Clinical Biochemistry, Vol. 32, No. 4, 249 –255, 1999 Copyright © 1999 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/99/$–see front matter
PII S0009-9120(99)00010-7
Development of an Enzyme-Linked Immunosorbent Assay, Using a Monoclonal Antibody Against ␣2-Macroglobulin, for the Diagnosis of Systemic Lupus Erythematosus NICOLA CAZZOLLA,1 LUCIANO SASO,2 JOSEPHINE GRIMA,1 MARIA GRAZIA LEONE,2 ELEONORA GRIPPA,2 C. YAN CHENG,1 and BRUNO SILVESTRINI2 The Population Council, 1230 York Avenue, New York, New York 10021, USA, and 2Department of Pharmacology of Natural Substances and General Physiology, “La Sapienza” University, P.le Aldo Moro 5, 00185 Rome, Italy
1
Objectives: To develop an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mab) directed against abnormally glycosylated serum ␣2-macroglobulin (␣2-M) from patients with systemic lupus erythematosus (SLE). Design and methods: Serum ␣2-M purified by HPLC from patients with SLE was injected in a Balb/c, CB6 F1 female mouse and hybrid cell lines were screened using ␣2-M Glu-C fragments derived from SLE and normal donors (NHS). A mab was selected and used to develop an ELISA by which sera from NHS (n ⫽ 14), SLE (n ⫽ 34), rheumatoid arthritis (n ⫽ 15), Sjo¨gren’s syndrome (n ⫽ 11), mixed connective tissue diseases (n ⫽ 12), and liver diseases (n ⫽ 11) were analyzed. Results: The affinity of the mab for ␣2-M from SLE, but not from the other diseases, was higher compared to NHS, as demonstrated by immunoblotting and ELISA. Conclusions: The ELISA was capable of recognizing changes of glycosylation of ␣2-M in SLE and may be useful for its differential diagnosis. Copyright © 1999 The Canadian Society of Clinical Chemists
KEY WORDS: ␣2-macroglobulin; glycosylation; ELISA; monoclonal antibody; rheumatic disease(s); systemic lupus erythematosus.
say (ELISA), using a monoclonal antibody (mab) directed against a specific epitope subject to changes upon abnormal glycosylation (7,9), was increased in selected rheumatic diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) (7). However, the diagnostic value of this assay appeared to be limited by the concomitant increase in concentration in inflammatory disorders since ␣1-AT is an APP in humans. We have, therefore, turned our attention to ␣2-M, a major APP in the rat (17) but not in the human (18,19). ␣2-M also appeared to be abnormally glycosylated in RA (6,10), SLE (6,10,11), and other rheumatic diseases (10). Here, we describe the preparation of a mab directed against ␣2-M purified from the serum of patients with active SLE and the development of an ELISA which could be of value for the diagnosis of SLE. Methods
Introduction t is known that several acute-phase proteins (APP) are abnormally glycosylated in different rheumatic diseases (1–11), a phenomenon that has a significant diagnostic potential for autoimmune conditions (2,5,7,12–16). In particular, we observed that the immunoreactivity of ␣1-antitrypsin (␣1-AT), measured by an enzyme linked immunosorbent as-
I
Correspondence: Prof. Bruno Silvestrini, Department of Pharmacology of Natural Substances and General Physiology, University of Rome “La Sapienza,” P.le Aldo Moro 5, 00185 Rome, Italy. E-mail:
[email protected] Manuscript received November 25, 1998; revised January 25, 1999; accepted January 26, 1999. CLINICAL BIOCHEMISTRY, VOLUME 32, JUNE 1999
