.2,94.56.3and94.91.8(lanesN,Oand I’) are from group II; Bud 114.4.2 (lane Q) is from group IV; Bud 46.15.1 (lane R) is from group I; and Bud 1.22.1 (lane S) and Bud 47.1.5,47.8.1 and 47.11.15 (lanes T, U and V) are from group III. The serum and hybridoma supernatants (lanes K to Q) blot proteins of 32 kDa (U 1RNP-A protein), 27/26 kDa (Sm-B/B), 12 kDa (Sm-D protein), while the serum only blots a 22 kDa (UlRNP-C protein).

Group III (Bud 47.8.1) antibodies bound Sm/RNP and ssDNA by ELISA. Greater than 50% inhibition was achieved with 5 pg of ssDNA and 25 yg of Sm/RNP but was not achieved with 25 pg of either Ro/SS-A or La/SS-B (Figure 3~). Group IV (Bud 114.4.2) antibodies bound Sm/RNP, Ro/SS-A, La/SS-B and ssDNA by ELISA. Greater than 50% inhibition was achieved with 5 ltg of ssDNA and 25 ltg of Sm/RNl?, Ro/SS-A and La/SS-B (Figure 3d). All of the antibodies in each of the groups were tested for inhibition with up to 25 ug of RNA and BSA, and no inhibition was observed (data not shown). Immunoblotting

Western immunoblotting was performed with the same afhnity purified antigens employed in ELISA, to confirm binding of the subcloned monoclonal antibodies to Sm/RNP, as well as to examine their reactivity to Ro/SS-A and La/SS-B proteins. Positive controls consisted of diluted human SLE serum containing IgM antibodies to Sm/RNP, Ro/SS-A or La/SS-B. Negative controls consisted of diluted normal human serum, HGM and GM4672 supernatant. The Sm/RNP proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated with HS, concentrated HS or the controls stated above (Figure 4). Diluted normal human sera, HGM or the GM4672 super-

Production and characterization of human antibodies

ABC

D

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Figure 5. Immunoblotting of cloned hybridoma monoclonal antibodies to Ro/SS-A. Ten Bg of affinity purified Ro/SS-A separated on SDS-PAGE was electrophoretically transferred to nitrocellulose and incubated with diluted (l/100) normal human sera (lane A), HGM (lane B), GM4672 supematant (lane C), diluted (l/100) anti-Ro/SS-A SLE sera (lane D) and HS. Bud 91.3.2 and Bud 91.3.7 HS (lanes E and F),Bud94.9.2andBud94.91.8HS(lanesGandH)arefromgroupII;Bud114.4.2and114.4.11HS(lanes I and J) are from group IV; Bud 46 HS (lane K) is from group I; Bud 1 HS (lane L), Bud 47.1.5 and Bud 47.8.1 HS (lanes M and N) are from group III.

natant did not bind to Sm/RNP proteins (Figure 4, lanes A, B and C). Known SLE anti-Sm and/or anti-RNP-positive sera contained IgM autoantibodies which bound to the UlRNP-A (32 kDa) and the UlRNP-C (22 kDa) proteins and to the Sm-B’/B (27/26 kDa), Sm-D (12 kDa) and Sm-E (10 kDa) proteins (Figure 4, lanes D to J). None of these IgM anti-Sm/RNP antibodies bound to the 68 kDa UlRNP protein or its presumed degradation product of 53 kDa identified on SDS-PAGE in the affinity purified Sm/RNP preparation. The same 32 kDa (UlRNP-A) protein immunoblotted by SLE serum IgM and the cloned hybridoma supernatant monoclonal antibodies from groups II and IV, but there was no observed reactivity with the 22 kDa (UlRNP-C) protein. Both serum IgM in group II and IV cloned hybridoma supernatants immunoblotted the 27/26 kDa (B’/B), 12 kDa (D) and 10 kDa (E) proteins of Sm (Figure 4). Cloned hybridoma supernatant antibodies from groups I and III did not immunoprecipitate any Sm or RNP antigens. Further, while IgG anti-UlRNP antibodies immunoblotted the 68 kDa (53 kDa degradation product), neither SLE serum, IgM nor hybridoma supernatant IgM antibody showed this reactivity (data not shown). The affinity purified bovine Ro/SS-A antigen preparation was run on SDSPAGE, transferred to nitrocellulose and incubated with concentrated HS or the controls stated above (Figure 5). Diluted normal human sera, HGM or GM4672 supernatant did not react with Ro/SS-A (Figure 5, lanes A, B and C). The diluted IgM anti-Ro/SS-A-positive SLE serum did blot to a 60 kDa Ro/SS-A protein (Figure 5, lane D). Group II and IV cloned hybridoma antibodies, which were antiRo/SS-A positive by ELISA, blotted to the 60 kDa Ro/SS-A protein (Figure 5, lanes

