TY - JOUR
AU1 - Sun, Yongjiang
AU2 - Fong, Kok-Yong
AU3 - Chung, Maxey C. M.
AU4 - Yao, Zhi-Jian
AB - Abstract Autoantibodies to double-stranded (ds) DNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Identifying dsDNA mimotopes is a way to discover diagnostic and therapeutic candidates for SLE. `Mono-specific' SLE anti-dsDNA antibodies were obtained by affinity purification using dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide library, we were able to identify a mimotope that has a motif peptide sequence of RLTSSLRYNP. This chemically synthesized peptide could be recognized by 88% (37 out of 42) of anti-dsDNA antibody-positive SLE sera with a cut-off point at mean + 3 SD of the negative control sera at OD492. The reaction of the peptide with SLE sera in ELISA was highly correlated with that of dsDNA (r = 0.809, P < 0.0001). Of particular interest, not only dsDNA but also single-stranded (ss) DNA and native RNA could inhibit the binding of the peptide with SLE sera, suggesting that the mimotope is shared by ds and ssDNAs as well as native RNA, whereas denatured RNA was not observed to inhibit the binding. The peptide was also able to elicit an immune response in rabbits and the anti-peptide rabbit serum was observed to cross-react with the peptide, ss and dsDNAs, and ss and dsDNAs could inhibit the binding of the anti-peptide serum and the peptide. However, the inhibition was not obtained with RNA. Our findings demonstrate the potential of the peptide mimic in diagnostic tests of SLE, and in the investigation of anti-DNA antibody origin and of DNA–anti-DNA antibody interaction. autoantibody, anti-double-stranded DNA antibody, mimotope, phage peptide library ANA antinuclear antibody, ds double-stranded, RPL random peptide library, SLE systemic lupus erythematosus, ss single-stranded, TU transducing unit, MAP multiple antigen peptides Introduction Autoimmune diseases are characterized by autoantibodies against a diverse range of self-antigens. Although the patterns of autoantibody have been considered as reflecting the immunopathological processes of the autoimmune disease, in many cases the precise contribution of the autoantibodies to the actual pathology is not known. Therefore, for a long time, the autoantigen and autoantibody approach had been the key points for the elucidation of the autoimmune pathological processes, and in seeking proper diagnostic/therapeutic procedures. Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease characterized by the deposition of autoantibodies and immune complex, which eventually leads to tissue damage. Since 1957, the antinuclear antibody (ANA) test had been one of the most important tests used to evaluate patients for the presence of connective tissue disorders (1). The ANA screening test detects primarily a group of antibodies against the nuclear antigens that are present in abundance, e.g. double-stranded (ds) DNA, Sm, nuclear ribonucleoprotein, SSA and SSB. Among them, the anti-dsDNA test is the most widely used serological hallmark for SLE, and its titer correlates positively with disease activity in both humans and mice. Moreover, antibodies to dsDNA are not only a diagnostic marker but also a pathogenic factor for SLE. A number of studies have shown that anti-DNA antibodies cross-react with a variety of antigens, including intracellular and extracellular components (2–4). Thus, direct binding of anti-DNA antibodies to glomerular basement membrane was suggested as the major step in the development of glomerulonephritis (5). On the other hand, although reports have demonstrated that anti-DNA antibodies have the characteristics of antibodies arising from an antigen-selected immune response (6–8), the actual role of DNA in eliciting the production of anti-DNA antibodies is not yet clear. It remains uncertain which antigen triggers the production of these antibodies. The technology of phage-displayed random peptide libraries (RPL) (9–11) has recently become a powerful technique for elucidating protein–protein/peptide interactions. Many publications have already shown the potency of this procedure by establishing the binding characteristics between antigen–antibody, signaling molecule–receptor and substrate–enzyme. In particular, this technology offers a number of advantages in the research area of autoimmune diseases. Firstly, the RPL screening method can be used to map any epitope (mimotope) without prior knowledge of the protein antigen. Secondly, since several reports have shown that polyclonal antibodies could be used as screening ligates, this means that autoimmune patient sera could be used as a source for isolating screening ligates, thus providing a unique advantage of linking the RPL approach to a disease-related antigenic structure directly. We present here the application of RPL in searching a peptide mimic of dsDNA antigenic structure by using anti-dsDNA antibodies obtained from SLE patient sera. We have successfully identified a peptide motif which could mimic the antigenicity of native human placenta dsDNA. Investigations in specificity revealed that this structure was shared by denatured human placenta single-stranded (ss) DNA, natural phage genomic ssDNA and native calf liver RNA. Moreover, when the peptide was used to immunize rabbits, the harvested anti-peptide rabbit serum could not only recognize the peptide itself, but also ss and dsDNAs. Methods Human sera Human sera used in this study were collected from SLE patients and non-SLE controls at National University Hospital, Singapore. All of 42 female SLE patients and 40 female non-SLE controls satisfied American College of Rheumatology (formerly American Rheumatology Association) revised criteria for the classification of SLE (1). The age of the SLE patient group ranged from 15 to 69 years with an average of 40 years. The control sera were from dengue fever patients ranging in age from 10 to 60 years with an average of 36 years. The presence and absence of antibodies to dsDNA in the SLE sera and control sera were assayed by the QUANTA Lite dsDNA ELISA test kit (INOVA Diagnostics, San Diego, CA). The assay procedures provided by the manufacturer were followed. Briefly, each serum was diluted 1:100 with sample diluent and introduced into dsDNA-coated ELISA microtiter wells (100 μl/well) in duplicate. After incubating for 30 min at room temperature, the wells were emptied and washed with wash buffer. Horseradish peroxidase-conjugated anti-human IgG (raised in goat) was then added and incubated for another 30 min at room temperature. The wells were then emptied, washed and colored by adding 3,3′,5,5′-tetramethylbenzidine (TMB) chromogen. After stopping the color reaction by adding 100 μl of ELISA stopping solution to each well, absorbance was read at 450 nm with a reference wavelength 620 nm. Preparation of dsDNA-coupled CNBr-activated Sepharose column CNBr-activated Sepharose 4B gel was purchased from Pharmacia (Uppsala, Sweden). The protocol provided by the manufacturer for treatment of the gel and for coupling of protein to the gel was used. Briefly, 1.5 g of the gel was soaked in 50 ml of 1 mM HCl for at least 30 min at room temperature and allowed to swell. The swollen gel was then washed 3 times with about 300 ml 1 mM HCl by suspending the gel in a beaker and filtering the gel suspension in a flask. Human placenta dsDNA (Sigma, St Louis, MO) was dissolved in 1×SSC (150 mM NaCl/15 mM sodium citrate, pH 7.0) at a concentration of 3 mg/ml and further diluted in coupling buffer (0.1 M Na2CO3/0.5 M NaCl, pH 8.3) to 0.5 mg/ml. The dried gel was resuspended in 6 ml of this dsDNA solution and incubated at 4°C overnight with constant rotation to keep the gel in suspension (~2 mg of dsDNA was retained with the gel by measuring the difference of OD260 before and after the incubation). After washing away the unbound dsDNA with coupling buffer, the gel was resuspended in 0.1 M Tris–HCl, pH 8.0 and incubated for 2 h at room temperature to block the uncoupled area. After the blocking incubation, the gel was successively washed with acetate buffer (0.1 M sodium acetate, 0.5 M NaCl), pH 4.0 followed by 0.1 M Tris–HCl, pH 8.0 (3 times), with 10 mM Tris, pH 7.5 (at least 8 times), with 0.1 N HCl–glycine, pH 2.5 (at least 8 times), with 0.1 M triethylamine, pH 11.5 (at least 8 times) and finally with 10 mM sodium phosphate buffer, pH 7.0 until the pH of filtrate was 7.0. The gel was then packed into a chromatography column (Pharmacia) and kept at 4°C for use. Preparation of the screening ligate The preparation of screening ligate has been described elsewhere (12), but with some modifications in this study. Briefly, 1 ml of pooled positive sera was loaded on a GammaBind Sepharose (Pharmacia) column. After incubating for 30 min at room temperature, the column was washed to remove unbound fractions with 0.1 M sodium phosphate buffer, pH 7.0 until the baseline reached zero. The bound IgG fraction was then eluted with 0.5 M acetic acid, pH 2.7 and neutralized immediately with 2 M Tris. The IgG-containing fractions were then pooled together and dialyzed against 10 mM phosphate buffer, pH 7.0. Subsequently, the `mono-specific' anti-dsDNA fraction was purified using the human placenta dsDNA-coupled CNBr-activated Sepharose 4B gel column prepared as described above. The column was washed extensively with washing buffer (10 mM sodium phosphate, pH 7.0) overnight before use. The dialyzed IgG fraction was then loaded onto the affinity column and incubated for 30 min at room temperature. After incubation, the unbound IgG fraction was washed away with the washing buffer, while the bound IgG was eluted with 0.1 M sodium phosphate buffer, pH 7.0. The affinity-purified anti-dsDNA IgG was dialyzed for 4 h against 0.1 M sodium borate buffer, pH 8.8 and biotinylated subsequently by the succinimide method (13). Biopanning of phage-displayed RPL by anti-dsDNA antibodies A 15mer peptide library displayed on gene VIII product of fd phage (14) was used in this study. This library was generously provided by Professor G. P. Smith (University of Missouri, Columbia, MO) The basic methods of screening were adapted from the procedures as described before (12,15). Three rounds of screening were performed with the biotinylated anti-dsDNA antibodies. For the first round of selection, 10 μg of biotinylated anti-dsDNA antibodies in 400 μl of 0.5×TBST (25 mM Tris, 75 mM NaCl and 0.05% Tween 20, pH 7.5) containing 1 mg/ml dialyzed BSA were immobilized onto a streptavidin-coated Petri dish by incubation for 2 h at room temperature. In subsequent rounds of selection, 1 μg of biotinylated anti-dsDNA antibodies was used. To saturate the biotin binding sites on the dish, 4 μl of 10 mM free biotin was added and incubated for 1 h at room temperature. The unbound antibodies and biotin were removed by washing 6 times with 0.5×TBST. Phage suspensions (5 μl of library for the first round and 100 μl of output phages for the second and third rounds and 4 μl of 10 mM biotin in 400 μl of 0.5×TBST) were added and incubated for 4 h at room temperature. After the removal of unbound phages by washing 10 times with 0.5×TBST, the bound phages were recovered using elution buffer (0.1 N HCl–glycine, pH 2.2, 1 mg/ml BSA, 0.1 mg/ml phenol red). The eluates were immediately neutralized using 2 M Tris. Eluted phages were amplified in Escherichia coli K91Kan and used as input in the subsequent rounds of selection. After three rounds of selection, phage clones harvested from the third round output were randomly selected and propagated in 1.5 ml of terrific broth [1.2% (w/v) bacto-tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 17 mM KH2PO4 and 72 mM K2HPO4] supplemented with 20 μg/ml tetracycline at 37°C for 20 h. The cultures were clarified at 2500 g for 10 min, and the supernatants were recovered for the selection of anti-dsDNA antibody binding clones and subsequent DNA sequencing. Test of biopanning enrichment and selection of phage clones reactive with anti-dsDNA antibodies by ELISA assay Preparation of ELISA plates. ELISA microtiter wells were coated overnight at 4°C with 100 μl/well affinity-purified anti-dsDNA antibodies at a concentration of 5 μg/ml in coating buffer (50 mM NaHCO3, pH 9.6). After incubation, the wells were washed 3 times with 0.5×TBST and blocked with Blotto (5% skimmed milk in 0.5×TBST) for 60 min at 37°C. After washing again, the well strips were stored at 4°C for use. Test of biopanning enrichment. Phages from the library and the output phages of three rounds were diluted with Blotto to 1×1011 transducing units (TU)/ml. The phage suspensions were introduced into the anti-dsDNA antibody-coated microtiter wells and incubated for 2 h at 37°C. The unbound phages were then removed and the wells were washed 3 times with 0.5×TBST. This was followed by incubation with biotinylated anti-fd phage antibodies (Sigma) diluted with Blotto at a ratio of 1/1000 for 2 h at 37°C. After another round of extensive washing, horseradish peroxidase-conjugated streptavidin (Sigma), 1/1000 diluted with Blotto, was then added and incubated for 1 h at 37°C. Finally, following another washing step, the plates were treated with o-phenylenediamine dihydrochloride (OPD) for color development. Absorbance was measured at 492 nm using 620 nm as reference wavelength in an ELISA reader (Dynex, Middlesex, UK). Selection of anti-dsDNA antibody-reactive phage clones. Phage supernatants prepared as described above were added to the wells (100 μl/well) of the ELISA selection plates and allowed to bind for 2 h at 37°C. The subsequent treatments for ELISA assay were exactly the same as described above. A phage clone bearing a 15mer phagotope DLHRYSWKTQGDDRE selected previously from the 15mer phage library by mAb MAb47 which is specific for dengue virus NS1 protein (unpublished data) was processed in parallel as a negative control and its