HUMAN
SERUM SAMPLES
Serum samples were obtained from 14 NHS and from patients with SLE (n ⫽ 34), RA (n ⫽ 15), SS (n ⫽ 11), MCTD (n ⫽ 12), and LD (n ⫽ 11). Diagnosis of SLE was based on the 1982 Revised Criteria for SLE (20) and RA was diagnosed according to Ropes et al. (21); the diagnoses of MCTD, SS, and LD were based on the available clinical, laboratory, and pathological data as previously described (22,23). BIOCHEMICALS 125
I-Bolton Hunter reagent [N-succinimidyl 3-(4hydroxy 5-[125I]iodophenyl) propionate, SA 27514400 Ci/mmol] was obtained from ICN Radiochemi249
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cals (Irvine, CA, USA). Hypoxanthine, aminopterin, thymidine, rabbit anti-mouse IgG-alkaline phosphatase conjugate, goat anti-mouse IgG, goat antimouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG ab, goat anti-mouse IgG3, goat antimouse IgM, concanavalin A (Con A), horseradish peroxidase, protein A-peroxidase, and bovine serum albumin (BSA, Cohn Fraction V, RIA grade) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tris, acrylamide, N,N⬘-diallyltartardiamide, N,N⬘-methylenebis(acrylamide), N,N,N⬘,N⬘-tetramethylethylenediamine, ammonium persulfate, and prestained high molecular weight protein standards were from GIBCO/BRL (Gaithersburg, MD, USA). Glycine, 2-mercaptoethanol, and sodium dodecyl sulfate (SDS) were from Bio-Rad. (Richmond, CA, USA). Powdered media of Ham’s F-12 Nutrient mixture /Dulbecco’s modified Eagle’s medium (1:1, v/v) [supplemented with gentamicin (20 mg/L), sodium bicarbonate (1.2 g/L), and 4-(2-hydroxyethyl)-l -piperazineethane sulfonic acid (15 mM)] were obtained from Irvine Scientific (Santa Ana, CA, USA). Nitrocellulose paper (0.45 m pore size) was from Schleicher & Schuell, Inc. (Keene, NH, USA). PREPARATION OF ␣2-M
A MONOSPECIFIC POLYCLONAL ANTIBODY
AGAINST
␣2-Macroglobulin (␣2-M) was purified from human serum obtained from patients with active SLE using procedures previously described for testicular ␣2-M (24). Briefly, about 10 mL of serum were loaded onto an Affi-Gel威 Blue column of 1.5 ⫻ 20 cm to remove albumin (25–27). The albumin-free sample was then equilibrated against 20 mM Tris, pH 7.4 at 22° C. ␣2-M was purified to apparent homogeneity by sequential anion-exchange, chromatofocusing, and gel permeation HPLC as detailed elsewhere (24). We routinely obtained about 2– 4 mg of highly purified ␣2-M from 10-mL serum by this procedure. The identity of the protein was confirmed by direct N-terminal protein sequencing (24,28,29) that yielded a partial N-terminal sequence of NH2-SVSGKPQYMVLVPSL, which was identical to the previously published human ␣2-M sequence (30). A monospecific polyclonal antibody was prepared in a New Zealand white female rabbit using an established procedure (31). The monospecificity of this antiserum was confirmed by crossed-immunoelectrophoresis and immunoblotting using human serum. PREPARATION
OF A MONOCLONAL ANTIBODY AGAINST
␣2-M
About 100 g of purified ␣2-M were suspended in protease buffer (125 mM Tris, pH 6.8 at 22° C containing 1 mM EDTA and 0.1% SDS, w/v) in a final volume of 100 L. It was then cleaved with a Staphylococcus aureus protease V8, a Glu-C specific endopeptidase, using an enzyme: substrate ratio of 20:1. Monoclonal antibodies were prepared by estab250
ET AL.