538

Cynthia J. L. Carruthers and David A. Bell

-43

A BCD

E FG

HIJ

KL

Figure 6. Immunoblotting of cloned hybridoma monoclonal antibodies to La/SS-B. Ten ug of affinity purified La/S%B separated by SDS-PAGE, was electrophoretically transferred to nitrocellulose and incubated with diluted (l/100) normal human sera (lane A), HGM (lane B), GM4672 (lane C), diluent (IaneD), diluted(1/1OO)anti-La/SS-B SLE sera(lanesE, FandG),andHS. Bud91 HS (1aneH)andBud 94 HS (lane I) are from group II; Bud 114 HS (lane J) is from group IV; Bud 46 HS (lane K) is from group I; and Bud 47 HS (lane L) is from group III.

E, F, G, H, I and J). The group I and III hybridoma antibodies that did not react with Ro/SS-A by ELISA did not blot the 60 kDa Ro/SS-A protein (Figure 5, lanes K, L, M and N). The affinity purified bovine La/SS-B antigen preparation was also employed in immunoblotting (Figure 6). Diluted normal human sera, HGM, GM4672 or diluent did not blot the La/SS-B protein (Figure 6, lanes A, B, C and D). Known IgM anti-La/SS-B-positive SLE serum controls blotted to the 43 kDa La/SS-B protein and to probable La/SS-B protein degradation products (Figure 6, lanes E, F and G). Hybridoma antibodies from group II and IV clones which bound La/SS-B by ELISA also blotted weakly to the La/SS-B protein and La protein degradation products (Fig-m& 6, lanes H, I and J). Group I and III antibodies which did not bind to La/SS-B by ELISA did not blot La/SS-B proteins (Figure 6, lanes K and L) when tested at similar antibody (IgM) concentrations as the group II and IV cloned hybridoma supernatants.

Dot blot analysis In order to investigate whether the lack of anti-Sm/RNP reactivity in Western immunoblotting of groups I and III ELISA-positive anti-Sm/RNl? antibodies could be attributed to the denaturing conditions employed in the SDS-PAGE, dot blot analysis was performed with native or denatured Sm/RNP proteins (Figure 7). 0.5 ug of untreated Sm/RNP diluted in 100 1.11 of TBS (row 1) or an equal amount Sm/RNP denatured by heating at 97°C (row 2) or mixing with 2% SDS (row 3) or mixing with 5% 2P-mercaptoethanol (row 4), were dot blotted onto nitrocellulose. Duplicate

Production and characterization of human antibodies

A

B

C

D

E

F

539

G

3 4 Figure 7. Dot blot analysis of cloned hybridoma monoclonal antibodies to Sm/RNP and treated Sm/RND. Bud HS were dot blotted to 0.5 pg of untreated Sm/RNP (row l), 0.5 pg of Sm/RNP heated for 2 min at 97°C (row 2), 0.5 pg of Sm/RNP with 2% SDS (row 3), and 0.5 pg of Sm/RNP with 5% 2 p mercaptoethanol (row 4). A positive SLE anti-Sm/RNP sera (lane A), GM4672 supematant (lane B), Bud 91.3.3(laneC),Bud94.13.1 (laneD),Bud 114.4.9(laneE),Bud46.15.3(laneF)andBud47.15.1 (1aneG) were incubated with the treated and untreated Sm/RNP.

