1 High Resolution Epitope Mapping of Human Autoimmune Sera against Antigens CENPA and KDM6B PEPperPRINT GmbH Heidelberg, 06/2014
2 Introduction This report describes the epitope mapping of autoimmune sera against a putative systemic sclerosis (SSc) biomarker identified by Protagen in SSc patient and control samples. Systemic sclerosis (SSc; also termed scleroderma) is a complex multisystem rheumatic disease in which autoimmunity, inflammation, fibrosis and vasculopathy lead to a complex pattern of organ-based complications with high mortality and morbidity. There is no diagnostic test providing the absence or presence of SSc. Like in the case of other connective tissue diseases, diagnosis of SSc is based on the combination of clinical and/or laboratory features e.g. SSc-related autoantibodies. The most frequently measured autoantibodies (anti-scl70, anti-centromere and anti-rnpiii) are associated with different subtypes of SSc or with specific organ involvements. The three antibodies are included in the new SSc classification criteria, but are only present in about 60-70% of SSc patients. This highlights the need for additional specific and sensitive diagnostic, prognostic and therapeutic response biomarkers in SSc. Therefore, Protagen applied its SeroTag process to discover new autoantibodies with high frequency in SSc. The SeroTag process is an automated bead-based, antigen array on the Luminex FlexMAP3D analyzer for measuring autoantibody reactivities against both established and putative autoantigens. Three putative SSc antigens were identified by Protagen, one of them being KDM6B (Lysine (K)-Specific Demethylase 6B). To further characterize the autoantibody responses against KDM6B, a high resolution epitope mapping with 15mer peptides and maximal peptide overlap of 14 amino acids was done by PEPperPRINT. In parallel, we mapped the epitopes of centromere protein A (CENPA) specific autoantibodies. CENPA is a well-known marker for SSc with an N-terminal main epitope PRRRS identified by Muro et al. 1 We compared 3 SSc patient sera and one healthy control serum and identified well defined polyclonal response with high signal-to-noise ratios against both autoantigens. Due to the high maximum peptidepeptide overlap, we further could identify two N-terminal neighbored epitopes within CENPA with a higher epitope resolution than before. Besides various other epitopes, we also identified a common epitope for the putative SSc antigen KDM6B. 1 Y. Muro et al., Clin. Exp. Immunol. 2000; 120:
3 Materials and Methods Microarray Content: Antigens CENPA and KDM6B were translated into 15 aa peptides with a peptide-peptide overlap of 14 amino acids resulting in 1,831 different peptides printed in duplicate (3,662 peptide spots in all). The corresponding peptide microarrays were further framed by Flag and HA control peptides (124 spots each). Samples: Washing Buffer: Blocking Buffer: Incubation Buffer: Assay Conditions: Three human sera from SSc patients (SSc1, SSc2, SSc3) including one healthy control serum (HC) PBS, ph 7.4 with 0.05% Tween 20 (2x1 min after each assay) Rockland blocking buffer MB-070 (60 min before the first assay) PBS, ph 7.4 with 0.05% Tween 20 and 10% Rockland blocking buffer Serum dilutions of 1:5000 in incubation buffer; incubation for 16 h at 4 C and shaking at 500 rpm Secondary Antibody: Goat anti-human IgG(H+L) conj. DyLight680; 30 min staining at RT and a dilution of 1:5000 Control Antibodies: Scanner: Microarray Data: Microarray Identifier: Monoclonal anti-ha (12CA5)-DyLight680, monoclonal anti-flag(m2)-dylight800; staining in incubation buffer for 1 h at RT and a dilution of 1:1000 LI-COR Odyssey Imaging System; scanning offset 1 mm, resolution 21 µm, scanning intensity green/red of 7/7 MicroarrayData_SSc1.xlsx, MicroarrayData_SSc2.xlsx, MicroarrayData_SSc3.xlsx, MicroarrayData_HC.xlsx, MicroarrayData_Summary.xlsx _02, _03, _05, _06 (two array copies each microarray)
4 Experimental and Data Analysis Pre-staining of one of the peptide arrays was done with the goat anti-human IgG(H+L) conj. DyLight680 antibody at a dilution of 1:5000 to investigate background interactions with the antigen-derived peptides that could interfere with the main assays. Subsequent incubation of the peptide microarrays with human sera SSc1, SSc2, SSc3 and HC at a dilution of 1:5000 in incubation buffer was followed by staining with the secondary antibody and read-out at a scanning intensity of 7 (red). HA and Flag control peptides framing the peptide arrays were finally stained as internal quality control to confirm the assay quality and the peptide microarray integrity (scanning intensities red/green: 7/7). Quantification of spot intensities and peptide annotation were done with PepSlide Analyzer and summarized in the Excel files MicroarrayData_SSc1.xlsx, MicroarrayData_SSc2.xlsx, MicroarrayData_SSc3.xlsx, MicroarrayData_HC.xlsx and MicroarrayData_Summary.xlsx. A software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal (see Raw Data tabs), and calculates the standard deviation of foreground median intensities (see Mapping Summary tabs). Based on averaged foreground median intensities, intensity maps were generated and binders in the peptide maps highlighted by an intensity color code with red for high and white for low spot intensities. We further plotted averaged spot intensities of all assays against the linked antigen sequences from the N-terminus of CENPA to the C-terminus of KDM6B to visualize overall spot intensities and signal to noise ratios (see Intensity Plot tabs). The intensity plots were correlated with peptide and intensity maps as well as with visual inspection of the microarray scans to identify peptides and consensus motifs that interacted with the human serum samples. In case it was not clear if a certain amino acid contributed to antibody binding, the corresponding amino acid letters were written in grey.
