Studies of human polyclonal and monoclonal antibodies binding to lupus autoantigens and cross-reactive antigens
A. Sharma,
D. Isenberg and
B. Diamond1
Centre for Rheumatology, University College London, London W1T 4NJ, UK and
1 Albert Einstein College of Medicine, New York, NY 1461, USA
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Abstract
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Background. Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease serologically characterized by production of a variety of autoantibodies. Antibodies to double-stranded (ds) DNA are considered to be a diagnostic marker in SLE and their presence often correlates with active disease. The murine R4A anti-dsDNA antibody was found to cross-react with a peptide, D/EWD/EYS/G (R4A peptide), identified by analysing decapeptides selected from a peptide library. The R4A peptide inhibited binding of antibody to dsDNA and antibody deposition in kidneys in vivo. In other previous work, mice immunized with the peptide in a decapeptide form bound to a polylysine backbone, multiple antigenic peptide, were found to develop both anti-DNA and anticardiolipin antibodies.
Methods. To determine if human anti-DNA antibodies bind R4A peptide, we investigated the binding of monoclonal and polyclonal anti-dsDNA and anticardiolipin antibodies to the R4A peptide from patients with SLE.
Results. DNA binding by four immunoglobulin (Ig) G and two IgM human monoclonal anti-DNA antibodies was inhibited by the R4A peptide. While monomeric peptide was unable to inhibit affinity-purified polyclonal anti-DNA antibodies, serum anti-DNA reactivity was inhibited by an octameric form of the peptide in 10 SLE patients.
Conclusions. Human anti-DNA reactivity includes the same fine specificity as that present in murine anti-DNA reactivity. Peptide binding might be a useful surrogate marker for SLE.
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Introduction
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Patients with systemic lupus erythematosus (SLE) demonstrate multiple autoantigenic specificities. In addition to having a diagnostic anti-DNA response, their sera also have antibodies to a variety of other nuclear, cytoplasmic and cell membrane antigens [15]. The role of anti-double-stranded (ds)DNA antibodies in disease, especially in glomerulonephritis, has been confirmed in studies in humans and mice [69]. Understanding the antigenic cross-reactivity of these antibodies has been of interest to investigators seeking to shed light on both potential triggering antigens and potential target antigens. Furthermore, surrogate antigens might be used to inhibit pathogenic antibodies from tissue deposition. In a study by Gaynor et al. [10], a peptide display library was used to select peptides bound by a pathogenic mouse monoclonal anti-dsDNA antibody, R4A, which deposits in the kidneys. A consensus sequence, DWEYS, was identified. When DWEYS peptide was administered intraperitoneally to mice given R4A antibody, it protected the mice from renal deposition of the R4A antibody. Furthermore, BALB/c mice immunized with a multimeric peptide containing the DWEYS consensus sequence on a polylysine backbone developed anti-dsDNA antibodies and other autoantibodies, such as anticardiolipin and antihistone antibodies, as well as glomerular deposition of immunoglobulin [11]. The consensus sequence is present in subunits of the glutamate receptor, NR2a and NR2b, and antibodies binding the peptide can mediate excitotoxic neuronal death [12].
In the present study, we asked whether human monoclonal and polyclonal anti-dsDNA and anticardiolipin antibodies bind to the DWEYS motif. In addition, we examined a set of 10 affinity-purified sera from patients with SLE to determine whether serum derived anti-dsDNA antibodies might also bind the consensus motif.
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Materials and methods
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Anti-dsDNA ELISA
Anti-DNA binding activity was tested by anti-dsDNA enzyme-linked immunosorbent assay (ELISA). Flat-bottom 96-well microtitre ELISA plates (Immulon 2; Costar, Corning, NY, USA) were coated with 50 µl/well of 100 µg/ml salmon sperm dsDNA (Calbiochem-Novabiochem, La Jolla, CA, USA). The wells were blocked and washed with PBS (phosphate-buffered saline)Tween (0.05%). Samples diluted in 0.1% BSA (bovine serum albumin) in PBS were added to plates for 1.5 h at 37°C. The plates were washed with PBSTween, and goat anti-human immunoglobulin (Ig) G (for IgG monoclonals) or goat anti-human IgM (for IgM monoclonals) linked to alkaline phosphatase (Southern Biotech, Birmingham, AL, USA) was added for 1 h at 37°C. After washing with PBSTween, the plates were developed with alkaline phosphatase substrate tablets (p-nitrophenyl phosphate) (Sigma, St Louis, MO, USA). Optical density was read at 405 nm with an ELISA reader.