reading was referenced as background binding. Those phage clones showing more than twice the reading of background binding at 492 nm were selected as positive clones. DNA sequencing Clarified culture supernatants of positive clones selected as mentioned above were transferred to new Eppendorf tubes and the phages were recovered by 20% (w/w) PEG/NaCl solution (16.7%/3.3 M). After incubation for 4 h at 4°C, phages were pelleted at 10,000 g for 10 min. The phage pellets were then re-suspended in 100 μl of TBS. Viral DNA was extracted from 30 μl of phage suspension with phenol/chloroform/water solution. DNA in the aqueous layer was then precipitated by ethanol and used as a template for cycling sequencing with the primer: 5′-CTGAAGAGAGTCAAAAGC-3′. The sequencing was carried out in an automated DNA sequencer (model 373) from Perkin-Elmer Applied Biosystems (Foster City, CA). The amino acid sequences of the inserts were deduced from the DNA sequences. Peptide synthesis Peptides were synthesized by Fmoc chemistry using an eight-branched multiple antigen peptides (MAP) resin. Fmoc amino acids and MAP resins were purchased from Bachem (San Jose, CA) and AnaSpec (Torrance, CA). Peptide synthesis was carried out in our laboratory in an automatic peptide synthesizer, model 433A (Perkin-Elmer Applied Biosystems). The motif peptide MAP-RLTSSLRYNP and a control peptide MAP-TLPNRSYLSR that is a randomly scrambled sequence of the motif peptide sequence were synthesized. The purity of the peptides was ≥90% checked by reverse-phase HPLC and the compositions were confirmed by amino acid analysis. Serum binding to synthetic peptides Motif peptide MAP-RLTSSLRYNP was first dissolved in DMSO at a concentration of 100 mg/ml (w/v) and then diluted to 10 mg/ml with water (stock solution). The stock solution was further diluted 100 times with coating buffer to 0.1 mg/ml for coating ELISA microtiter wells. Control peptide MAP-TLPNRSYLSR was dissolved in coating buffer at a concentration of 0.1 mg/ml. The peptide solutions were added to microtiter wells (100 μl/well) and incubated at 4°C overnight. The wells were washed and blocked with Blotto. The sera diluted 1/100 with Blotto were added to the peptide-coated wells and incubated for 2 h at room temperature. After washing, horseradish peroxidase-conjugated anti-human IgG was added and incubated for 1 h at room temperature. The plates were then washed and colored by OPD chromogen as described above. Competitive binding inhibition assay Human placenta DNA, ss fd phage genomic DNA and calf liver RNA were 2-fold serially diluted starting from 400 μg/ml. fd phage DNA was extracted using ssPHAGE DNA Spin kit (BIO101, La Jolla, CA), and the other two DNA and RNA were purchased from Sigma. A set of dilutions of human placenta DNA and a set of dilutions of calf liver RNA were heated at 95°C for 3 min and rapidly cooled on ice to obtain denatured ssDNA and RNA. To be comparable, all dilutions of DNAs and RNAs were cooled on ice before mixing equally in volume with a cold dilution of a panel of SLE sera. The final concentrations of DNAs and RNAs were from 200 to 0.39 μg/ml and the dilution of SLE sera was 1/100. After mixing together, they were left at 4°C for 2 h and then transferred to a motif peptide-coated ELISA plate in duplicate (100 μl/well) for 30 min at 4°C. After subsequent incubation with horseradish peroxidase-conjugated anti-human IgG for 30 min at room temperature, the plates were processed as described above. The percentage inhibition of sera binding to peptide by the inhibitors was calculated according to the following equation: 100×[1 – (OD492 with inhibitor – background OD492)/(OD492 without inhibitor – background OD492)]. Immunization The immunization was performed by mixing equal volumes of Freund's adjuvant with peptide MAP-RLTSSLRYNP solution at a dose of 0.5 mg of peptide per injection. The emulsion was injected s.c. at multiple points in the back region of the rabbits. Complete Freund's adjuvant was used in the first injection, whereas incomplete adjuvant was used in booster injections. Booster injections were carried out on days 14, 28, 42 and 56 after the first injection. Blood samples were taken before immunization and on the days when the booster injections were performed. The last sample of serum was obtained on day 70. ELISA binding reactivity and competitive inhibition assay of anti-peptide rabbit serum Anti-peptide rabbit serum was tested for its ELISA binding reactivities with both motif and control peptide-coated plates and the QUANTA Lite ds and ssDNA ELISA plates. The rabbit sera taken before and after the peptide immunization were 2-fold serially diluted with Blotto from 1/100 to 1/6400, and added to the peptide- and DNA-coated wells separately. Each dilution was assayed in duplicate. The sera were allowed to bind for 1 h at room temperature. After washing away the sera with 0.5×TBST, horseradish peroxidase-conjugated anti-rabbit IgG, 1/1000 diluted with Blotto, was added and incubated for 30 min at room temperature. The wells were washed, and colored by OPD chromogen and absorbance was measured as described above. To demonstrate the specificity of anti-peptide serum, competitive binding inhibition ELISA assay was also performed with anti-peptide rabbit serum and the inhibitors DNAs and RNAs as with SLE sera described above. The experiment and the concentrations of inhibitor DNAs and RNAs remained the same, but the final dilution of anti-peptide serum was 1/300 in Blotto and the secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG, 1/1000 diluted in Blotto. Results The ligate and biopanning Based on our previous observations (12,15), preparing the screening ligate as `monospecific' as possible is the key step for a successful selection of target ligand(s) using RPL, especially when the screening ligates are polyclonal antibodies. To obtain such `monospecific' quality of screening ligate, SLE patient sera with anti-dsDNA antibodies were pooled and the IgG fraction was separated using a GammaBind column. The purified IgG was then subject to affinity purification with a Sepharose column coupled with human placenta dsDNA. After affinity purification, a commercial dsDNA ELISA kit (QUANTA Lite) was applied to evaluate the specific reactivity of anti-dsDNA antibodies. Figure 1 shows the specific ELISA reactivity of anti-dsDNA antibodies before and after affinity purification. When 0.5 μg of antibody protein was added to each well, a 5.16-fold increase in OD492 (1.24/0.24 = 5.16) was observed. It was lower compared with that obtained previously with hyper-immune rabbit serum (12), suggesting that the enhancement of specificity by affinity purification may vary depending on different ligate and ligand and affinity-purification procedures. This purified anti-dsDNA antibody was biotinylated and used as ligate for screening. An important indication for a successful biopanning is the enrichment of phage