research poster biomarkers lupus 48x36 (5)
1
Methods The comparison data for three of the six lupus nephritis panel analytes (MCP-1, NGAL, and adiponectin) is shown below. Samples were run both on ELISA and Luminex simplex assays to determine how well the ELISA data correlated with Luminex data prior to assembling the analytes into a panel. Pearson correlation coefficients were 0.88 (p=0.04) for NGAL, 0.89 (p<0.00001) for MCP-1 and 1.0 (p<0.00001) for adiponectin. • The levels of the renal biomarkers are measured by Luminex Magpix assays and compared to ELISA data for 16 randomly chosen samples. • The multiplex assay we generated was compared to commercial ELISA kits for human NGAL (BioPorto Diagnostics, Denmark), human MCP-1, and human adiponectin (the latter two from R&D Systems, Minneapolis, MN). • Initially all kits were used according to their individual manufacturers recommendations. NGAL and MCP-1 bead sets continue to follow the manufacturers protocol; however, due to dilution constraints, we used a different dilution factor as our standard for adiponectin, keeping the other two assays regular standard curve intact. • Data from these were compared to the data generated from the multiplex on the same day. To assemble the three bead sets into one assay, we used the ProcartaPlex Human Basic Kit (eBioscience, Vienna, Austria) for the liquid medium. • ELISAs were compared to these singleplex magnetic bead assays, prior to combining the assays into a triplex. • Data was analyzed using Spearman rank order. Potential Biomarkers for Predicting Lupus Nephritis Activity in Children OBJECTIVE. The purpose of this study is to compare the results of multiplex assays to published results of enzyme-linked immunosorbent assays (ELISAs) for a panel of six biomarkers, hemopexin, ceruloplasmin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein-1 (MCP-1), and adiponectin. Previous studies have identified these biomarkers to have the potential to predict lupus nephritis activity. This study aims to develop multiplex assays for established panels of these SLE nephritis biomarkers. METHODS. The levels of the renal biomarkers are measured by multiplex assays and compared to published ELISA data. This was initially done by comparing magnetic bead assays with ELISA data from the same day. Magnetic bead assays were combined with minimal changes to manufacturers recommendations for further comparison with the corresponding ELISAs. RESULTS. The data for three of the six analytes shows that there was not a significant difference between ELISA data and the data collected from the multiplex assays for NGAL, MCP-1, and adiponectin (Pearson correlation coefficients were 0.88 (p=0.04) for NGAL, 0.89 (p<0.00001) for MCP-1 and 1.0 (p<0.00001) for adiponectin.) CONCLUSION. This allows us to begin creating an established multiplex panel, to which we can compare future samples. This will make the process of determining biomarker levels faster and more cost effective, so that we can better predict lupus activity. As we continue with this study, we will test the remaining three analytes. Although a promising start, further research with a larger sample size is required to validate the Luminex multiplex technology. Results Discussion References Introduction Caroline B. Christian, Christopher Haffner, Qing Ma, Kasha Wiley, Michael R. Bennett, Hermine I. Brunner Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine Singleplex vs. ELISA 1. Brunner, Hermine I., Michelle Mueller, Cynthia Rutherford, Murray H. Passo, David Witte, Alexei Grom, Jaya Mishra, and Prasad Devarajan. "Urinary Neutrophil Gelatinase–associated Lipocalin as a Biomarker of Nephritis in Childhood-onset Systemic Lupus Erythematosus." Arthritis & Rheumatism Arthritis Rheum 54.8 (2006): 2577- 584. Web. 2. Suzuki, Michiko, Kristina Wiers, Elizabeth B. Brooks, Kenneth D. Greis, Kathleen Haines, Marisa S. Klein-Gitelman, Judyann Olson, Karen Onel, Kathleen M. O'neil, Earl D. Silverman, Lori Tucker, Jun Ying, Prasad Devarajan, and Hermine I. Brunner. "Initial Validation of a Novel Protein Biomarker Panel for Active Pediatric Lupus Nephritis." Pediatr Res Pediatric Research 65.5 (2009): 530-36. Web. 3. Brunner, Hermine I., Michael R. Bennett, Rina Mina, Michiko Suzuki, Michelle Petri, Adnan N. Kiani, Joshua Pendl, David Witte, Jun Ying, Brad H. Rovin, and Prasad Devarajan. "Association of