wells were incubated with an SLE IgM anti-Sm/RNP positive diluted serum control (lanes A) or negative GM4672 supernatant control (lanes B) or hybridoma antibody. SLE serum IgM bound to untreated and denatured Sm/RNP while the GM4672 supernatant did not bind to either undenatured or denatured Sm/RNP. Group II HS in lanes C and D (Bud 91.3.3 and 94.13.1 respectively) bound to denatured or undenatured Sm/RNP with the same intensity. In contrast, a group I HS (Bud 46.15.3) and group III HS (Bud 47.15.1) in lanes F and G respectively bound to undenatured Sm/RNP but did not bind to denatured Sm/RNP. Therefore, the reactivity to Sm/RNP proteins by the cloned antibodies from groups II and IV was unaffected by denaturing the Sm/RNP, while groups I and III cloned antibodies which were negative by Western immunoblotting were only reactive to Sm/RNP proteins in their native but not in their denatured state. Immunoprecipitation

experiments

Concentrated antibody supernatants from representative clones of each group were present at a sufficient concentration to perform immunoprecipitation experiments with HeLa cell extracts. These experiments, kindly performed by Dr John Harley, University of Oklahoma, Health Sciences Center, USA, employed known IgG serum antibodies to Sm/RNP, Ro/SS-A or La/SS-B. U, and U, RNA were precipitated with the HS from clones 114.5.7, but no RNA species was precipitated with the HS from any other clones (data not shown). Thus, while Western immunoblotting of HS from clones in group II showed binding to Sm/RNP proteins even under denaturing conditions and expressed many of the same properties as the HS from clones in group IV, only the latter exhibited immunoprecipitation to RNA species typical of these Sm/RNPs. While the denaturing conditions employed in these immunoprecipitation experiments may provide an explanation for the failure of HS from clones in group I and group II to bind to U RNA under the conditions of these experiments, this does not provide an explanation for the failure of HS clones of group II to behave in this manner. Further, although the HS from clones from groups II and IV bound to both Ro/SS-A and La/SS-B proteins by Western immunoblotting, these HS failed to immunoprecipitate the hY RNAs associated with these proteins.

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Cynthia J. L. Carruthers and David A. Bell

Table

1. Summary

of reactivity

of anti-SmlRNP antibodies

cloned

hybridoma

monoclonal

Dot blot? Group

1 (IgMW

II (IgML)

III (IgMK) IV (IgMK)

ELISA

Inhibition*

Sm/RNP Sm/RNP Ro/SS-A La/SS-B Sm/RNP ssDNA Sm/RNP Ro/SS-A La/SS-B ssDNA

Sm/RNP Sm/RNP

ssDNA 9 Sm/RNP ssDNA$

Sm/RNP

Western immunoblot

Native

Denatured

R.F.

NEG. Sm/RNP$ Ro/SS-A La/SS-B

POS. POS.

NEG. POS.

NEG. POS.$

NEG.

POS.

NEG.

NEG.

Sm/RNPJ Ro/SS-A La/SS-B

POS.

POS.

POS.II

*500/b inhibitionof anti-Sm/RNP ELISA vs Sm/RNP coated wells. TDot blot analysis with either native Sm/RNP or denatured (97”Cx2 sm/RNP. $Reactivity vs UlRNP A; Sm-B/B, D, E proteins. SReactivity in ELISA vs IgG, IgG Fab, IgG Fc. IIReactivity in ELISA vs IgG, IgG Fc.