Quantitation of human IgG and IgM anti-DNA monoclonals and affinity-purified anti-DNA antibodies was performed using a standard IgG and IgM quantitation ELISA [13].
Peptide inhibition ELISA
For inhibition ELISAs, an additional step of preincubation of the antibody (411 µg/ml) with varying concentrations of peptide, DWEYS (0.510 mM) or octameric peptide (DWEYSVWSLN-MAP) (0.22.16 mM) (Invitrogen, Paisley, UK) was performed. The millimolar concentrations of the octameric peptide are based on the molecular weight of the decapeptide only and not on the molecular weight of decapeptide plus backbone. After incubation at 37°C for 2 h and overnight at 4°C, antibody and peptide were transferred to a dsDNA-coated ELISA plate and the assay was continued as described above. Similar concentrations of monomeric irrelevant peptides, RNKANDYTTEYS and SANKANDYTTE-MAP (multiple antigenic peptide) (Invitrogen), were used as controls in the inhibition experiments.
Two IgM and seven human IgG monoclonal anti-DNA antibodies were assayed. These were derived from culture supernatants of hybridoma cell lines made by fusing peripheral blood cells from SLE patients to cells of the humanmurine heteromyeloma cell line CBF-7 [14].
RT84 and RT129 are human IgM
anti-DNA monoclonal antibodies; RH14 and B3 anti-DNA antibodies are IgG1
antibodies [15, 16]; 35.12 is an IgG2
antibody [17] and 32.B9 and DIL-6 are IgG3
antibodies; D5 is an IgG1
antibody [18], 33.C9 is an IgG2
antibody, RSP3, RSP18 and RSP57 are IgM
human anticardiolipin antibodies and RSP13 is an IgM
anticardiolipin antibody [19]. These were obtained from heterohybridomas. Patients with SLE (patients CC, NH, KB, KC, AP, AC, NN, MG, PN and PG) with high titres of anti-dsDNA antibodies were chosen for the study. All the patients were female. Their mean age was 34.8 yr (S.D. 13.13 yr). Of the patients, five were Caucasian, three were Afro-Caribbean, one was Asian and one was mixed (Asian/Caribbean). All the patients met four or more of the revised SLE classification criteria of the American College of Rheumatology [20].
The linear range of binding of anti-dsDNA antibodies to dsDNA at varying dilutions (1:101:320) from serum samples from patients with SLE was determined (Fig. 4A
). In order to investigate the possible peptide inhibition of dsDNA binding, serum samples from patients with SLE at a dilution within the linear range of binding (1:50) were incubated with different concentrations of DWEYS (0.510 mM) and DWEYSVWLSN-MAP (0.22.16 mM) peptide for inhibition assays.
A similar dilution of normal samples was used as a control. The mean age of the 11 normal controls was 42.90 (S.D. 9.67) yr and the female:male ratio was 6:5.

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FIG. 1. Inhibition of IgM anti-DNA monoclonal antibodies by DWEYS peptide. Inhibition of binding of two human IgM anti-DNA monoclonals (RT84 and RT129) to dsDNA by DWEYS was assayed by inhibition ELISA. An irrelevant peptide was used as a control.
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Affinity purification of anti-DNA antibodies
Anti-DNA antibodies were purified from the SLE sera using DNA cellulose chromatography, as described by Suenaga et al. [21]. A Hitrap Protein G column (Pharmacia, Piscataway, NJ, USA) was used to purify IgG from SLE serum. One millilitre of serum diluted 1:2 in PBS was passed through a protein G column. IgG was eluted using 0.1 M glycineHCl buffer. Eluted IgG was dialysed against PBS overnight at 4°C and concentrated using Centricon concentrators (Amicon, Bedford, MA, USA).
Purified IgG was loaded onto a DNA cellulose column (Sigma) to purify anti-DNA antibodies. IgG was passed through the column two or three times to ensure maximal binding. The column was then washed extensively with TBS-EDTA. The bound anti-DNA IgG was eluted using 6 M urea, 2 M NaCl and dialysed sequentially against 4 M urea, 1 M NaCl, 2 M urea, 0.5 M NaCl, 1 M urea, 0.25 M NaCl in TBSEDTA (Tris buffered saline) and finally against PBS. Recovered anti-DNA antibodies were concentrated using Centricon concentrators. The activity and specificity of the affinity-purified anti-DNA antibody was confirmed by anti-DNA ELISA.