clones reactive with screening ligates. We used an affinity ELISA to monitor the enrichment of anti-dsDNA antibody-binding phages. After three successive rounds of biopanning, a constant amount of phages (1×1011 TU), harvested from each round of biopanning, was added to the ELISA plate coated with purified anti-dsDNA antibodies. The result showed that phage clones that were reactive with anti-dsDNA antibodies were progressively enriched between rounds of biopanning (Fig. 2). Selection of anti-dsDNA antibody-reactive phage clones and the putative motif From the third round output of biopanning, phage clones were randomly inoculated for microculture and evaluated by their binding to the screening ligate. At this step, a criterion was used to select phage clone, only those with 2 times higher ELISA readings than background were selected as putative positive clones. According to this selective criterion, 11 clones were selected and sequenced (Table 1). A putative motif, RLTSSLRYNP, was obtained by aligning their deduced amino acid sequences. Most of clones (10 of 11) contribute more than four residues to the motif sequence. Meanwhile, three negative clones that did not satisfy the criterion were also selected and sequenced. They showed random sequences with no significant alignment with this motif. Binding of SLE sera with motif peptide RLTSSLRYNP in ELISA To test the presumed mimic structure, we chemically synthesized a peptide corresponding to the motif sequence RLTSSLRYNP and a control peptide TLPNRSYLSR. To increase binding reactivity with human sera, the peptides were synthesized as eight-branch MAP. The SLE sera and the control sera were tested by commercial QUANTA Lite ds and ssDNA ELISA kits. All of the sera in the SLE group were positive with antibodies to dsDNA while the control sera tested negative. The specificity of the motif peptide was demonstrated by ELISA assay. The motif peptide was reactive with the SLE sera only but not with the control sera and the SLE sera were not reactive with the control peptide (Fig. 3). The differences in means of OD492 readings between the SLE sera and the control sera when they bound to motif peptide and between motif peptide and control peptide when they bound with SLE sera were statistically compared by Student's t-test, and the differences were very significant (two-tailed probability P < 0.0001). If taking the mean of the control sera group + 3 SD as the cut-off point (0.087, Fig. 3), the observed specificity of the control sera group was 100% and the sensitivity of SLE sera group was 88% (37 positive sera out of 42). That the SLE sera bound with motif peptide but not with control peptide suggests that this binding reactivity is sequence specific. It also demonstrated the specificity of the motif. When ELISA readings of the SLE sera bound with the dsDNA-coated plates are plotted against the corresponding results of SLE sera bound with the motif peptide-coated plates, a close correlation in ELISA binding reactivity (correlation coefficient r = 0.809, two-tailed probability P < 0.0001) was observed (Fig. 4). This feature could make the motif peptide a diagnostic candidate for SLE. Binding of SLE sera with motif peptide was competitively inhibited by DNA and RNA Inhibition ELISA assay was carried out to investigate if the motif peptide MAP-RLTSSLRYNP mimics an antigenic structure of dsDNA and if the above demonstrated peptide-based binding with SLE sera indeed mimics the binding property of dsDNA with anti-dsDNA antibody. Human placenta dsDNA, which was used as ligand in affinity purification for screening ligate, was used as inhibitor and the assay result showed that human placenta dsDNA inhibited the binding of anti-dsDNA antibody with motif peptide (Fig. 5). This inhibition effect suggests that MAP-RLTSSLRYNP mimics an antigenic structure of dsDNA and the binding property of dsDNA with anti-dsDNA antibody. However, it has been indicated that the bulk of SLE anti-dsDNA antibodies are cross-reactive, they not only recognize dsDNA but also ssDNA, and anti-dsDNA antibodies that bind only dsDNA are relatively rare (16,17). Thus the specificity of motif peptide as a dsDNA mimic is an interesting feature to demonstrate. Denatured human placenta ssDNA, ss phage genomic DNA and calf liver RNA were used to demonstrate the specificity by inhibition ELISA assay. The results showed that the binding of SLE sera with motif peptide was also inhibited by both denatured and natural ssDNAs, suggesting that the motif peptide is also a ssDNA mimic. It is noteworthy that denatured human placenta ssDNA exerted more inhibition than the same concentrations of native human placenta dsDNA, and natural ss phage genomic DNA exerted more inhibition than both human placenta ss and dsDNAs (Fig. 5). One explanation for these results could be that more binding sites, normally masked by the dsDNA structure, are exposed to the antibody after denaturation, and as phage genomic DNA is a much smaller molecule than human placenta DNA, phage ssDNA will give a higher molar concentration when they are prepared at the same concentration (in w/v) and this possibly exhibits more biding sites to the antibody. Inhibition was also observed with native calf liver RNA, although not high; however, interestingly, this inhibition disappeared after denaturation of the RNA (Fig. 5). The observations that RNA inhibited the binding of anti-DNA antibody to DNA and the ssDNA showed higher inhibition than dsDNA have been previously reported (18). Immunogenic motif peptide MAP-RLTSSLRYNP and anti-peptide serum Since motif peptide MAP-RLTSSLRYNP showed highly sensitive and specific binding reactivity with SLE sera in ELISA, it is very interesting to demonstrate its antigenic and immunogenic properties on animals. If the motif peptide is indeed a mimic of dsDNA antigenic determinant, the antibody against this peptide should recognize both the peptide and dsDNA. Therefore, we used rabbits to demonstrate the immunogenicity of the motif peptide. The anti-peptide rabbit serum showed high ELISA binding reactivities with the motif peptide and also with dsDNA (Fig. 6A and B), suggesting that the peptide does mimic an antigenic determinant of dsDNA and this antigenic determinant is shared by ssDNA as mentioned above since the serum could also recognize ssDNA (Fig. 6B). On the other hand, the anti-peptide serum did not bind to the control peptide (Fig. 6A), suggesting that the serum is specific for the motif peptide sequence. Inhibition ELISA assay was then performed to determine that the recognition of anti-peptide serum to DNA is indeed due to the same antigenicity of the peptide and DNA that drove the productive immunization. Native (ds) and denatured (ss) human placenta DNAs and ss phage genomic DNA were found to inhibit the binding of anti-peptide serum with the peptide, but both native and denatured calf liver RNAs were not (Fig. 7), despite native calf liver