Noninvasively Measured Renal Protein Biomarkers with Histologic Features of Lupus Nephritis." Arthritis & Rheumatism 64.8 (2012): 2687-697. Web. 4. Rovin, B. H., and X. Zhang. "Biomarkers for Lupus Nephritis: The Quest Continues." Clinical Journal of the American Society of Nephrology 4.11 (2009): 1858-865. Web. 5. Abulaban, Khalid M., and Hermine I. Brunner. "Biomarkers for Childhood-Onset Systemic Lupus Erythematosus." Curr Rheumatol Rep Current Rheumatology Reports 17.1 (2014): n. pag. Springer Link. Web. 6. Bennett, Michael R., Edward Nehus, Christopher Haffner, Qing Ma, and Prasad Devarajan. "Pediatric Reference Ranges for Acute Kidney Injury Biomarkers." Pediatric Nephrology Pediatr Nephrol 30.4 (2014): 677-85. SpringerLink. Web. 7. Dossus, Laure, Susen Becker, David Achaintre, Rudolf Kaaks, and Sabina Rinaldi. "Validity of Multiplex-based Assays for Cytokine Measurements in Serum and Plasma from “non-diseased” Subjects: Comparison with ELISA." Journal of Immunological Methods 350.1-2 (2009): 125-32. ScienceDirect. Web. 8. Dupont, Nefertiti C., Kehui Wang, Pathik D. Wadhwa, Jennifer F. Culhane, and Edward L. Nelson. "Validation and Comparison of Luminex Multiplex Cytokine Analysis Kits with ELISA: Determinations of a Panel of Nine Cytokines in Clinical Sample Culture Supernatants." Journal of Reproductive Immunology 66.2 (2005): 175-91. ScienceDirect. Web. • Systemic lupus erythematosus (SLE) affects approximately 1.5 million Americans. Many of these individuals develop kidney disease (lupus nephritis). • There is a known connection between lupus activity and renal function in patients with a lupus nephritis diagnosis. • The current biomarkers used to predict lupus activity and damage are expensive, invasive, and insensitive. • Previous research has identified six promising renal biomarkers, hemopexin, ceruloplasmin, NGAL, KIM-1, MCP-1, and adiponectin, which are being assembled into a multiplex assay. • Previous work has been done using ELISAs; however, the major disadvantages of the ELISAs are cost, time, and required sample volumes. • Multiplex assays minimize these factors by saving antibody, sample, and cost per analyte, and include a greater dynamic range. • By comparing the multiplex assays of MCP-1, NGAL, and adiponectin to ELISA data we are able to begin developing multiplex assays for established panels • Our data indicates that there was not a significant difference between the data from multiplex assays and ELISA methods described previously in publication • This information has the potential to simplify the process of testing samples so that we can better predict changes in lupus nephritis activity. • There are limitations of this study to consider when reviewing the data. 1. Human error may allow for slight variations in singleplex data. 2. Also, data was collected from a relatively small population, in which random chance is more likely to play a role. 3. The study collected information only from patients who were under the age of 18, so this information may not be consistent in adult populations. • Another key concern with working with multiplex bead assays is the possibility of the “matrix effect,” which is why we chose to use singleplex assays for our initial comparisons. • A multiplex composed of these three analytes has also been conducted; however, the results have not been compared to ELISA data from the same day. • As we continue with this study, we will examine hemopexin, ceruloplasmin, and KIM-1, by comparing them to the accepted ELISA data. • Future research we plan to conduct will include translational studies incorporating this new information after the completed assay is validated. • In conclusion, these results are promising for patients suffering from lupus nephritis, but further research is required to validate this technology and rule out chance factors. Abstract
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Transcript of research poster biomarkers lupus 48x36 (5)
Methods
The comparison data for three of the six lupus nephritis panel analytes (MCP-1, NGAL, and adiponectin) is shown below. Samples were run both on ELISA and Luminex simplex assays to determine how well the ELISA data correlated with Luminex data prior to assembling the analytes into a panel. Pearson correlation coefficients were 0.88 (p=0.04) for NGAL, 0.89 (p<0.00001) for MCP-1 and 1.0 (p<0.00001) for adiponectin.