Anticardiolipin ELISA
Cardiolipin (50 µg/ml) in ethanol was coated onto 96-well flat-bottom Immulon 2 plates overnight at 4°C. The plates were blocked with 200 µl/well of 10% fetal calf serum (FCS) in PBS and incubated for 2 h at room temperature (RT). Serum samples diluted in PBS with 10% FCS were added to the wells (50 µl/well) and incubated for 2 h at RT.
The plates were washed four times with PBS and 50 µl/well of goat anti-human heavy- and light-chain Ig (IgG+IgM+IgA) conjugated to alkaline phosphatase (Southern Biotech) was added. After 2 h of incubation at RT, plates were washed four times with PBS and once with carbonatebicarbonate buffer. p-Nitrophenyl phosphate substrate (Sigma) was added and the plates were developed at RT and read at 405 nm with a Titertek ELISA reader (ICN flow Thame, OXON, UK).
For inhibition ELISAs, antibody was preincubated with varying concentrations of peptide [DWEYS (0.610 mM) or octameric peptide (DWEYSVWLSN-MAP) (0.12.16 mM)] for 2 h at 37°C and overnight at 4°C, antibody and peptide were transferred to a cardiolipin-coated ELISA plate, and the assay was continued as described above. Similar concentrations of monomeric RNKANDYTTEYS and SANKANDYTTE-MAP were used as controls in the inhibition experiments.
Sera from SLE patients with anticardiolipin antibodies but no anti-phospholipid syndrome symptoms (patients PF, CB, AJ, AD, LN, LS and SR) were used for inhibition assays using DWEYS peptide and DWEYSVWLSN-MAP peptide. All patients except CB also had anti-DNA antibodies. The normal sera described above were used as a control.
The linear range of binding of anticardiolipin antibodies to cardiolipin at varying dilutions (1:251:3200) from serum samples from patients with SLE with anticardiolipin antibodies was determined (Fig. 6A
). Serum samples from patients with SLE with anticardiolipin antibodies but no antiphospholipid syndrome were incubated at a dilution within the linear range of binding (1:50) with different concentrations (0.610 mM) of DWEYS and (0.12.16 mM) of DWEYSVWLSN-MAP peptide for inhibition assays.

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FIG. 2. Inhibition of IgG anti-DNA monoclonal antibodies by DWEYS. Inhibition of binding of seven human IgG anti-DNA monoclonals (RH14, B3, 32B9, 33C9, 35.12, D5 and DIL-6) to dsDNA by DWEYS was assayed by inhibition ELISA. An irrelevant peptide was used as a control.
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Results
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Inhibition of human IgM and IgG anti-dsDNA monoclonals by DWEYS (R4A peptide)
Inhibition ELISAs were performed using peptide to inhibit autoreactivity of anti-DNA and anticardiolipin antibodies. As shown in Fig. 1
, peptide inhibited DNA binding by RT84 and RT129, two IgM anti-DNA antibodies. At 5 mM of peptide, 64% inhibition of RT84 and 94% inhibition of RT129 was observed. Similar concentrations of monomeric irrelevant peptide did not show any inhibition.
Monoclonal IgG anti-DNA antibodies were also tested for binding to peptide. As seen in Fig. 2, 4
mM peptide inhibited DIL-6 binding to dsDNA by 37% and B3 binding to dsDNA by 3540%. Approximately 20% inhibition of DNA binding by 32B9 and 33C9 was obtained at 4 mM peptide. The peptide did not inhibit DNA binding by RH14, D5 and 35.12. As a control for peptide inhibition assays, concentrations of dsDNA from 1 to 200 µg/ml were used to inhibit anti-dsDNA antibodies. Increasing inhibition of anti-dsDNA antibodies was observed with increasing concentration of dsDNA; at 200 µg/ml there was approximately 60% inhibition for all antibodies. IgG monoclonal anti-dsDNA antibodies did not show any inhibition by irrelevant peptide Thus, some IgM and IgG monoclonal anti-dsDNA antibodies do cross-react with the peptide.

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FIG. 3. Inhibition of DNA binding of affinity-purified IgG anti-DNA antibodies from sera of SLE patients by DWEYS. IgG anti-DNA antibodies from sera of patients with SLE were affinity-purified on protein G and DNA cellulose columns. Inhibition of binding of IgG affinity-purified anti-DNA antibodies to dsDNA by DWEYS was assayed by inhibition ELISA.