RNA showing some inhibition on the binding of the motif peptide with SLE sera (Fig. 5). Accordingly, these specific inhibition effects suggest that the motif peptide presented the same antigenicity as DNA to the immune system of the rabbits. Discussion There are two original considerations when we decided to investigate anti-dsDNA antibody binding structure from SLE patient sera with phage-displayed RPL. Firstly, it is of interest to investigate whether it is possible to identify peptide mimic(s) for a natural DNA molecule. Antibody to dsDNA is found in the sera of 60–70% of SLE patients (17) and has thus served as an important marker for the diagnosis of this ailment. Although not all anti-dsDNA antibodies are potentially pathogenic, it is found that some idiotypes, including human polyclonal and monoclonal as well as murine monoclonal IgG anti-dsDNA and anti-ssDNA antibodies, are capable of binding to glomerular antigens, and formed immune deposits in vivo and in vitro (2–5). The formation of these deposits is believed to be a key step in the renal damage of SLE patients. Therefore, defining the binding of anti-DNA antibodies to DNA in affinity, specificity and interaction mode is particularly important for understanding pathogenesis of SLE. The identification of peptide mimics for dsDNA that are capable of inhibiting the binding of dsDNA with antibodies to dsDNA is not only a potential drug discovery route for SLE, but also a valuable tool for studying the dsDNA–anti-dsDNA antibody complex. Recently, a peptide surrogate for dsDNA, which was able to inhibit the glomerular deposition of a murine monoclonal anti-dsDNA antibody in vivo, has been identified from RPL using murine monoclonal anti-dsDNA antibodies as ligate (19). However, epitopes selected with murine mAb reflect the repertories of immune response of mouse rather than human. Only antibodies in human sera are the natural response of human immune system to immunogens. Therefore, epitopes selected by using patient sera as ligate would be a more direct and effective way for discovering the candidates of diagnostic and/or therapeutic agents. In fact, there is now an increasing trend in the use of disease-specific sera (20,21) and body fluids (22,23) from patients in disease-specific epitope mapping with RPL. In this study, we have identified a peptide mimic for dsDNA using RPL and SLE patient sera. This peptide mimic, with the sequence RLTSSLRYNP, exhibited a close correlation (correlation coefficient, r = 0.809, P < 0.0001) with dsDNA in the binding reactivities with human serum antibody to dsDNA (Fig. 4). This result suggests the possibility of using the peptide mimic in the diagnosis of SLE. By using the peptide mimic in an ELISA format, it was established that autoantibodies to dsDNA were detected in 37 of the 42 SLE+ sera (Fig. 3). More importantly and interestingly, the peptide mimic was able to bind competitively SLE anti-DNA antibodies in vitro not only with dsDNA but also with ssDNA (Fig. 5). These findings, on the one hand, suggest that the peptide mimic is an antigenic structure shared by both ss and dsDNAs, and, on the other hand, reflect that the motif peptide binding SLE anti-dsDNA antibodies are cross-reactive. This type of antigenic structures is believed to encompass exposed bases as well as the phosphodiester backbone (16,17). With the same spices of DNA, human placenta DNA, more binding structures were exhibited to the antibody on denatured ssDNA than on native dsDNA. This feature most probably results from the conformational changes of ds to ssDNA that allow the antibody to bind exposed regions of single strandedness (24) that are normally masked by the dsDNA helix structure. Alternatively, the small molecule of phage ssDNA exhibited more binding sites than the large molecule of human placenta ss and dsDNAs when they were applied at the same amount, suggesting that the binding site occurs variably on both species of DNAs and the number of the binding site is not directly proportional to the length of DNA molecule. Another interesting finding is the inhibition on motif peptide and SLE sera binding by native calf liver RNA. It has been reported that only anti-DNA antibodies eluted from the kidney of MRL-lpr/lpr mice bound with RNA, the antibodies in MRL-lpr/lpr mouse serum did not; moreover, the most characteristic feature of nephritogenic autoantibody was polyreactivity—the ability of individual antibodies to bind to multiple antigens (18). We did not address the inhibition by other autoantigens other than polynucleotide antigens in this study and the pathogenic potential of the idiotypic anti-dsDNA antibodies that bind the motif peptide needs to be further investigated. The potential of the motif peptide to be a drug candidate for SLE could be true only when the peptide binding anti-dsDNA antibodies are tested as a pathogenic idiotype. It is certain, however, that this peptide mimic provides us with a unique opportunity to investigate the antigenic structure of DNA and the DNA–anti-DNA antibody interaction. The origins of antibodies to DNA in SLE are not yet clear. Native mammalian DNA is poorly immunogenic and has never been found to be able to induce anti-DNA antibodies (25,26). Although some bacterial DNAs have been demonstrated to be immunogenic in normal mouse, they do not elicit production of antibodies that cross-react with mammalian DNA (27,28). Complexes of ssDNA and Escherichia coli dsDNA non-covalently bound to a cationic protein such as methylated serum albumin have been reported to be able to induce antibodies with specificity only for ssDNA and Escherichia coli dsDNA respectively, but not for native mammalian DNA, and the complexes when prepared with mammalian dsDNA have not been immunogenic (29,30). Similarly, although anti-DNA antibodies produced after polyoma BK virus infection and immunization were specific for native DNA, these induced anti-DNA antibodies were still different from autoimmune anti-DNA antibodies (31,32). Until recently, anti-dsDNA antibodies have been successfully induced by a complex of native calf thymus DNA and a highly immunogenic DNA binding peptide, Fus1 (33). The structural and functional characteristics of the induced anti-DNA antibodies were similar in all aspects to autoimmune anti-DNA antibodies (33,34). The common feature in these studies is that DNA seemed more immunogenic in non-autoimmune animals only when complexed with immunogenic protein and peptide. On the other hand, nevertheless, pneumococcal polysaccharide antigens have been described to be able to elicit an immune response that produced some idiotypic antibodies cross-reactive with DNA (35). More recently, a peptide surrogate for dsDNA, which was identified from a phage peptide library using a murine monoclonal anti-dsDNA antibody (19), has induced the production of anti-dsDNA antibodies in non-autoimmune BALB/c mice. Moreover, the immunized mice also developed