• The levels of the renal biomarkers are measured by Luminex Magpix assays and compared to ELISA data for 16 randomly chosen samples.
• The multiplex assay we generated was compared to commercial ELISA kits for human NGAL (BioPorto Diagnostics, Denmark), human MCP-1, and human adiponectin (the latter two from R&D Systems, Minneapolis, MN).
• Initially all kits were used according to their individual manufacturers recommendations. NGAL and MCP-1 bead sets continue to follow the manufacturers protocol; however, due to dilution constraints, we used a different dilution factor as our standard for adiponectin, keeping the other two assays regular standard curve intact.
• Data from these were compared to the data generated from the multiplex on the same day. To assemble the three bead sets into one assay, we used the ProcartaPlex Human Basic Kit (eBioscience, Vienna, Austria) for the liquid medium.
• ELISAs were compared to these singleplex magnetic bead assays, prior to combining the assays into a triplex.
• Data was analyzed using Spearman rank order.
Potential Biomarkers for Predicting Lupus Nephritis Activity in Children
OBJECTIVE. The purpose of this study is to compare the results of multiplex assays to published results of enzyme-linked immunosorbent assays (ELISAs) for a panel of six biomarkers, hemopexin, ceruloplasmin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein-1 (MCP-1), and adiponectin. Previous studies have identified these biomarkers to have the potential to predict lupus nephritis activity. This study aims to develop multiplex assays for established panels of these SLE nephritis biomarkers.
METHODS. The levels of the renal biomarkers are
measured by multiplex assays and compared to published ELISA data. This was initially done by comparing magnetic bead assays with ELISA data from the same day. Magnetic bead assays were combined with minimal changes to manufacturers recommendations for further comparison with the corresponding ELISAs. RESULTS. The data for three of the six analytes shows that there was not a significant difference between ELISA data and the data collected from the multiplex assays for NGAL, MCP-1, and adiponectin (Pearson correlation coefficients were 0.88 (p=0.04) for NGAL, 0.89 (p<0.00001) for MCP-1 and 1.0 (p<0.00001) for adiponectin.) CONCLUSION. This allows us to begin creating an established multiplex panel, to which we can compare future samples. This will make the process of determining biomarker levels faster and more cost effective, so that we can better predict lupus activity. As we continue with this study, we will test the remaining three analytes. Although a promising start, further research with a larger sample size is required to validate the Luminex multiplex technology.
Results
Discussion
References
Introduction
Caroline B. Christian, Christopher Haffner, Qing Ma, Kasha Wiley, Michael R. Bennett, Hermine I. BrunnerCincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine
Singleplex vs. ELISA
1. Brunner, Hermine I., Michelle Mueller, Cynthia Rutherford, Murray H. Passo, David Witte, Alexei Grom, Jaya Mishra, and Prasad Devarajan. "Urinary Neutrophil Gelatinase–associated Lipocalin as a Biomarker of Nephritis in Childhood-onset Systemic Lupus Erythematosus." Arthritis & Rheumatism Arthritis Rheum 54.8 (2006): 2577-584. Web.2. Suzuki, Michiko, Kristina Wiers, Elizabeth B. Brooks, Kenneth D. Greis, Kathleen Haines, Marisa S. Klein-Gitelman, Judyann Olson, Karen Onel, Kathleen M. O'neil, Earl D. Silverman, Lori Tucker, Jun Ying, Prasad Devarajan, and Hermine I. Brunner. "Initial Validation of a Novel Protein Biomarker Panel for Active Pediatric Lupus Nephritis." Pediatr Res Pediatric Research 65.5 (2009): 530-36. Web.3. Brunner, Hermine I., Michael R. Bennett, Rina Mina, Michiko Suzuki, Michelle Petri, Adnan N. Kiani, Joshua Pendl, David Witte, Jun Ying, Brad H. Rovin, and Prasad Devarajan. "Association of Noninvasively Measured Renal Protein Biomarkers with Histologic Features of Lupus Nephritis." Arthritis & Rheumatism 64.8 (2012): 