Summary

of reactivity of cloned anti-SmlRNP

or 2% SDS or 5% 2ME)

IgM antibodies

Table 1 provides a summary of the reactivity by ELISA, Western immunoblotting and dot blot analysis of the antibodies from each of the four antigen reactivity groups for comparison purposes. Thus, groups I and II hybridoma antibodies bound to and were inhibited predominantly by Sm/RNP in ELISA, while the antibodies in groups III and IV bound to Sm/RNl? and ssDNA but were inhibited more effectively by ssDNA than Sm/RNP. Anti-Sm/RNP reactivity of antibodies in groups II and IV bound to Sm/RNP proteins under the denaturing conditions of Western immunoblotting while the anti-Sm/RNP reactivity of antibodies from groups I and III could only be revealed by dot blot analysis under non-denaturing conditions. The ability to bind to Ro/SS-A and La/SS-B either by ELISA or by Western immunoblotting correlated directly with the existence of rheumatoid factor activity. Discussion

These studies provide the first direct evidence that the B cells of normal humans have the genetic potential, expressed here under the conditions of somatic cell hybridization, to produce anti-Sm/RNP antibodies. The pattern of binding of these antibodies is similar to the polyclonal antibody response which is typical of SLE patients with anti-Sm antibodies which target proteins typical of both Sm(B’/B, D, E) and UlRNP(A). The stable subclones produced from these hybridomas in many instances revealed concentrations of IgM antibodies similar to those reported by others using EBV transformation of circulating SLE lymphocytes [27]. Some of these antibodies from the B cells derived from the selective cloning of hybridomas

Production and characterization of human antibodies

541

with reactivity to Sm/RNP proteins also showed additional unexpected reactivity to other structurally unrelated antigens including Ro/SS-A, La/SS-B and ssDNA. The pattern of reactivity of these antibodies permitted these cloned B cells to be segregated for purposes of comparison into four unique groups, as summarized in Table 1. Anti-Sm/RNP antibodies in groups II and IV appeared to target Sm/RNP antigenic determinants which retained their stability under denaturing conditions while those from groups I and III were only able to target Sm/RNP molecules under conditions which retained their native conformational state. While the HS from cloned B cells of groups II and IV bound to Sm/RNP, Ro/SS-A and La/SS-B proteins under the denaturing conditions of Western immunoblotting, only the HS from the B cell clones of group IV immunoprecipitated the U, and U, RNA associated with the Sm/RNP particle. Further, none immunoprecipiated hY RNA associated with the Ro/SS-A and/or La/SS-B particle. We assume that this failure to immunoprecipitate RNA from the remaining clones is a reflection of the relatively low aflinity of the IgM antibodies produced by these clones and/or the relative insensitivity of the assay system for the detection of binding by these types of antibodies. Failure to detect U RNA by immunoprecipitation under denaturing conditions with the HS of clones I and II could be interpreted as a further indication of the conformational instability of the epitopes targeted by these antibodies. An additional unexpected finding of these studies was the ability of some of these antiboides to target IgG or the Fab or Fc fragments of IgG. This was identified only among those anti-Sm/RNP selected clones which also targeted Ro/SS-A and La/SS-B (groups II and IV). The targets of these anti-Sm/RNP antibodies retained conformational stability under denaturing conditions since these antibodies also revealed binding to IgG and IgG Fab and Fc fragments in Western immunoblotting (data not shown). This suggests that common conformational determinants shared by IgG, Sm/RNP, Ro/SS-A and La/SS-B cannot explain this polyreactivity. We have also noted in other studies that hybridoma monoclonal antibodies selected for binding to Ro/SS-A frequently reveal binding to IgG or its Fab or Fc fragments on immunoblotting. While these data suggest the existence of some common structure(s) shared by the targeted epitopes present in Ro/SS-A, La/SS-B and IgG, we know of no direct evidence to support this contention. Mamula et al. [ 171, however, have previously demonstrated that serum IgG anti-Ro/SS-A autoantibodies from R SLE patients could bind IgG including Fab and/or Fc. Since these molecules could also absorb out the IgG anti-Ro/SS-A reactivity to the 60 kDa bovine protein, other different but closely located binding sites for these molecules exist in the antibody variable region, or these molecules may have some structurally homologous epitopes. The unusual reactivity of these cloned hybridoma monoclonal antibodies is not without precedent. For example, polyspecificity of normal human IgM monoclonal antibodies has been reported previously by us [ 1 l] and by other investigators [lo] and has generally been attributed to the relatively low afhnity of IgM compared with IgG antibodies or to the inherent degeneracy of the primary B cell repertoire. We have observed in other studies that cloned human hybridomas selected for anti-Ro/SS-A reactivity and the IgM anti-Ro/SS-A antibodies in SLE patients frequently show reactivity to other RNA protein antigens (Sm/RNl?, La/SS-B) [lg]. A common genetic origin has also been suggested for anti-DNA and anti-Sm antibodies [19]