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Inhibition of polyclonal IgG affinity-purified anti-dsDNA antibodies from lupus sera by DWEYS
Binding of IgG polyclonal affinity-purified anti-dsDNA antibodies to peptide was investigated by inhibition ELISA. Samples from PG and AP showed 2025% at 10 mM DWEYS. The remaining samples showed less than 10% inhibition at 10 mM DWEYS (Fig. 3
). An irrelevant peptide which was used as a control did not show any inhibition.

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FIG. 5. Inhibition of IgM anticardiolipin monoclonal antibodies by DWEYS peptide. Inhibition of binding of four human IgM anticardiolipin monoclonals to cardiolipin by DWEYS was assayed by ELISA. An irrelevant peptide was used as a control.
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Inhibition of anti-dsDNA antibodies from SLE sera by R4A map peptide
Inhibition of serum anti-dsDNA activity by an octameric form of the peptide was investigated in 10 SLE patients. As shown in Fig. 4B, 2
mM R4A MAP peptide inhibited 2075% of DNA binding by anti-dsDNA antibodies in serum from 10 SLE patients. Increasing inhibition of anti-dsDNA antibodies was observed with increasing concentration of octameric peptide. As shown in Figs 4C and D
(see also Tables 1
and 2
), inhibition of both IgG and IgM antibodies by octameric peptide was observed. There was no inhibition of anti-dsDNA antibotdies by control irrelevant MAP peptide. Similar dilutions of normal sera (1:50) used in the inhibition experiments did not show any binding to dsDNA.


Inhibition of human IgM anticardiolipin monoclonals by DWEYS
As shown in Fig. 5
, a 5 mM concentration of DWEYS peptide inhibited monoclonal anticardiolipin antibodies. RSP3 was inhibited by 75%, RSP13 by 76%, RSP18 by 67% and RSP57 by 72%. Monoclonal anticardiolipin antibodies were not inhibited by irrelevant peptide.
Inhibition of polyclonal anticardiolipin antibodies from patients with SLE with no clinical symptoms of antiphospholipid syndrome by DWEYS and DWEYSVWLSN-MAP peptide
Inhibition of polyclonal anticardiolipin antibodies from patients with SLE by varying concentrations of DWEYS (0.610 mM) was investigated, and no inhibition was observed (data not shown). Control irrelevant peptide did not show any inhibition.
Inhibition with DWEYSVWLSN-MAP peptide (0.12.16 mM) was also investigated. DWEYSVWLSN-MAP peptide at 2.16 mM inhibited binding of anticardiolipin antibodies by 6580% (Fig. 6B
). Increasing inhibition of anticardiolipin antibodies was observed with increasing concentration of octameric peptide. MAP peptide inhibited both polyclonal IgG and IgM anticardiolipin antibodies from patients with SLE [Fig. 6C and D
(see also Tables 3
and 4
)]. Control irrelevant MAP peptide did not show any inhibition.
Similar dilutions of normal sera (1:50), used in the inhibition experiments, did not show any binding to cardiolipin.
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Discussion
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Anti-dsDNA antibodies, which occur spontaneously in the serum of patients with SLE, are of interest both immunologically and clinically. The presence of serum IgG anti-dsDNA antibodies correlates with nephritis both in mice and in humans. For example, Okamura et al. [22] have shown a close relationship between renal disease and IgG anti-dsDNA titres but not IgM anti-dsDNA titres or antibodies to single-stranded DNA of either isotype. Previous studies [10] have suggested that surrogate antigens, such as peptides derived from phage display peptide libraries, could be instrumental in inhibiting these pathogenic antibodies and preventing their deposition in kidneys.
In studies by Gaynor et al. [10], peptide sequences that react with R4A, a pathogenic monoclonal anti-DNA antibody, were identified using phage display libraries. The dominant motif was D/EWD/EYS/G. The DWEYS peptide inhibits dsDNA binding by R4A and also inhibits R4A deposition in the kidneys of SCID mice. Mice immunized with an octameric form of peptide develop anti-DNA and anticardiolipin antibodies, with over 50% of both specificities inhibitable by peptide [11]. The binding of monoclonal and polyclonal anti-dsDNA and anticardiolipin antibodies from patients with SLE to the five-amino-acid peptide DWEYS (R4A peptide) has now been explored. DNA binding of four IgG and two IgM human monoclonal anti-DNA antibodies was inhibited by the peptide. Binding of four human monoclonal IgM anticardiolipin antibodies to cardiolipin was also inhibited by peptide.