2687-697. Web.4. Rovin, B. H., and X. Zhang. "Biomarkers for Lupus Nephritis: The Quest Continues." Clinical Journal of the American Society of Nephrology 4.11 (2009): 1858-865. Web.5. Abulaban, Khalid M., and Hermine I. Brunner. "Biomarkers for Childhood-Onset Systemic Lupus Erythematosus." Curr Rheumatol Rep Current Rheumatology Reports 17.1 (2014): n. pag. Springer Link. Web.6. Bennett, Michael R., Edward Nehus, Christopher Haffner, Qing Ma, and Prasad Devarajan. "Pediatric Reference Ranges for Acute Kidney Injury Biomarkers." Pediatric Nephrology Pediatr Nephrol 30.4 (2014): 677-85. SpringerLink. Web.7. Dossus, Laure, Susen Becker, David Achaintre, Rudolf Kaaks, and Sabina Rinaldi. "Validity of Multiplex-based Assays for Cytokine Measurements in Serum and Plasma from “non-diseased” Subjects: Comparison with ELISA." Journal of Immunological Methods 350.1-2 (2009): 125-32. ScienceDirect. Web.8. Dupont, Nefertiti C., Kehui Wang, Pathik D. Wadhwa, Jennifer F. Culhane, and Edward L. Nelson. "Validation and Comparison of Luminex Multiplex Cytokine Analysis Kits with ELISA: Determinations of a Panel of Nine Cytokines in Clinical Sample Culture Supernatants." Journal of Reproductive Immunology 66.2 (2005): 175-91. ScienceDirect. Web.9. Elshal, Mohamed F., and J. Philip Mccoy. "Multiplex Bead Array Assays: Performance Evaluation and Comparison of Sensitivity to ELISA." Methods 38.4 (2006): 317-23. ScienceDirect. Web.
• Systemic lupus erythematosus (SLE) affects approximately 1.5 million Americans. Many of these individuals develop kidney disease (lupus nephritis).
• There is a known connection between lupus activity and renal function in patients with a lupus nephritis diagnosis.
• The current biomarkers used to predict lupus activity and damage are expensive, invasive, and insensitive.
• Previous research has identified six promising renal biomarkers, hemopexin, ceruloplasmin, NGAL, KIM-1, MCP-1, and adiponectin, which are being assembled into a multiplex assay.
• Previous work has been done using ELISAs; however, the major disadvantages of the ELISAs are cost, time, and required sample volumes.
• Multiplex assays minimize these factors by saving antibody, sample, and cost per analyte, and include a greater dynamic range.
• By comparing the multiplex assays of MCP-1, NGAL, and adiponectin to ELISA data we are able to begin developing multiplex assays for established panels of kidney disease markers.
• The expectation is that this will allow doctors to better predict lupus nephritis activity, and thus treat it more effectively.
• Our data indicates that there was not a significant difference between the data from multiplex assays and ELISA methods described previously in publication
• This information has the potential to simplify the process of testing samples so that we can better predict changes in lupus nephritis activity.
• There are limitations of this study to consider when reviewing the data.
1. Human error may allow for slight variations in singleplex data.2. Also, data was collected from a relatively small population, in
which random chance is more likely to play a role. 3. The study collected information only from patients who were
under the age of 18, so this information may not be consistent in adult populations.
• Another key concern with working withmultiplex bead assays is the possibility of the “matrix effect,” which is why we chose to use singleplex assays for our initial comparisons.• A multiplex composed of these three analytes has also been conducted; however,the results have not been compared to ELISA data from the same day. • As we continue with this study, we willexamine hemopexin, ceruloplasmin, and KIM-1, by comparing them to the acceptedELISA data. • Future research we plan to conduct will include translational studies incorporatingthis new information after the completed assay is validated. • In conclusion, these results are promisingfor patients suffering from lupus nephritis,but further research is required to validatethis technology and rule out chance factors.
Abstract