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Cynthia J. L. Carruthers and David A. Bell

despite the fact that these antibodies appear to be expressed disco-ordinately in disease. Spontaneously arising hybridoma antibodies from MRL mice, which bind both DNA and Sm, have been identified and these antibodies often express shared idiotypes [20]. Of greater interest is the recent observation of Bloom et al. [21] that the V region genes encoding anti-Sm and anti-DNA in MRL mice are highly homologous. Structural elements of the 70 kDa UlRNP associated antigen have also been identified in the VL sequence of an MRL/lpr mouse antibody expressing H130, a recurrent idiotype of anti-DNA antibodies in this strain [22]. Polyspecificity of autoantibodies to structurally different molecules could also be explained by antigen binding by different V region sites of the same antibody molecule. Radic et al. [23], through transfection experiments, have recently demonstrated the separate contribution of Ig H and L chains to autoimmune polyspecific autoantibodies in man. There is also evidence that the antigen binding site of some IgM rheumatoid factors may be located on the light chain V region [26]. In the present study, our observation that the cloned anti-Sm/RNP antibodies from groups III and IV could be inhibited quite readily with ssDNA may indicate that closely related or overlapping Ig V region gene segments are responsible for encoding these IgM antibodies with the property of binding to both ssDNA and Sm/RNP antigens. Finally, our results revealing the reactivity of normal human monoclonal antibodies with Sm/RNP proteins should be compared to the results supported by two other groups employing human :monoclonal antibodies targeting these antigens. Chen et al. [24] identified an IgM antibody with Sm/RNP reactivity, resulting from the EBV transformation of circulating lymphocytes from a patient with mixed connective tissue disease. Thisantibody targeted the 68 kDa polypeptide of UlRNP and also immunoprecipitated UlRNA. Its reactivity to the 68 kDa protein was also inhibited with other Ul Sm/RNP polypeptides (A, B’/B, D) but could be inhibited by only 50 y0 with relatively high concentrations of the homologous 68 kDa polypeptide. In the present studies, we were unable to demonstrate reactivity to this 68 kDa UlRNP polypeptide with any of our monoclonal antibodies. A human IgM K hybridoma monoclonal anti-Sm antibody which was identified following somatic cell hybridization with GM4672 and the circulating lymphocytes of an SLE patient was studied by Takei et al. [25] and Sanz et al. [B]. This antibody targeted the B’/B and D proteins of Sm but its reactivity with other RNA proteins or DNA was not discussed. The existence in the normal B cell repertoire of antibodies that target both the B and D peptides of Sm, as suggested by the present studies and by those of Takei et al., are also consistent with the hypothesis proposed by Eisenberg et al. [28] that such antibodies may have a negative regulatory function. The passive administration of antibodies with its specificity suppressed a full anti-Sm antibody response when administered to MRL/lpr mice while those targeting only the D peptide had an enhancing effect on anti-Sm antibodies. The existence in the normal B cell repertoire of antibodies targeting both the D and B peptides of Sm is predicted by this hypothesis since these antibodies may have the potential to suppress the emergence of a full autoantibody response to the Sm particle. It remains to be determined whether all such ‘suppressive antibodies’ are encoded by germline V region Ig genes. In summary, the foregoing studies reveal that anti-Sm/RNP antibodies with quite different binding properties can be derived from the B lymphocytes of normal