On the basis of the greater inhibition of IgM than of IgG monoclonal antibodies, it may be speculated that the affinity maturation which may occur in the anti-DNA response could alter peptide binding, with IgM antibodies more likely to bind. Somatic diversification may lead to a loss of peptide reactivity in some antibodies. With only two IgM antibodies included in this study, this conclusion is speculative. The binding of polyclonal anti-dsDNA antibodies (both from sera of patients with SLE and affinity-purified anti-dsDNA antibodies) to dsDNA was not inhibited by monomeric peptide; however, binding was inhibited by octameric peptide. We found a range of 2075% of inhibition of binding of polyclonal anti-dsDNA antibodies to dsDNA by octameric peptide, a range of 2590% of inhibition of binding of polyclonal IgG anti-dsDNA antibodies and a range of 3085% of inhibition of polyclonal IgM anti-dsDNA antibodies to dsDNA by octameric peptide. As no difference in cross-reactivity to peptide was observed in polyclonal IgM anti-DNA antibodies compared with polyclonal IgG anti-DNA antibodies, it may be that affinity maturation does not alter peptide reactivity or that the enhanced avidity of the multimeric peptide overcomes the decreased affinity of the IgG antibodies.
Because studies in mice have demonstrated that both anti-DNA and anticardiolipin antibodies cross-react with peptide, we assayed anticardiolipin antibodies for binding to peptide in inhibition studies. Like anti-DNA reactivity, binding of polyclonal anticardiolipin antibodies to cardiolipin was not inhibited by the monomeric peptide but binding was inhibited by octameric peptide. We found a range of 6580% inhibition of binding of polyclonal anticardiolipin antibodies to cardiolipin by octameric peptide, a range of 2090% inhibition of binding of polyclonal IgG anticardiolipin antibodies and a range of 5082% inhibition of polyclonal IgM anticardiolipin antibodies to cardiolipin by octameric peptide. Thus, the DWEYS peptide is recognized by some human anti-dsDNA and anticardiolipin antibodies.
Multivalent peptide is required to inhibit binding of human polyclonal autoantibodies to both dsDNA and cardiolipin, perhaps because DNA and cardiolipin are also multivalent antigens. Also, one can speculate that the octameric form of the peptide provides a conformational epitope which influences the binding such that polyclonal anti-DNA and anticardiolipin antibodies are inhibited by this octameric peptide but not by the monomer. Studies mimicking a conformational B-cell epitope of the heat-shock protein PfHsp70-1 antigen of Plasmodium falciparum using a multiple antigenic peptide have shown that a (MAP) form of this sequence is instrumental in identification of a new B-cell epitope not detected by ELISA with linear peptides. The polymer was strongly recognized by sera from monkeys or humans, whereas the monomer was not [23]. One can also speculate that multimeric peptide constructs such as MAPs may imitate natural autoimmune multimeric stimuli, such as viral proteins displayed on the cell surface, self-proteins concentrated in apoptotic blebs, endogenous immune complexes, and proteins with repeated epitopes, resulting in probable higher binding of octameric peptides by autoantibodies compared with the monomeric peptide.
The demonstration that peptide mimics for dsDNA and cardiolipin can be isolated now permits us to consider the potential of these peptides in diagnosis and therapy.
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Acknowledgments
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Antibodies 32.B9, 33.C9 and 35.12 were kindly provided by T. Winkler and J. Kalden. This work was supported by the National Institutes of Health and by the Lupus Research Institute.
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Notes
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Correspondence to: D. Isenberg, Centre for Rheumatology, University College London, 4th Floor, Arthur Stanley House, 4050 Tottenham Street, London W1T 4NJ, UK. E-mail: d.isenberg{at}ucl.ac.uk 
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References
|
---|
- Theofilopoulos AN, Dixon FJ. Etiopathogenesis of murine SLE. Immunol Rev 1981;55:179216.[ISI][Medline]
- Steinberg AD, Smolen JS, Twomey J. Disorders of immune regulation. In: Pathophysiology of human immunologic disorders. Baltimore (MD): Urban and Schwarzenberg Publications, 1982:173.