5 Pre-Staining with Secondary Ab Goat anti-human IgG(H+L) conj. DyLight680 antibody, 1:5000 After 10 min pre-swelling in standard buffer and 60 min in blocking buffer, one of the peptide arrays was initially incubated with the secondary goat anti-human IgG(H+L) conj. DyLight680 antibody at a dilution of 1:5000 for 60 min at room temperature to analyze background interactions with the antigen-derived peptides. At scanning intensity of 7, we did not observed any background due to non-specific binding of the secondary antibody. Data quantification with PepSlide Analyzer was neither possible nor required, since the absence of any spot pattern hampered alignment of the microarray grid.
6 SSc1, Microarray Scans SSc1, 1:5000 Control Staining One of the CENPA/KDM6B peptide microarrays was incubated with human serum SSc1 at a dilution of 1:5000 in incubation buffer followed by staining with the secondary the secondary goat anti-human IgG(H+L) conj. DyLight680 antibody and read-out at a scanning intensity of 7. The CENPA-derived peptides were placed in the top three rows of double spots, the KDM6B-derived peptides covered the rest of the peptide microarray. In both antigens, we observed strong and well-defined interactions with some clear epitope-like spot patterns formed by rows of neighbored peptides with a consensus motif. The final staining of the HA and Flag control peptides framing the peptide arrays gave rise to the expected and well-defined spot pattern and validated the overall peptide microarray integrity.
7 SSc1, Intensity Plot CENPA KDM6B Data quantification was followed by generation of peptide and intensity maps as well as of an intensity plot with the CENPA peptides on the left and the KDM6B peptides on the right of the plot. We observed a very strong interaction with some CENPA peptides as well as moderate to strong interactions with some KDM6B epitopes.
8 RRRSRKPEAPRR RSPSPTPTPGPSR SSc1, CENPA Intensity Plot GPSRRGPSLGAS The intensity plot of the CENPA peptides highlighted the very strong response of serum SSc1 and excellent signal to noise ratios. We observed at least three different epitopes close to the N-terminus of CENPA; a third epitope could be overlaid by the two main epitopes RSPSPTPTPGPSR and GPSRRGPSLGAS. A weak epitope, albeit still at moderate spot intensities, was based on peptides with the consensus motif RRRSRKPEAPRR.
9 LPGTTTSSSSSS LPAPLPPSHGSS SSQFSTSGGPWAR REKLNPPTPSIYL SSc1, KDM6B Intensity Plot PQPSASSSSQF The intensity plot of the KDM6B peptides showed a moderate to strong response of serum SSc1 against 5 main epitopes. From the N- to the C-terminus, the corresponding peptides were based on consensus motifs LPAPLPPSHGSS, LPGTTTSSSSSS, PQPSASSSSQF, SSQFSTSGGPWAR and REKLNPPTPSIYL. The slightly blurry spot morphologies of peptides with the consensus motif LPGTTTSSSSSS and the weaker spot intensities may hint at a non-specific contribution to binding of serum antibodies.
10 SSc2, Microarray Scans SSc2, 1:5000 Control Staining One of the CENPA/KDM6B peptide microarrays was incubated with human serum SSc2 at a dilution of 1:5000 in incubation buffer followed by staining with the secondary the secondary goat anti-human IgG(H+L) conj. DyLight680 antibody and read-out at a scanning intensity of 7. The CENPA-derived peptides were placed in the top three rows of double spots, the KDM6B-derived peptides covered the rest of the peptide microarray. In both antigens, we observed strong and well-defined interactions with some clear epitope-like spot patterns and a pronounced similarity to serum SSc1. The final staining of the HA and Flag control peptides framing the peptide arrays gave rise to the expected and well-defined spot pattern and validated the overall peptide microarray integrity.
