Journal of Clinical Laboratory Analysis 6:343-350 (1992)
Chemically Humanized Murine Monoclonal Antibody Against a Cell Nuclear Antigen: Usefulness in Autoimmune Diagnostics Junko Miyachi,’ Kazuyuki Doi,* Kazuyuki K i t a m ~ r aTomofumi ,~ Jitsukawa,’ and Hiroshi Watanabe’ Laboratories for ‘immunology, ’Chemistry and 3Cel/ Biology, Pharma Research Laboratories, Hoechst Japan Limited, Kawagoe, Saitama 350,Japan The cellular nuclear antigen SS-B/La is known to be a major antigenic target to an autoantibody in patients with Sjogren’s syndrome and systemic lupus erythematosus. It is useful to detect an anti-SS-B/La antibody from patients’ sera in a clinical point of view. We purified SS-B/La from rabbit thymus acetone powder by affinity chromatography with a murine anti-SS-B/La monoclonal antibody (1C3-H7). An enzyme-linked immunosorbent assay method, in which SS-B/La was used to coat a plate, was also successfully established. It is difficult to obtain a large volume of patient’s serum with high antibody titer and high specificity as a positive control. We investigated whether or not a positive conKey words:
SS-B/La, autoantibody, autoantigen, enzyme-linked irnmunosorbent assay, positive control, monoclonal antibody
INTRODUCTION Anti-nuclear antibodies are known to occur in virtually all patients with systemic lupus erythematosus (SLE) and in a large portion of patients with other rheumatic diseases. The study of these antibodies has assumed a central role in rheumatology, because there is an important clinical correlation between anti-nuclear antibodies and rheumatic diseases that make these antibodies useful for the diagnosis. SS-B/La is a small nuclear RNA protein which precipitates autoantibodies produced in many patients with SLE and Sjogren’s syndrome (SS). It is also known that SS-B/La has a molecular weight (M.W.) of 48 kD, and it is a ubiquitous protein transiently associated not only with RNA polymerase 111 transcripts (1-10) but also with U1 RNA (1 I), RNA polymerase I1 transcript, and the vesicular stomatitis virus leader RNA ( 12). Some studies have shown that the SS-B/La protein from a HeLa cell consists of two structural domains that are relatively protease resistant (13- 15). One of them is a peptide of M.W. 28 kD from a methionine-rich region designated as an X domain and the other is a peptide of M.W. 23 kD from a phosphorylated region designated as a Y domain. It is suggested that there are at least two epitopes on a SS-B/La antigen 0 1992 Wiley-Liss, Inc.
trol from human could be replaced by a murine monoclonal antibody to SS-B/La. The 1C3H7 was conjugated with a human IgG Fc’ fragment using N-y-maleimidobutyryloxysuccinimide as a cross-linker. The chemically humanized murine monoclonal antibody (1 C3Fc’) was recognized by antiserum specific for human IgG Fc fragment. 1C3-Fc’ reacted to SS-B/La but not to other antigens. Furthermore, the titration curve of this conjugate ran parallel with those of patients’ sera specific for SS-B/La. It is concludedthat a chemically humanized murine monoclonal antibody is useful as a positive control in place of a human patient’s serum. o 1992wiiey-~iss,inc.
and the sera from the majority of patients with anti-SS-B/La react with these two epitopes. Clinical importance of anti-SS-B/La antibodies has been reported in patients with SS (16). Specific control sera from patients are required for establishing the diagnostic test kits. However, it is difficult to obtain monospecific patients’ sera and to standardize them as controls because of a polyclonal nature of anti-nuclear antibodies in patients’ sera. In this report, successful conjugation of a murine monoclonal antibody (IC3-H7) against a SS-B/La antigen with a human IgG Fc’ fragment is described. This chemically humanized murine monoclonal antibody allows the use of a well-standardized specific control in the diagnostic tests.
MATERIALS AND METHODS Materials A SS-B/Laantigen was purified from rabbit thymus acetone powder which was purchased from Pel-Freeze Co. (Rogers, ~
Received December 2, 1991; accepted May 3, 1992 Address reprint requests to Junko Miyachi, Laboratory for Immunology, Pharma Research Laboratories, Hoechst Japan Limited, 1-3-2 Minami-dai, Kawagoe, Saitama 350, Japan.
Miyachi et al.