lished procedures (32,33) as modified in this laboratory (7). Briefly, 50 g of cleaved ␣2-M were emulsified with an equal volume of Freund’s complete adjuvant and injected intradermally at three separate sites into a female mouse (Balb/c, CB6 F1 strain; Jackson Laboratories, Bar Harbor, ME, USA). Six weeks later, the mouse received a booster injection of 50 g of the same immunogenic material, emulsified with Freund’s incomplete adjuvant. Splenectomy was performed 3 days later and spleen cells were fused with mouse myeloma cells (X63/Ag 8.653), using polyethylene glycol as previously described (7). A solid-phase enzyme-linked immunoblot assay was developed for screening of hybrid cell lines for antibodies in which ␣2-M Glu-C fragments derived from normal individuals, patients with SLE and RA were resolved by analytical PAGE in the presence of sodium dodecyl sulfate (SDS) onto 15% T SDS-polyacrylamide gel (about 5 g protein/lane), transferred onto nitrocellose paper, and incubated with the hybridoma media. Positive clones were recognized using rabbit anti-mouse Ig-alkaline phosphatase conjugate. Therefore, hybridoma media derived from each well were screened with Glu-C fragments derived from normal donors, and patients with SLE and RA to determine if it reacted differentially. The immunoglobulin class of the monoclonal antibody was determined by radial immunodiffusion with goat anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, and IgM. ELISA
FOR HUMAN
␣2-MACROGLOBULIN
All assays were performed on 96-well titer plates (MicroTest III U-bottom Flexible Assay plates, Becton Dickinson, Oxnard, CA, USA) at 22° C unless otherwise specified and all samples were run in triplicate. The assay was calibrated using a pool of human serum samples (HSSP-003), obtained from patients with SLE, RA, MCTD, and SS. This pool was serially diluted with PBS-Tris buffer (10 mM sodium phosphate, 10 mM Tris, pH 7.4 at 22° C, containing 150 mM NaCl,) so that a final volume of 50 L contained 0.001, 0.003, 0.005, 0.008, 0.01, 0.03, 0.05, and 0.1 L of HSSP-003. Samples were denatured at 100° C for 5 min, 0.5 L of 2-mercaptoethanol was then added onto each well and the proteins were coated onto the plate for 1 h at 65° C. Non-specific binding sites of the plate were then blocked with 300 L of BSA-PBS buffer [10 mM sodium phosphate, pH 8.0 at 22° C, containing 150 mM NaCl and 1% BSA (w/v),] for 1 h. The plates were washed once with PBS-Tris buffer. Thereafter, 100 L of the mab 263-6-II, was added onto each well at a working dilution of 1:1000 and incubated with the antigens for 3 h. The plates were washed sequentially with PBS (10 mM sodium phosphate, pH 7.4 at 22° C, containing 150 mM NaCl), PBS containing Tween-20 (0.05%, v/v), and PBS. The amount of antibody-bound antigen was quantified using rabbit anti-mouse IgG-alkaline phosphatase conjugate as previously described (7). The minimal CLINICAL BIOCHEMISTRY, VOLUME 32, JUNE 1999
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detectable dose was 0.0022 L of serum (HSSP-003) per assay well and the 50% displacement of the competition curve was at 0.012 L of serum (HSSP003) equivalents. The inter- and intra-assay coefficients of variation (CVs), determined at curve middle points, were 18% and 9%, respectively. RADIOIMMUNOASSAY
FOR
␣2-MACROGLOBULIN
Radioimmunoassay (RIA) for human serum ␣2-M was performed essentially as previously described for rat ␣2-M (34) except that the monospecific polyclonal antibody prepared against human ␣2-M was used at a working dilution of 1:10,000. All assays were run in triplicates including unknown samples. The HSP-003 human serum pool was used to calibrate standard curves. Briefly, highly purified ␣2-M (about 3 g) was labeled with 125I-Bolton Hunter by established procedure (35). Each assay tube contained an appropriate volume of standard or unknown sample, 100 L of antiserum, 100 L of [125I]-human ␣2-M (about 15,000 cpm) and RIA buffer [10 mM sodium phosphate, pH 7.4 at 22° C, containing 150 mM NaCl, 0.5% BSA (w/v) and 0.05% NaN3 (w/v)], to a final volume of 500 L. The assay tubes were vortexed briefly and then incubated at 4° C for 36 h. Thereafter, 25 L of washed Staphylococcus aureus formalin fixed cells were added and the tubes were incubated for 60 min at 4° C. Supernatants were removed and the radioactivity in the pellets were counted for radioactivity. The minimal detectable dose was at 0.03 L eq/assay tube and 50% displacement was at 0.09 L eq. The inter- and intra-assay coefficients of variation (CVs), determined at the curve middle points, were 12% and 8%, respectively. GENERAL
METHODS