- Dziarski R. Autoimmunity: polyclonal activation or antigen induction? Immunol Today 1988;9:3402.[CrossRef][ISI][Medline]
- Klinman DM, Steinberg AD. Systemic autoimmune disease arises from polyclonal B cell activation. J Exp Med 1987;165:175560.[Abstract]
- Klinman DM, Steinberg AD. Systemic lupus erythematosus and overlap syndromes. In: Samter M, Talmage DW, Frank DW, Austin MM, Claman K, eds. Immunological diseases. Boston: Little Brown,1988:1335.
- Vlahakos D, Foster MH, Ucci AA, Barrett KJ, Datta SK, Madaio MP. Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo. J Am Soc Nephrol 1992;2:134554.[Abstract]
- Madaio MP, Carlson J, Cataldo J, Ucci A, Migliorini P, Pankewycz O. Murine monoclonal anti-DNA antibodies bind directly to glomerular antigens and form immune deposits. J Immunol 1987;138:28839.[Abstract/Free Full Text]
- Suzuki N, Harada T, Mizushima Y, Sakane T. Possible pathogenic role of cationic anti-DNA autoantibodies in the development of nephritis in patients with systemic lupus erythematosus. J Immunol 1993;151:112836.[Abstract/Free Full Text]
- Lefkowith JB, Gilkeson GS. Nephritogenic autoantibodies in lupus: current concepts and continuing controversies. Arthritis Rheum 1996;39:894903.[ISI][Medline]
- Gaynor B, Putterman C, Valadon P, Spatz L, Scharff MD, Diamond B. Peptide inhibition of glomerular deposition of an anti-DNA antibody. Proc Natl Acad Sci USA 1997;94:195560.[Abstract/Free Full Text]
- Putterman C, Diamond B. Immunization with a peptide surrogate for double-stranded DNA (dsDNA) induces autoantibody production and renal immunoglobulin deposition. J Exp Med 1998;188:2938.[Abstract/Free Full Text]
- DeGiorgio LA, Konstantinov KN, Lee SC, Hardin JA, Volpe BT, Diamond B. Novel mechanism for brain injury in SLE: a subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor. Nat Med 2001;7:118993.[CrossRef][ISI][Medline]
- Iliev A, Spatz L, Ray S, Diamond B. Lack of allelic exclusion permits autoreactive B cells to escape deletion. J Immunol 1994;153:35516.[Abstract/Free Full Text]
- Grunow R, Jahn S, Porstmann T et al. The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus. J Immunol Methods 1988;106:25765.[CrossRef][ISI][Medline]
- Ehrenstein MR, Longhurst CM, Latchman DS, Isenberg DA. Serological and genetic characterization of a human monoclonal immunoglobulin G anti-DNA idiotype. J Clin Invest 1994;93:178797.[ISI][Medline]
- Ravirajan CT, Rahman MA, Papadaki L et al. Genetic, structural and functional properties of an IgG DNA-binding monoclonal antibody from a lupus patient with nephritis. Eur J Immunol 1998;28:33950.[CrossRef][ISI][Medline]
- Winkler TH, Jahn S, Kalden JR. IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus. Clin Exp Immunol 1991;85:37985.[ISI][Medline]
- Ehrenstein MR, Hartley B, Wilkinson LS, Isenberg DA. Comparison of a monoclonal and polyclonal anti-idiotype against a human IgG anti-DNA antibody. J Autoimmun 1994;7:349367.[CrossRef][ISI][Medline]
- Ravirajan CT, Kalsi J, Wiloch HW et al. Antigen-binding diversity of human hybridoma autoantibodies derived from splenocytes of patients with SLE. Lupus 1992;1:15765.[ISI][Medline]
- Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:12717.[ISI][Medline]
- Suenaga R, Evans M, Hatfield M, Abdou NI. Study of anti-DNA antibodies prepared by DNA cellulose or Cibacron blue chromatography. J Immunol Methods 1986;93:13140.[CrossRef][ISI][Medline]
- Okamura M, Kanayama Y, Amastu K et al. Significance of enzyme linked immunosorbent assay (ELISA) for antibodies to double stranded and single stranded DNA in patients with lupus nephritis: correlation with severity of renal histology. Ann Rheum Dis 1993;52:1420.[Abstract]
- Dat MH, Behr C, Jouin H, Baleux F, Mercereau-Puijalon O, Dubois P.2000. Studies mimicking a conformational B cell epitope of the heat shock protein PfHsp70-1 antigen of Plasmodium falciparum using a multiple antigenic peptide. Parasite Immunol 1000;22:53544.[CrossRef][ISI][Medline]
Submitted 28 June 2002;
Accepted 9 October 2002