11 SSc2, Intensity Plot CENPA KDM6B Data quantification was followed by generation of peptide and intensity maps as well as of an intensity plot with the CENPA peptides on left and the KDM6B peptides on right of the plot. We observed a very strong interaction with some CENPA peptides as well as two clear interactions with KDM6B-dervied peptides at moderate spot intensities.
12 TPTPGPSRRGPS SSc2, CENPA Intensity Plot RSPSPTPTPGPSR GPSRRGPSLGAS The intensity plot of the CENPA peptides highlighted the very strong response of serum SSc2 with excellent signal to noise ratios. We observed at least two different epitopes close to the N-terminus of CENPA; a third possible epitope like TPTPGPSRRGPS could be overlaid by the two main epitopes RSPSPTPTPGPSR and GPSRRGPSLGAS that were also identified in serum SSc1.
13 SSc2, KDM6B Intensity Plot SPQPSASSSSQF PAPLPPSHGSS YQYQLALERYEWNEV The intensity plot of the KDM6B peptides showed a moderate to strong response of serum SSc2 against two main epitopes. From the N- to the C-terminus, the corresponding peptides were based on consensus motifs PAPLPPSHGSS and SPQPSASSSSQF that were also identified in serum SSc1. A rather uncommon single peptide interaction based on peptide YQYQLALERYEWNEV was found close to the C-terminus of KDM6B.
14 SSc3, Microarray Scans SSc3, 1:5000 Control Staining One of the CENPA/KDM6B peptide microarrays was incubated with human serum SSc3 at a dilution of 1:5000 in incubation buffer followed by staining with the secondary the secondary goat anti-human IgG(H+L) conj. DyLight680 antibody and read-out at a scanning intensity of 7. The CENPA-derived peptides were placed in the top three rows of double spots, the KDM6B-derived peptides covered the rest of the peptide microarray. Only in antigen CENPA we observed a strong and well-defined interaction with clear epitope-like spot patterns. In antigen KDM6B, however, some weaker responses also hinted at autoantibodies against the new putative SSc antigen KDM6B. The final staining of the HA and Flag control peptides framing the peptide arrays gave rise to the expected and well-defined spot pattern and validated the overall peptide microarray integrity.
15 SSc3, Intensity Plot CENPA KDM6B Data quantification was followed by generation of peptide and intensity maps as well as of an intensity plot with the CENPA peptides on the left and the KDM6B peptides on the right of the plot. We observed a very strong interaction with some CENPA peptides as well as one clear interactions with KDM6B-dervied peptides at moderate spot intensities.
16 TPTPGPSRRGPS SSc3, CENPA Intensity Plot SPSPTPTPGPSR GPSRRGPSLGAS The intensity plot of the CENPA peptides highlighted the very strong response of serum SSc3 with excellent signal to noise ratios. We observed at least two different epitopes close to the N-terminus of CENPA; a third possible epitope like TPTPGPSRRGPS could be overlaid by the two main epitopes SPSPTPTPGPSR and GPSRRGPSLGAS that were also identified in sera SSc1 and SSc2.
17 WARERRAGEEPV SSc3, KDM6B Intensity Plot QPPLPAPLPPSHGSS The intensity plot of the KDM6B peptides showed a moderate response of serum SSc3 against one main epitope. The corresponding interaction was based on peptide QPPLPAPLPPSHGSS similar to epitope PAPLPPSHGSS identified in sera SSc1 and SSc2. A second very weak interaction was observed for peptides with the consensus motif WARERRAGEEPV that was not identified in sera SSc1 and SSc2.
18 HC, Microarray Scans HC, 1:5000 Control Staining One of the CENPA/KDM6B peptide microarrays was incubated with human serum HC at a dilution of 1:5000 in incubation buffer followed by staining with the secondary the secondary goat anti-human IgG(H+L) conj. DyLight680 antibody and read-out at a scanning intensity of 7. The CENPA-derived peptides were placed in the top three rows of double spots, the KDM6B-derived peptides covered the rest of the peptide microarray. At a dilution of , we did not observe any remarkable interaction above the microarray background. The few hardly visible spot features exhibited very weak intensities and blurry spot morphologies that are indicative for non-specific interactions. The final staining of the HA and Flag control peptides framing the peptide arrays gave rise to the expected and well-defined spot pattern and validated the overall peptide microarray integrity.
19 HC, Intensity Plot CENPA KDM6B Data quantification was followed by generation of peptide and intensity maps as well as of an intensity plot with the CENPA peptides on the left and the KDM6B peptides on the right of the plot. We only observed few very weak hardly visible interactions with single KDM6B peptides that likely resulted from non-specific binding of serum antibodies. Please note that the scale of the y-axis was adjusted to the lower spot intensities.