USA). A SS-NRo antigen and histones were purified from calf thymus acetone powder which was also purchased from PelFreeze Co. (Rogers, USA). ds-DNA was purified from calf thymus DNA purchased from Calbiochem (La Jolla, USA). A human carcinoembryonic antigen (CEA) was purchased from The Binding Site Ltd. (Birmingham, UK). Patients’ sera were supplied by Dr. Takasaki of Juntendo University (Tokyo, Japan). ELISA plates were purchased from A/S Nunc (Kamstrup, Denmark). Horseradish peroxidase (HRP0)-labeled goat anti-human IgG (Fc-specific) and HRPO-labeled rabbit anti-mouse immunoglobulins (Igs) were purchased from Cappel Co. (Marvern, USA) and DAKO Co. (Glostrup, Denmark), respectively. A human IgG Fc fragment was purchased from Green-Cross Co. (Tokyo, Japan), N-y-maleimidobutyryloxysuccinimide (GMBS) was from Calbiochem (La Jolla, USA), and N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB) was from Pierce Co. (Rockford, USA). Murine monoclonal antibodies, not only anti-SS-B/La monoclonal antibodies (1C3-H7, 4D5-E5, and 2G7-D4) but also other monoclonal antibodies, that is, anti-SS-A/Ro (lG10-D4), anti-Sm (55DF2), and anti-CEA (CA208), were also prepared in our laboratory. Anti-proliferating cell nuclear antigen (PCNA) monoclonal antibodies (19A2 and 19F4) were previously reported by Dr. Ogata et al. (17).
Preparation of Anti-SS-B/La Monoclonal Antibodies Seven week-old female BALB/c mice were immunized intraperitoneally with 10 k g of the SS-B/La antigen that was affinity-purified with a patient’s serum IgG fraction which contained an anti-SS-B/La antibody, in complete Freund’s adjuvant, followed by further injections at 2 week intervals. Spleen cells from BALB/c mice whose sera had the highest antibody activities were prepared 3 days after the final immunization. They were fused with P3-Ul mouse myeloma cells to make hybridomas. The murine monoclonal antibodies were purified from the culture supernatants of hybridoma cells by protein A-sepharose chromatography.
Purificationof Antigens A SS-B/La antigen was purified as follows. A phosphatebuffered saline (PBS)-extractable fraction of rabbit thymus acetone powder was treated with 25-75% saturated ammonium sulfate. After dialysis, this fraction was applied onto cyanogen bromide (CNBr)-activated sepharose 4B gel (Pharmacia, Uppsala, Sweden) coupled with one (1C3-H7) of antiSS-B/La monoclonal antibodies. The fraction which reacted with lC3-H7 was eluted with 3 M potassium thiocyanate (KSCN) after washing with 0.4 M KSCN and then dialyzed against PBS. Similarly, a SS-A/Ro antigen was purified by affinity chromatography of a 30-60% saturated ammonium sulfate fraction of calf thymus acetone powder with an anti-SS-A/Ro monoclonal antibody (lGlO-D4). The antigen fraction which binds to 1G 10-D4 was eluted with 3 M KSCN after washing with 0.1 M NaCI-PBS. Histones were extracted with 0.5 N
sulfuric acid from calf thymus acetone powder, followed by dialysis against distilled water. ds-DNA was obtained by treatment of DNA with S 1 nuclease (Takara, Tokyo, Japan) and then the fraction was applied onto a hydroxyapatite column (F-2025, Mitsui-touatsu Chemicals, Tokyo, Japan).
Reactivity of the Purified SS-BILa Antigen With Panel Sera ELISA was performed to evaluate the specificity of the purified SS-B/La antigen. The purified SS-BILa antigen was used to coat on an ELISA plate at 50 nglwell. After 3 washings with POD washing solution (Behringwerke, Germany), 100-fold-diluted patients’ sera were added into each well. The plate was again washed after incubation and HRPO-labeled goat anti-human IgG (Fc-specific) was added. ChromogenTMB (Behringwerke, Marburg, Germany) was used as a substrate for color reaction, then this reaction was terminated by addition of 0.5 M sulfuric acid, followed by measuring the absorbance at 4501650 nm wave length (Behring ELISA Processor 11, Behringwerke, Marburg, Germany).
Reactivityof Anti-SS-B/La Monoclonal Antibodies With the Purified Antigen The purified SS-B/La antigen was treated with 0.5 kg/ml of trypsin for 30 min at 37°C. The digested SS-B/La antigen was applied to a 12.5% SDS-polyacrylamide gel to fractionate electrophoretically, followed by transfer of the antigen to a nitrocellulose filter at 60 V for 2 hr. The filter was washed after blocking with 5% FCS-PBS, and the separated fragments were reacted with anti-SS-B/La monoclonal antibodies (1C3-H7,4D5-E5, and 2G7-D4) and patients’ sera which were diluted 200-fold. The detection was performed by o-dianisidine (Sigma, St. Louis, USA).
