Shalini Chaturvedi1, Derick Siegel2, Carrie L Wagner3, Jaehong Park1, Helgi van de Velde4, Jessica Vermeulen5, Man-Cheong Fung6, Manjula Reddy1, Brett Hall1,7, Kate Sasser1. 1. Oncology, Translational Research, Janssen Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania. 2. Oncology, Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania. 3. Immunology Development, Janssen Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania, USA. 4. Oncology Development, Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium. 5. Oncology Development, Janssen Pharmaceutical Companies of Johnson & Johnson, Leiden, the Netherlands. 6. Oncology, Janssen Pharmaceutical Companies of Johnson & Johnson, Raritan, New Jersey. 7. Current address: MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, USA.
Abstract
AIM: Interleukin-6 (IL-6), a multifunctional cytokine, exists in several forms ranging from a low molecular weight (MW 20-30 kDa) non-complexed form to high MW (200-450 kDa), complexes. Accurate baseline IL-6 assessment is pivotal to understand clinical responses to IL-6-targeted treatments. Existing assays measure only the low MW, non-complexed IL-6 form. The present work aimed to develop a validated assay to measure accurately total IL-6 (complexed and non-complexed) in serum or plasma as matrix in a high throughput and easily standardized format for clinical testing. METHODS: Commercial capture and detection antibodies were screened against humanized IL-6 and evaluated in an enzyme-linked immunosorbent assay format. The best antibody combinations were screened to identify an antibody pair that gave minimum background and maximum recovery of IL-6 in the presence of 100% serum matrix. A plate-based total IL-6 assay was developed and transferred to the Meso Scale Discovery (MSD) platform for large scale clinical testing. RESULTS: The top-performing antibody pair from 36 capture and four detection candidates was validated on the MSD platform. The lower limit of quantification in human serum samples (n = 6) was 9.77 pg l(-1) , recovery ranged from 93.13-113.27%, the overall pooled coefficients of variation were 20.12% (inter-assay) and 8.67% (intra-assay). High MW forms of IL-6, in size fractionated serum samples from myelodysplastic syndrome and rheumatoid arthritis patients, were detected by the assay but not by a commercial kit. CONCLUSION: This novel panoptic (sees all forms) IL-6 MSD assay that measures both high and low MW forms may have clinical utility.
AIM: Interleukin-6 (IL-6), a multifunctional cytokine, exists in several forms ranging from a low molecular weight (MW 20-30 kDa) non-complexed form to high MW (200-450 kDa), complexes. Accurate baseline IL-6 assessment is pivotal to understand clinical responses to IL-6-targeted treatments. Existing assays measure only the low MW, non-complexed IL-6 form. The present work aimed to develop a validated assay to measure accurately total IL-6 (complexed and non-complexed) in serum or plasma as matrix in a high throughput and easily standardized format for clinical testing. METHODS: Commercial capture and detection antibodies were screened against humanized IL-6 and evaluated in an enzyme-linked immunosorbent assay format. The best antibody combinations were screened to identify an antibody pair that gave minimum background and maximum recovery of IL-6 in the presence of 100% serum matrix. A plate-based total IL-6 assay was developed and transferred to the Meso Scale Discovery (MSD) platform for large scale clinical testing. RESULTS: The top-performing antibody pair from 36 capture and four detection candidates was validated on the MSD platform. The lower limit of quantification in human serum samples (n = 6) was 9.77 pg l(-1) , recovery ranged from 93.13-113.27%, the overall pooled coefficients of variation were 20.12% (inter-assay) and 8.67% (intra-assay). High MW forms of IL-6, in size fractionated serum samples from myelodysplastic syndrome and rheumatoid arthritispatients, were detected by the assay but not by a commercial kit. CONCLUSION: This novel panoptic (sees all forms) IL-6MSD assay that measures both high and low MW forms may have clinical utility.
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