antibodies against some other lupus antigens and Ig deposition was present in renal glomeruli (36). We could also demonstrate in this study that a peptide surrogate identified from a phage peptide library using anti-dsDNA antibodies in SLE sera has the same antigenic structure with DNA and native RNA, and induced anti-DNA antibody production in rabbits. These studies suggest that DNA antigen is not indispensable for the production of anti-DNA antibodies. Therefore, which antigen induces spontaneous production of anti-DNA antibodies in the autoimmunity of SLE remains unclear. As such, our second goal of the project is to attempt to identify the `genuine autoantigen(s)' that may elicit the production of anti-DNA antibodies in SLE. Since the RPL approach can map and identify epitopes of antigens without prior knowledge of their structures, the peptide motifs/sequence(s) selected by biopanning could provide clues for a known antigen by using an online homology search program (e.g. Blitz). The competition with DNA for binding anti-DNA antibodies in SLE sera and the ability to induce immune response of producing anti-DNA antibodies (Figs 6 and 7) suggest that the motif peptide mimics an antigenic structure of SLE autoantigen, that may be or may not be DNA. To our knowledge, this is the first report of the identification of a peptide mimic for DNA by polyclonal antibodies separated directly from SLE patient sera. Motif peptide RLTSSLRYNP exhibits homology with a chromosome-associated polypeptide, but it does not show any similarity with antigens of suspected infectious agents, such as pneumococcal (35) or Epstein–Barr viral antigens (37,38). An online similarity search is useful only for linear epitopes; furthermore, the amino acid residues of a linear epitope must be well defined. Otherwise, an online search is usually fruitless. In the case of motif peptide RLTSSLRYNP, it may represent a mimotope rather than a linear epitope of `genuine autoantigen(s)'. If this is the case, then it is difficult to identify the causative antigen(s) for SLE using this approach. On the other hand, we are further characterizing this motif for defining more critical residues and for shortening the sequence if possible. Thus, we could not rule out the possibility that the pathogenesis is still microbial in origin. With more sero-epidemiological studies of SLE and more peptide epitopes/mimotopes of dsDNA, it may enhance the chance of identifying potential putative antigens for anti-DNA antibodies. Table 1. The deduced amino acid sequence of inserts selected from RPL Deduced amino acid sequences  Motif  Motif residues are underlined.  From positive clones    RRTPDPAVSPWQLTY    RDGQRLTSSKTMLPY    KLTSSLRYNSPPLCF  RLTSSLRYNP  KSTSSLRDNSPPVCF    KLTSSLRCNCPPLRF    THDLSSRASSSLSYN    QAPRLMSSLSYFPQS    DHRSPPWLTSLLTIS    DTWPTARLTSSMQYI    HTYTSHLRYVPPISL    GHRYTSSVSLTEACP    From negative clones    SGTSHSASTTSKWFL    NLLSVAPFWPLNDSL    SDPDQWPFWRANEYG    Deduced amino acid sequences  Motif  Motif residues are underlined.  From positive clones    RRTPDPAVSPWQLTY    RDGQRLTSSKTMLPY    KLTSSLRYNSPPLCF  RLTSSLRYNP  KSTSSLRDNSPPVCF    KLTSSLRCNCPPLRF    THDLSSRASSSLSYN    QAPRLMSSLSYFPQS    DHRSPPWLTSLLTIS    DTWPTARLTSSMQYI    HTYTSHLRYVPPISL    GHRYTSSVSLTEACP    From negative clones    SGTSHSASTTSKWFL    NLLSVAPFWPLNDSL    SDPDQWPFWRANEYG    View Large Fig. 1. View largeDownload slide Enhancement of screening ligate in specific reactivity with dsDNA after affinity purification. Anti-dsDNA antibody reactivities in total serum IgG before affinity purification (BP), and in the unretained fraction (UF) and in retained fraction (RF) after affinity purification were compared by ELISA assay on QUANTA Lite dsDNA plates (0.5 μg of IgG/well). The anti-dsDNA antibody reactivity was highly enhanced after affinity purification. Fig. 1. View largeDownload slide Enhancement of screening ligate in specific reactivity with dsDNA after affinity purification. Anti-dsDNA antibody reactivities in total serum IgG before affinity purification (BP), and in the unretained fraction (UF) and in retained fraction (RF) after affinity purification were compared by ELISA assay on QUANTA Lite dsDNA plates (0.5 μg of IgG/well). The anti-dsDNA antibody reactivity was highly enhanced after affinity purification. Fig. 2. View largeDownload slide Enrichment of phages reacted with anti-dsDNA antibodies by biopanning. After three rounds of biopanning, phage particles from the phage library and the output of each round allowed to bind to affinity-purified anti-dsDNA antibody immobilized on microtiter plates (1×1011 TU/well). Biotinylated anti-phage antibody and streptavidin-conjugated horseradish peroxidase were then applied to this ELISA assay. PL, 15mer phage library; 1st, first round of output; 2nd, second round of output; 3rd, third round of output. Phages with the specificity to anti-dsDNA antibody were enriched after three rounds of biopanning. Fig. 2. View largeDownload slide Enrichment of phages reacted with anti-dsDNA antibodies by biopanning. After three rounds of biopanning, phage particles from the phage library and the output of each round allowed to bind to affinity-purified anti-dsDNA antibody immobilized on microtiter plates (1×1011 TU/well). Biotinylated anti-phage antibody and streptavidin-conjugated horseradish peroxidase were then applied to this ELISA assay. PL, 15mer phage library; 1st, first round of output; 2nd, second round of output; 3rd, third round of output. Phages with the specificity to anti-dsDNA antibody were enriched after three rounds of biopanning. Fig. 3. View largeDownload slide Reaction of SLE sera and control sera with MAP peptides. SLE sera and control sera at a dilution of 1/100 were incubated with motif peptide MAP-RLTSSLRYNP and control peptide MAP-TLPNRSYLSR (with SLE sera only) immobilized on microtiter plates for ELISA assay. (A) ELISA readings of 42 SLE sera when bound to motif peptide. Thirty-seven out of 42 (88%) SLE sera bound with motif peptide when using mean + 3 SD of OD readings of control sera bound to motif peptide as a cut-off point (the dashed line). (B) ELISA readings of 40 control sera when bound to motif peptide. None of control sera bound with motif peptide. The mean of OD readings of SLE sera was statistically different from that of control sera (P < 0.0001 by Student's unpaired t-test) when they bound to motif peptide. (C) ELISA readings of 42 SLE sera when bound to control peptide. None of SLE sera bound significantly with control peptide. The means of OD readings of SLE sera bound to motif peptide and control peptide were also very significantly different (P < 0.0001 by Student's paired t-test). Fig. 3. View largeDownload slide Reaction of SLE sera and control sera with MAP peptides. SLE sera and control sera at a dilution of 1/100 were incubated with motif peptide MAP-RLTSSLRYNP and control peptide MAP-TLPNRSYLSR (with SLE sera only) immobilized