SDS-PAGE was performed as previously described (36,37). Lectin- and immunoblots were performed as detailed elsewhere (6,38 – 40). Protein estimation was performed by Coomassie blue-dye binding assay (41), as modified by Macart and Gerbaut (42), using BSA as a standard. Densitometric scanning, to determine the relative staining intensity on immunoblots and lectin blots, was performed by an EC910 densitometer (E-C Apparatus Corp., Petersburg, FL, USA) interfaced to a Hewlett-Packard (Model HP3394A) integrator at the wavelength of 600 nm. A slit size of 0.2 ⫻ 3 mm and a scan rate of 1.6 cm/min were used. Results and discussion CHARACTERIZATION OF THE POLYCLONAL AGAINST ␣2-MACROGLOBULIN
ANTIBODY
The purity of ␣2-M (about 3 mg), extracted from 10 mL of serum from patients with active SLE, was confirmed by SDS-PAGE by Coomassie blue staining (Figure 1, lane 3, containing 5 g of purified CLINICAL BIOCHEMISTRY, VOLUME 32, JUNE 1999
Figure 1 — Characterization of purified ␣2-macroglobulin and its monoclonal antibody for immunoassays. About 5 g of ␣2-macroglobulin (␣2-M), purified by HPLC were fractionated by SDS-PAGE onto a 10% T SDS-polyacrylamide gel (lane 3). Lane 2 contained the starting material for the Superose 12 column and lane 1 2.5 g protein each of the following prestained markers (BRL): myosin, 200,000; phosphorylase b, 97,000; BSA, 68,000; ovalbumin, 45,000; and carbonic anhydrase, 29,000. The gel was stained with Coomassie blue R-250.
␣2-M, vs. lane 2, containing an aliquot of the starting material). The monospecificity of the polyclonal antibody prepared against this purified protein was verified by both immunoblotting and crossed-immunoelectrophoresis (data not shown), which yielded a single immunostained band and a single immunoprecipitation arc, respectively. These analyses revealed that these reagents are suitable to be used for immunoassay development. CHARACTERIZATION OF THE MONOCLONAL AGAINST ␣2-MACROGLOBULIN
ANTIBODY
When the hybridoma media were screened using peptide fragments of ␣2-M cleaved by Glu-C endopeptidase and immunoblots, 35 culture wells out of 960 were positive and were subcloned by limiting dilutions, until one mab, designated 263-6-II, was identified which was selected based on its high reactivity for the peptide fragments of ␣2-M isolated from SLE patients (Figure 2A). It was noted that this ␣2-M mab detected peptide fragments of ␣2-M designated b and e, which were not found in NHS when protein fragments were visualized by immunoblots (Figure 2A). Moreover, 251
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ET AL.
Figure 2 — Characterization of the monoclonal antibody, 263-6-II, against human ␣2-macroglobulin. (A) Serum ␣2-M (about 5 g), from normal donors (NHS) and systemic lupus erythematosus (SLE) patients, was cleaved by Staphylococcus aureus V8 and the resulting peptides were fractionated onto a 15% T SDS-polyacrylamide gel. The protein fragments were immunostained with the monoclonal antibody (mab) 263-6-II. It was noted that the mab had higher affinity for the fragments derived from SLE compared to those from NHS. (B) When 0.05 L of serum samples from NHS (lanes 1,2) and patients with SLE (lanes 3,4) were resolved by SDS-PAGE and stained with the mab 263-6-II, a higher immunoreactivity of ␣2-M of SLE was observed, as confirmed by densitometric analysis (C).
fragments a and d yielded more intense staining in ␣2-M derived from SLE patients than from NHS. The mab, which belonged to the IgM class as demonstrated by radial immunodiffusion (data 252
not shown), was monospecific for ␣2-M as proved by immunoblotting using serum samples derived from normal donors (NHS, Figure 2B, lanes 1,2) and patients with SLE (Figure 2B, lanes 3,4). CLINICAL BIOCHEMISTRY, VOLUME 32, JUNE 1999
␣2-MACROGLOBULIN AND SYSTEMIC LUPUS ERYTHEMATOSUS
Figure 4 — Analysis of serum samples by ELISA and RIA for ␣2-macroglobulin. Serum samples from 14 normal donors (NHS) and from patients with systemic lupus erythematosus (SLE, n⫽34), rheumatoid arthritis (RA, n ⫽ 15), Sjo¨gren’s syndrome (SS, n ⫽ 11), mixed connective tissue disease (MCTD, n ⫽ 12), and liver diseases (LD, n ⫽ 11) were analyzed by ELISA using the mab 263-6-II (A) and by RIA (B), using a polyclonal antibody. The immunoreactivity was expressed as L equivalent of the same serum standard pool (HSSP-003) per milliliter of serum sample. Statistical analysis was performed with the software package Sigma-Stat (Jandel): *p ⬍ 0.05 by KruskalWallis ANOVA on ranks: ns, non significantly different. Data are reported as median with the boundaries of the boxes indicating the 25th and the 75th percentiles and the whiskers the 10th and the 90th percentiles.