Production and characterization of human antibodies

543

humans. These apparently natural IgM antibodies show some binding properties also reported with IgG antibodies to RNA proteinsantigens obtained in SLE mice and in human SLE. The analysis of the V region genes used to encode these antibodies in relation to those used to encode anti-Sm/RNP antibodies from patients with disease, as well as the genes used to encode antibodies to unrelated antigens such as DNA, is currently underway in this laboratory. These studies should provide further insight into the molecular genetic mechanisms responsible for the emergence of antibodies with these different specificities in human disease and may clarify in human B cells the contribution of H and L chain variable regions to these different specificities. Acknowledgement

~

The authors wish to acknowledge the assistance and advice of Drs Ewa Cairns and Helene Massicotte and to thank them for carefully reviewing the manuscript. We thank Dr Harley and his associates at the University of Oklahoma Health Science Center for performing the immunoprecipitation experiments with our hybridoma supernatants. The authors also thank Tina Krulc for preparing the manuscript. This work was supported by a Research Grant from The Canadian Arthritis Society. References 1. Tan, E. M. 1989. Interactions between autoimmunity and molecular and cell biology. J. Clin. Invest. 84: l-6 2. Sharpe, G. C., W. S. Irvin, E. M. Tan, R. G. Gould, and H. R. Holman. 1972. Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am.J. Med. 52: 148-159 3. Harely, J., E. Alexander, W. Bias, 0. Fox, T. Provost, M. Reichlin, H. Yamagata, and F. Arnett. 1986. Anti-R0 (SS-A) and Anti-La (SS-B) in patients with Sjiigren’s Syndrome. Arthritis Rheum. 29: 196-206 4. Reichlin, M. and J. Harley. 1987. Antibodies to Ro (SS-A) and the heterogeneity of systemic lupus erythematosus. J. Rheumatol. 14: 112-l 17 5. Cairns, E., P. C. Dwong, V. Misener, P. Ip, D. A. Bell, and K. A. Siminovitch. 1989. Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. J. Zmmunol. 143: 685-691 6. Dersimonian, H., R. S. Schwartz, K. J. Barrett, and B. D. Stollar. 1987. Relationship of human variable region heavy chain VH germ line genes and genes encoding anti-DNA autoantibodies. J, Zmmunol. 139: 2496-2501 7. Eisenberg, R. A., S. Y. Craven, R. W. Warren, and P. I. Cohen. 1987. Stochastic control of anti-Sm autoantibodies in MRL-lpr/lpr mice.J. Clin. Invest. 80: 691-697 8. Sanz, I., H. Dang, M. Takei, N. Talal, and J. D. Capra. 1989. VH sequence of a human anti-Sm autoantibody. J. Zmmunol. 142: 883-887 9. Bellamy, N., E. Cairns, and D. A. Bell. 1983. Immunoregulation in rheumatoid arthritis: evaluation of T lymphocyte function in the control of polyclonal immunoglobulin synthesis in vitro. J. Rheumatol. 10: 19-27 10. Shoenfeld, Y., S. C. Hsu-Lin, J. E. Gariels, L. E. Silberstein, B. C. Furie, B. D. Stollar, and R. S. Schwartz. 1982. Production of autoantibodies by human-human hybridomas. J. Gin. Invest. 70: 205-208 11. Cairns, E., J. Block, and D. A. Bell. 1984. Anti-DNA autoantibody-producing hybridomas of normal human lymphoid cell origin. J. Clin. Invest. 74: 880-887 12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685