on microtiter plates for ELISA assay. (A) ELISA readings of 42 SLE sera when bound to motif peptide. Thirty-seven out of 42 (88%) SLE sera bound with motif peptide when using mean + 3 SD of OD readings of control sera bound to motif peptide as a cut-off point (the dashed line). (B) ELISA readings of 40 control sera when bound to motif peptide. None of control sera bound with motif peptide. The mean of OD readings of SLE sera was statistically different from that of control sera (P < 0.0001 by Student's unpaired t-test) when they bound to motif peptide. (C) ELISA readings of 42 SLE sera when bound to control peptide. None of SLE sera bound significantly with control peptide. The means of OD readings of SLE sera bound to motif peptide and control peptide were also very significantly different (P < 0.0001 by Student's paired t-test). Fig. 4. View largeDownload slide Correlation of motif peptide and dsDNA in ELISA reactivity. The 42 SLE sera were diluted 1/100 in Blotto and added in duplicate to motif peptide-coated plates and QUANTA Lite dsDNA plates separately. The reaction of SLE sera with motif peptide and dsDNA was assayed using ELISA. The ELISA reactivity of anti-dsDNA antibody with the peptide and dsDNA was highly correlated (r = 0.809, P < 0.0001). The dots represent OD readings measured at 492 nm. Fig. 4. View largeDownload slide Correlation of motif peptide and dsDNA in ELISA reactivity. The 42 SLE sera were diluted 1/100 in Blotto and added in duplicate to motif peptide-coated plates and QUANTA Lite dsDNA plates separately. The reaction of SLE sera with motif peptide and dsDNA was assayed using ELISA. The ELISA reactivity of anti-dsDNA antibody with the peptide and dsDNA was highly correlated (r = 0.809, P < 0.0001). The dots represent OD readings measured at 492 nm. Fig. 5. View largeDownload slide Inhibition of binding of SLE sera to motif peptide by DNA and RNA. A pool of SLE sera at a final dilution of 1/100 was incubated at 4°C for 2 h with varying concentrations of inhibitors as indicated in the legends. The final concentrations of the inhibitors were from 200 to 0.39 μg/ml. Denatured human placenta ssDNA and calf liver RNA were obtained by heating at 95°C for 3 min and rapidly cooling on ice. The mixtures were then introduced to motif peptide-coated ELISA plates and further incubated at 4°C for 30 min for an inhibition ELISA assay as described in Methods. The binding of the sera with the peptide was inhibited by the ss and dsDNAs, and native RNA, but not by denatured RNA. Fig. 5. View largeDownload slide Inhibition of binding of SLE sera to motif peptide by DNA and RNA. A pool of SLE sera at a final dilution of 1/100 was incubated at 4°C for 2 h with varying concentrations of inhibitors as indicated in the legends. The final concentrations of the inhibitors were from 200 to 0.39 μg/ml. Denatured human placenta ssDNA and calf liver RNA were obtained by heating at 95°C for 3 min and rapidly cooling on ice. The mixtures were then introduced to motif peptide-coated ELISA plates and further incubated at 4°C for 30 min for an inhibition ELISA assay as described in Methods. The binding of the sera with the peptide was inhibited by the ss and dsDNAs, and native RNA, but not by denatured RNA. Fig. 6. View largeDownload slide Reaction of anti-peptide rabbit serum with motif and control peptides and DNAs. Anti-peptide rabbit serum and pre-immune rabbit serum were 2-fold serially diluted starting from 1/100, and incubated with MAP-RLTSSLRYNP- and MAP-TLPNRSYLSR-coated plates (A), and with QUANTA Lite dsDNA and ssDNA plates (B) for ELISA assays. Anti-peptide serum showed high ELISA binding reactivities with the motif peptide and the ss and dsDNAs, but not the control peptide. Fig. 6. View largeDownload slide Reaction of anti-peptide rabbit serum with motif and control peptides and DNAs. Anti-peptide rabbit serum and pre-immune rabbit serum were 2-fold serially diluted starting from 1/100, and incubated with MAP-RLTSSLRYNP- and MAP-TLPNRSYLSR-coated plates (A), and with QUANTA Lite dsDNA and ssDNA plates (B) for ELISA assays. Anti-peptide serum showed high ELISA binding reactivities with the motif peptide and the ss and dsDNAs, but not the control peptide. Fig. 7. View largeDownload slide Competitive binding between the peptide and DNAs with anti-peptide rabbit serum. Anti-peptide rabbit serum at a final dilution of 1/300 was incubated at 4°C for 2 h with varying concentrations of inhibitors as indicated. The final concentrations of the inhibitors were from 200 to 0.39 μg/ml. The mixtures were then added to motif peptide-coated ELISA plate and further incubated at 4°C for 30 min for inhibition ELISA assay as described in Methods. The binding of anti-peptide serum with the peptide was competitively inhibited by native (ds) and denatured (ss) human placenta DNA and natural ss phage genomic DNA. Inhibition was not observed with native and denatured calf liver RNA. Fig. 7. View largeDownload slide Competitive binding between the peptide and DNAs with anti-peptide rabbit serum. Anti-peptide rabbit serum at a final dilution of 1/300 was incubated at 4°C for 2 h with varying concentrations of inhibitors as indicated. The final concentrations of the inhibitors were from 200 to 0.39 μg/ml. The mixtures were then added to motif peptide-coated ELISA plate and further incubated at 4°C for 30 min for inhibition ELISA assay as described in Methods. The binding of anti-peptide serum with the peptide was competitively inhibited by native (ds) and denatured (ss) human placenta DNA and natural ss phage genomic DNA. Inhibition was not observed with native and denatured calf liver RNA. 4 Present address: Institute of Dermatology, National Skin Centre, 1 Mandalay Road, Singapore 308205, Republic of Singapore 5 Present address: National Center for Human Genome Research, BDA, #3-707 North Yongchang Road, Beijing 100176, China Transmitting editor: A. Kelso We thank Ms Mao Yan Ying for the excellent immunization work. References 1 Tan, E. M., Cohen, A. S., Fries, J. F., Masi, A. T., McShane, D. J., Rothfield, N. F., Schaller, J. G., Talal, N. and Winchester, R. J. 1982. The 1982 revised criteria for the classification of systemic lupus erythematosus (SLE). Arthritis Rheum.  25: 1271. Google Scholar 2 Sun, K.-H., Liu, W.-T., Tang, S.-J., Tsai, C.-Y., Hsieh, S.-C., Wu, T.-H., Han, S.-H. and Yu, C.-L. 1996. The expression of acidic ribosomal phosphoproteins on the surface membrane of different tissues in autoimmune and normal mice which are the target molecules for anti-double-stranded DNA antibodies. Immunology  87: 362. Google Scholar 3 Jacob, L., Lety, M.-A., Louvard, D. and Bach, J.-F. 1985. Binding of a monoclonal anti-DNA autoantibody to identical protein(s) present at the surface of several human cell types involved in lupus pathogenesis. J. Clin. Invest.  