Chemical Humanizationof 1C3-H7 A murine monoclonal antibody against a SS-B/La antigen designated as 1C3-H7 was incubated with 5 , 25, 250, and 1,000-fold excess amount of GMBS or SIAB in the mole ratio for 2 hr at room temperature, followed by gel-filtration (PD-10 Sephadex (3-25, Pharmacia, Uppsala, Sweden). On the other hand, a human IgG Fc fragment (Green-Cross, Tokyo, Japan) was reduced by incubating with dithiothreitol (DTT) for 90 min at room temperature to obtain a Fc’ fragment, and the extra DTT was removed by PD-10. GMBS- or SIAB-treated 1C3-H7 and a Fc’ fragment were mixed and stirred to prepare the conjugate (lC3-Fc’) at 5°C overnight.
Estimation of Murine IgG in the 1C3-Fc’ Conjugate Rabbit anti-mouse immunoglobulins (diluted 1/ I ,000) (Dako, Glostrup, Denmark) was used to coat each well of an ELISA plate. Murine IgG as a standard and the 1C3Fc’ conjugate were added into these wells and incubated. After washing, HRPO-labeled rabbit anti-mouse immuno-
Use of 1C3-Fc' for Autoimmune Diagnostics
globulins was added, and the plate was washed again after incubation. TMB was then added for coloring.
Reactivity of the 1C3-Fc' Conjugateto a SS-B/La Antigen A humanized murine monoclonal antibody (1C3-Fc') and patients' sera were serially diluted and added into wells of an ELISA plate with SS-B/La to compare the reaction curves. After 3 washings, HRPO-labeled rabbit anti-mouse immunoglobulins or HRPO-labeled goat anti-human IgG (Fc specific) was added, and incubated for 1 hr at room temperature. Consecutively, the ELISA plate was washed, and then TMB was added for coloring. L
Western Blot Analysis of the 1C3-Fc' Conjugate Components of the 1C3-Fc' conjugate were separated on polyacrylamide-gradient gel (PAG PLATE 4/ 15, Daiichi Pure Chemicals, Tokyo, Japan) electrophoretically. The separated components were stained with Coomassie brilliant blue. On the other hand, transfer was performed at 60 V for 2 hr to a nitrocellulose filter. After blocking, 500-fold-diluted HRPO-labeled goat anti-human IgG (Fc-specific) or HRPO-labeled rabbit antimouse immunoglobulins was reacted with the filter. The nitrocellulose filter was then washed and stained with o-dianisidine.
Inhibition Assays to Examine Reaction Specificity The 1C3-Fc' conjugate was mixed with a SS/B/La antigen and other nuclear antigens as inhibitors, that is , SS-NRo, RNP, histones, and ds-, ss-DNA, and incubated for 2 hr at room temperature; these mixtures were added consecutively into wells of an ELISA plate with a SS-B/La antigen to detect the remained reactivity. After incubation for 1 hr, the plate was washed, HRPO-labeled anti-human IgG (Fc-specific) was added, and the plate was again incubated. TMB was then added for coloring. On the other hand, an inhibition test of binding of the conjugate to a SS-B/La antigen by 1C3-H7 or other monoclonal antibodies (2G7-D4, 1G10-D4, and CA208) was also performed. The method was same as the previous one in principle. In brief, monoclonal antibodies against nuclear antigens were added to an ELISA plate with SS-B/La. After incubation for 3 hr and washing, the 1C3-Fc' conjugate was added into the wells and incubated for 1 hr. The bound conjugate was detected with HRPO-labeled anti-human IgG (Fc-specific).
Fig. 1. The reactivity of a SS-BiLa antigen with specific patients' sera as demonstrated by ELISA.
SS-B/La antibody but not with the sera with other specificities by ELISA (Fig. 1). Furthermore, this SS-B/La antigen reacted with murine anti-SS-B/La monoclonal antibodies (IC3-H7, 2G7-D4, and 4D5-E5) but not with other murine monoclonal antibodies, that is, anti-PCNA (19A2 and 19F4), anti-Sm (55DF2), and anti-SS-A/Ro (lGlO-D4) (data not shown).
Clinical Evaluation of the SS-B/La Antigen The purified SS-B/La antigen was coated on ELISA plates to evaluate its clinical significance in antibody detection (Fig. 2). Sera from 364 patients with autoimmune diseases and from 105 normal subjects were tested by this method whether or not they contained an anti-SS-B/La antibody. Thirty-nine percent (86/221) of the sera with SLE, 12% (5/42) of those with RA, and 58% (29/50) of those with SS displayed positive levels of an anti-SS-B/La antibody. In this evaluation, the cut-