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13. Elkon, K. B. and I’. W. Jankowski. 1985. Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-l proteins defined by two-dimensional gel electrophoresis and immunoblotting. J. Imnaunol. 134: 3819-3824 14. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc. i?utZ. Acad. Sci. USA 76: 4350-4354 15. Forman, M. S., M. Nakamura, T. Mimori, C. Gelpi, and J. Hardin. 1985. Detection of antibodies to small nuclear ribonucleotide proteins and small cytoplasmic ribonucleo proteins using unlabelled cell extracts. Arthritis Rheum. 28: 135c-1361 16. Caller, H. A. and B. S. Collar. 1983. Statistical analysis of repetitive subcloning by the limiting dilution technique with a view toward ensuring hybridoma monoclonality. Hybridoma 2: 91-96 17. Mamula, M. J., 0. F. Fox, H. Yamagata, and J. B. Harley. 1986. The Ro/SS-A autoantigen as an immunogen: some anti Ro/SS-A antibodies bind IgG. J. Exp. Med. 86: 889-1901 18. Massicotte, H. and D. A. Bell. 1987. Human-human hybridomas with anti-Ro(SS-A) activity. Arthritis Rheum. 30: S77 (Abstr.) 19. Migliorini, I’., B. Ardman, J. Kaburaki, and R. S. Schwartz. 1987. Parallel sets of autoantibodies in MRL-lpr/lpr mice. An anti-DNA, anti-SmRNP, anti-gp70 network. J. Exp. Med. 165: 483-499 20. Pisetsky, D. S., S. 0. Hoch, C. L. Klatt, M. A. O’Donnell, and J. D. Keene. 1985. Specificity and idiotypic analysis of monoclonal anti-Sm antibody with anti-DNA activity. J. Immunol. 135: 408@-4089 21. Bloom, D., J. L. Davignon, R. A. Isenberg, P. L. Cowen, D. Pisetsky, M. Retter, M. Schlomchik, M. Weigert, and S. Clarke. 1990. Arthritis Rheum. 33:S4 (Abstr) 22. Puccetti, A., T. Koizum, P. Migliorini, J. Andre-Schwartz, K. J. Barrett, and R. S. Schwartz. 1990. An immunoglobulin light chain from a lupus-prone mouse induces auto-antibodies in normal mice. J. Exp. Med. 171: 1919-1930 23. Radic, M. Z., M. A. Mascelli, J. Erikson, H. Shan, and M. Weigert. 1991. Ig H and L chain contribution to autoimmune specificities. J. Zmmunol. 146: 176-182 24. Chen, J., Y. Takeda, W. E. Vanderstece, G. C. Sharp, I. Petterson, A. Rosen, H. Wigzell, and R. J. Wang. 1988. Human auto-antibodies secreted by immortalized lymphocyte cell line against the 68K polypeptide of the Ul small nuclear ribonucleoprotein. Arthritis Rheum. 31: 1265-1271 25. Takei, M., H. Dang, R. J. Wang, and N. Talal. 1988. Characteristics of a human monoclonal anti-Sm auto-antibody expressing an interspecies idiotype. J. Zmmunol. 140: 3108-3113 26. Weisbert, R. H., A. C. Wong, D. Noritake, A. Kacena, G. Chen, C. Ruland, E. Chin, S. Irvin, Y. Chen, and J. D. Rosenblatt. 1991. The rheumatoid factor reactivity of a human IgG monoclonal autoantibody is encoded by a variant VKII L chain gene. J. Zmmunol. 147: 2795-2801 27. Manheimer-Lory, A. J., A. Davidson, D. Watkins, N. R. Hannigan, and B. A. Diamond. 1991. Generation and analysis of cloned IgM- and IgG-producing human B cell lines expressing an anti-DNA-associated idi0type.J. C&z. Invest. 87: 1519-1525 28. Eisenberg, R. A., D. S. Pisetsky, S. Y. Craven, J. P. Grudier, M. A. O’Donnell, and P. L. Cohen. 1990. Regulation of the anti-Sm autoantibody response in systemic lupus erythematosus mice by monoclonal anti-Sm antibodies. J. C&z. Invest. 85: 86-92

Production and characterization of human hybridoma monoclonal anti-Sm and anti-U1RNP antibodies spontaneously produced from normal human lymphocytes.

A panel of human:human hybridomas secreting monoclonal anti-Sm/RNP antibodies was established by the fusion of normal human tonsillar lymphocytes to t...
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