20 HC, CENPA Intensity Plot The intensity plot of the CENPA peptides did not show any antibody response of control serum HC.
21 HC, KDM6B Intensity Plot The intensity plot of the KDM6B peptides showed very weak signals of serum HC against single peptides CSVNINIGPGDCEWF, DYLTGSWWPILDDLY and YQYQLALERYEWNEV. Due to the blurry spot morphologies along with the very low spot intensities, however, all interactions were regarded as non-specific.
22 Data Comparison CENPA KDM6B To compare antibody responses of all serum samples, we combined the intensity plots of all assays and leveled them to provide a clearer data overview. We observed a pronounced similarity of sera SSc1, SSc2 and SSc3 with a very strong response against the CENPA-derived peptides and a moderate to strong response against the KDM6B peptides. Serum HC was obviously not reactive against one of the antigens.
23 RSPSPTPTPGPSR TPTPGPSRRGPS Data Comparison CENPA Peptides SPSPTPTPGPSR GPSRRGPSLGAS The intensity plots of the CENPA peptides highlighted the very strong and virtually identical response of sera SSc1, SSc2 and SSc3 with the two main epitopes SPSPTPTPGPSR and GPSRRGPSLGAS and a possible third but overlaid epitope TPTPGPSRRGPS. Serum SSc1 showed a slight difference in the intensity ratio of both main epitopes and gave rise to an additional N-terminal epitope RSPSPTPTPGPSR.
24 LPGTTTSSSSSS SSQFSTSGGPWAR REKLNPPTPSIYL SPQPSASSSSQF Data Comparison KDM6B Peptides QPPLPAPLPPSHGSS WARERRAGEEPV PAPLPPSHGSS YQYQLALERYEWNEV LPAPLPPSHGSS The intensity plots of the KDM6B peptides highlighted the moderate to strong and partly similar response of sera SSc1, SSc2 and SSc3 with the main epitopes LPAPLPPSHGSS and SPQPSASSSSQF. While SPQPSASSSSQF was only observed in sera SSc1 and SSc2, epitope LPAPLPPSHGSS was common in all positive serum samples. Other epitopes like REKLNPPTPSIYL or SSQFSTSGGPWAR were only identified in one of the samples.
25 Conclusion The PEPperMAP Type 1 Epitope Mappings of human sera SSc1, SSc2, SSc3 and HC were done against antigens CENPA and KDM6B that were translated into 15 aa peptides with peptide-peptide overlaps of 14 aa. The peptide microarrays with the antigen-derived peptides were incubated with the human sera at a dilution of 1:5000 followed by staining with the secondary goat anti-human IgG(H+L) conj. DyLight680 antibody and read-out with a LI-COR Odyssey Imaging System. Quantification of spot intensities and peptide annotation were done with PepSlide Analyzer. Sera SSc1, SSc2 and SSc3 showed a strong response with excellent signal-to-noise ratios against epitopes in both antigens. Two CENPA epitopes SPSPTPTPGPSR and GPSRRGPSLGAS as well as KDM6B epitope LPAPLPPSHGSS were observed in all three serum samples as common epitopes. Some other epitopes like RSPSPTPTPGPSR (SSc1) in CENPA or SPQPSASSSSQF (SSc1 and SSc2) were only identified in one or two of the positive sera. In contrast to the excellent signal-tonoise ratios of the positive samples, serum HC only showed a slight background with few hardly visible single peptide interactions that most likely resulted from non-specific interactions with serum antibodies. In accordance with the study of Muro et al., 1 we observed a clear and very strong autoantibody response against the N-terminus of CENPA. Due to the high epitope resolution, however, we were able to identify at least two neighbored but different epitopes instead of the reported single epitope PRRRS, what underlined the benefit of high resolution PEPperMAP Type 1 Epitope Mappings. For further validation, the investigation of sequence conservation and the determination of the actual epitope length we recommend an in-depth analysis of some of the suggested epitopes by a PEPperMAP Full Substitution Scan with all amino acids at all amino acid positions. On request PEPperPRINT can also provide HPLC purified peptides for competition assays and affinity purification.
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Aviva Custom Antibody Service and Price Mouse Monoclonal Antibody Service Package Number Description Package Contents Time Price Customer provides antigen protein $6,174 Monoclonal package1 (From protein
User Manual Ordering information: (for detailed kit content see Table 2) EdU Flow Cytometry Kits for 50 assays: Product number EdU Used fluorescent dye BCK-FC488-50 10 mg 6-FAM Azide BCK-FC555-50 10 mg
A Positive ANA Test. A Positive ANA test. Lupus Society of Illinois 525 W. Monroe Street, Suite 900 Chicago, Illinois 60661 Robert S. Katz, M.D. Professor of Medicine Rush University Medical Center Northwestern