Figure 3 — Specificity of the monoclonal antibody, 2636-II for human ␣2-macroglobulin. Displacement curves were generated using either a pool of human serum (HSSP-003) (A) or purified human ␣2-M (C) and compared to monkey and rabbit serum and ram rete testis fluid (RTF) (A) as well as turtle, ram, chicken, hamster, and cat serum (B).
Besides, when this blot was densitometrically scanned at 600 nm, it was noted that the immunoreactivity of the protein of SLE was significantly higher compared to NHS (Figure 2C). Using this mab, we developed a specific enzymelinked immunosorbent assay as shown in Figure 3: it was noted that a pool of crude human serum CLINICAL BIOCHEMISTRY, VOLUME 32, JUNE 1999
(HSSP-003) generated a displacement that was parallel to that obtained using a highly purified ␣2-M (Figures 3A and 3C). In addition, this mab did not cross-react with homologous proteins contained in rat, monkey, ram, rabbit, turtle, chicken, and cat serum (Figures 3A and 3B). The indirect setup for the assay was preferred over the probably more precise and sensitive sandwich format for its simplicity and reproducibility in other laboratories, provided the availability of the mab only. Besides, we made use of our previous experience with ␣1-antitrypsin (7), which could be efficiently coated, probably due, like ␣2-M, to its relative abundance in the serum, on condition that denaturation at 100° C, in the pres253
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ence of the reducing agent 2-mercaptoethanol, was performed. ANALYSIS OF SERUM ␣2-MACROGLOBULIN
SAMPLES BY
ELISA
AND
RIA
ET AL.
4.
FOR
When we analyzed 97 serum samples from NHS and patients with SLE, RA, SS, MCTD, and LD, by ELISA using the mab 263-6-II, it was noted that the immunoreactivity of ␣2-M was elevated in active SLE compared to NHS (Figure 4A). However, when these samples were analyzed by radioimmunoassay (RIA), using a polyclonal antibody, no significant difference were noted (Figure 4B), indicating that the mab is able to recognize an epitope(s) that is specifically exposed in SLE. In conclusion, a mab, designated 263-6-II, was developed, which preferentially recognized ␣2-M in SLE patients. This reagent, belonging to the IgM class, appeared to be monospecific for human ␣2-M, and showed higher affinity for the protein present in the serum of patients with SLE than that of NHS, as demonstrated by immunoblotting, using either purified ␣2-M or serum. When serum samples from normal donors and patients with SLE, RA, SS, MCTD, and LD were analyzed by ELISA using this mab, a significant increase of the immunoreactivity of ␣2-M was observed in SLE but not in the other groups, indicating that this assay could be useful for the differential diagnosis of SLE. Because the basal level of ␣2-M were not significantly different in these samples as verified by radioimmunoassay using a monospecific polyclonal antibody, which is in agreement with previous observations illustrating ␣2-M is not an acute phase protein (18,19), we speculate that this increase could be due to abnormal glycosylation of the protein, which was previously reported in this disease (6,10,11). Additional studies, using a larger sample population should be performed to evaluate the value of this assay for the diagnosis of SLE, an inflammatory condition which is currently diagnosed on the basis of limited clinical and serologic features with poor prognosis merit (43). Acknowledgements
5.
6. 7.
8.
9. 10.
11.
12.
13. 14.
15.
This work was partially supported by a grant from the Noopolis Foundation. 16.
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