75: 315. Google Scholar 4 Madaio, M. P., Carlson, J., Cataldo, J., Ucci, A., Migliorini, P. and Pankewycz, O. 1987. Murine monoclonal anti-DNA antibodies bind directly to glomerular antigens and form immune deposits. J. Immunol.  138: 2883. Google Scholar 5 Ehrenstein, M. R., Katz, D. R., Griffiths, M. H., Papadaki, L., Winkler, T. H., Kalden, J. R. and Isenberg, D. A. 1995. Human IgG anti-DNA antibodies deposit in kidneys and induce proteinuria in SCID mice. Kidney Int.  48: 705. Google Scholar 6 Diamond, B., Katz, J. B., Paul, E., Aranow, C., Lustgarten, D. and Scharff, M. D. 1992. The role of somatic mutation in the pathogenic anti-DNA response. Annu. Rev. Immunol.  10: 731. Google Scholar 7 Marion, T. N., Tillman, D. M., Jou, N. T. and Hill, R. J. 1992. Selection of immunoglobulin variable regions in autoimmunity to DNA. Immunol. Rev.  128: 123. Google Scholar 8 Radic, M. Z. and Weigert, M. 1994. Genetic and structural evidence for antigen selection of anti-DNA antibodies. Annu. Rev. Immunol.  12: 487. Google Scholar 9 Scott, J. K. and Smith, G. P. 1990. Searching for peptide ligands with an epitope library. Science  249: 386. Google Scholar 10 Devlin, J. J., Panganiban, L. C. and Devlin, P. E. 1990. Random peptide libraries: A source of specific protein binding molecules. Science  249: 404. Google Scholar 11 Cwirla, S. E., Peters, E. A., Barrett, R. W. and Dower, W. J. 1990. Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl Acad. Sci. USA  87: 6378. Google Scholar 12 Yao, Z. J., Chan, M. C., Kao, M. C. C. and Chung, M. C. M. 1996. Linear epitopes of sperm whale myoglobin identified by polyclonal antibody screening of random peptide library. Int. J. Peptide Prot. Res.  48: 477. Google Scholar 13 Bayer, E. A. and Wilchek, M. 1980. The use of the avidin–biotin complex as a tool in molecular biology. Methods Biochem. Anal.  26: 1. Google Scholar 14 Smith, G. P. and Scott, J. K. 1993. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol.  217: 228. Google Scholar 15 Yao, Z. J., Kao, M. C. C. and Chung, M. C. M. 1995. Epitope identification by polyclonal antibody from phage-displayed random peptide library. J. Protein Chem.  14: 161. Google Scholar 16 Tan, E. M. 1989. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv. Immunol.  44: 93 Google Scholar 17 Ferrell, P. B. and Tan, E. M. 1985. Systemic lupus erythematosus. In Rose, N. R. and Mackay, I. R., eds, The Autoimmune Diseases, p. 29. Academic Press, San Diego, CA. Google Scholar 18 Pankewycz, O. G., Migliorin, P. and Madaio, M. P. 1987. Polyreactive autoantibodies are nephritogenic in murine lupus nephritis. J. Immunol.  139: 3287. Google Scholar 19 Gaynor, B., Putterman, C., Valadon, P., Spatz, L., Scharff, M. D. and Diamond, B. 1997. Peptide inhibition of glomerular deposition of an anti-DNA antibody. Proc. Natl Acad. Sci. USA  94: 1955. Google Scholar 20 Yao, Z. J., Ong, L. H., Chan, L. and Chung, M. C. M. 1998. Identifying antigenic region of hepatitis B surface antigen by patient's serum with random peptide library. Prot. Peptide Lett.  5: 33. Google Scholar 21 Folgori, A., Tafi, R., Meola, A., Felici, F., Galfre, G., Cortese, R., Monaci, P. and Nicosia, A. 1994. A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO J.  13: 2236. Google Scholar 22 Rand, K. H., Houck, H., Denslow, N. D. and Heilman, K. M. 1998. Molecular approach to find target(s) for oligoclonal bands in multiple sclerosis. J. Neurol. Neurosurg. Psychiat.  65: 48. Google Scholar 23 Dybwad, A., Førre, Ø., Natvig, J. B. and Sioud, M. 1995. Structural characterization of peptides that bind synovial fluid antibodies from RA patients: a novel strategy for identification of disease-related epitopes using a random peptide library. Clin. Immunol. Immunopathol.  75: 45. Google Scholar 24 Emlen, W., Pisetsky, D. S. and Taylor, R. P. 1986. Antibodies to DNA. A perspective. Arthritis Rheum.  29: 1417. Google Scholar 25 Madaio, M. P., Hodder, S., Schwartz, R. S. and Stollar, B. D. 1984. Resposiveness of autoimmune and normal mice to nucleic acid antigens. J. Immunol.  132: 872. Google Scholar 26 Marion, T. N., Krishnan, M. R., Desai, D. D., Jou, N.-T. and Tillman, D. M. 1997. Monoclonal anti-DNA antibodies: structure, specificity, and biology. Methods  11: 3. Google Scholar 27 Pisetsky, D. S., Grudier, J. P. and Gilkson, G. S. 1990. A role for immunogenic DNA in the pathogenesis of systemic lupus erythematosus. Arthritis Rheum.  33: 153. Google Scholar 28 Gilkeson, G. S., Pippen, A. M. and Pisetsky, D. S. 1995. Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA. J. Clin. Invest.  95: 1398. Google Scholar 29 Stollar, B. D. 1989. Immunochemistry of DNA. Int. Rev. Immunol.  5: 1. Google Scholar 30 Gilkeson, G. S., Grudier, J. P., Karounos, D. G. and Pisetsky, D. S. 1989. Induction of anti-double stranded DNA antibodies in normal mice by immunization with bacterial DNA. J. Immunol.  142: 1482. Google Scholar 31 Flaegstad, T., Fredriksen, K., Dahl, B., Traavik, T. and Rekvig, O. P. 1988. Inoculation with BK virus may break immunological tolerance to histone and DNA antigens. Proc. Natl Acad. Sci. USA  85: 8171. Google Scholar 32 Rekvig, O. P., Fredriksen, K., Brannsether, B., Moens, U., Sundsfjord, A. and Traavik, T. 1992. Antibodies to eukaryotic, including autologous native DNA are produced during BK virus infection, but not after immunization with non-infectious BK DNA. Scand. J. Immunol.  36: 487. Google Scholar 33 Desai, D. D., Krishnan, M. R., Swindle, J. T. and Marion, T. N. 1993. Antigen specific induction of antibodies against native mammalian DNA in nonautoimmune mice. J. Immunol.  151: 1614. Google Scholar 34 Krishnan, M. K. and Marion, T. N., 1993. Structural similarity of antibody variable regions from immune and autoimmune anti-DNA antibodies. J. Immunol.  150: 4948. Google Scholar 35 Grayzel, A., Solomon, A., Aranow, C. and Diamond, B. 1991. Antibodies elicited by pneumococcal antigens bear an anti-DNA-associated idiotype. J. Clin. Invest.  87: 842. Google Scholar 36 Putterman, C. and Diamond, B. 1998. Immunization with a peptide surrogate for double-stranded DNA (dsDNA) induces autoantibody production and renal immunoglobulin deposition. J. Exp. Med.  188: 29. Google Scholar 37 James, J. A., Kaufman, K. M., Farris, A. D., Taylor-Albert, E., Lehman, T. J. A. and Harley, J. B. 1997. An increased prevalance of Epstein–Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus. J. Clin. Invest.  100: 3019. Google Scholar 38 Incaprera, M., Rindi, L., Bazzichi, A. and Garzelli, C. 1998. Potential role of the Epstein–Barr virus in systemic lupus erythematosus autoimmunity. Clin. Exp. Rheum.  16: 289. Google Scholar © 2001 Japanese Society for Immunology
TI - Peptide mimicking antigenic and immunogenic epitope of double-stranded DNA in systemic lupus erythematosus
JF - International Immunology
DO - 10.1093/intimm/13.2.223
DA - 2001-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/peptide-mimicking-antigenic-and-immunogenic-epitope-of-double-stranded-kWx285Tapx
SP - 223
EP - 232
VL - 13
IS - 2
DP - DeepDyve
ER -