Development and validation of panoptic Meso scale discovery assay to quantify total systemic interleukin-6

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.

Keywords: cancer; interleukin-6; panoptic assay; rheumatoid arthritis.

Figure 1

Capture antibodies screened for IL-6 capture in combination with four different detection antibodies. Each candidate antibody was used as a capture antibody to assess 125 pg ml^–1 human recombinant IL-6. Detection antibodies from commercially available ELISA kits were used to determine which antibody pair would result in the best detection of IL-6 in human serum. The approach was repeated for human plasma samples, with similar results (data not shown). IL-6, interleukin 6; ELISA, enzyme-linked immunosorbent assay; OD, optical density; mAb, monoclonal antibody; det Ab, detection antibody; Det Ab-1, Det Ab-2, Det Ab-3, Det Ab-4

Figure 2

Comparison of capture antibodies in the second round of screening. Capture antibodies that passed the broad first round screening were further evaluated in standard ELISA format. Thermo-Pierce 5IL6 (mAb-12; circled) consistently emerged as a superior capture antibody and was included in all ELISAs to normalize ELISA plate runs. ELISA, enzyme-linked immunosorbent assay; OD, optical density; mAb, monoclonal antibody

Figure 3

Comparison of top-performing detection and capture antibodies. (A) Four detection antibodies from commercial IL-6 kits were screened and compared for their ability to recognize human recombinant IL-6. Thermo-Pierce 5IL6 was used as a capture antibody. Invitrogen detection antibody (505E23C7) was selected as the detection antibody for future screenings. (B) The three top-performing capture monoclonal anti-IL-6 antibodies (Thermo-Pierce 5IL6, Ebiosciences 16YOR5/66 and Epitomics EBI-R14-19) were compared in a single analysis. The detection antibody was Invitrogen ELISA detection antibody 505E23C7. IL-6, interleukin 6; hIL6, human interleukin 6; ELISA, enzyme-linked immunosorbent assay; OD, optical density; det Ab, detection antibody, R&D det mAb, Thermo-Pierce det mAb, PeproTech det mAb, Invitrogen det mAb, Thermo-Pierce 5IL6, Ebiosciences 16YOR5/66, Epitomics EBI-R14-19

Figure 4

Final antibody pair selection for determining the best pair that gives the maximum spread of values. Capture/detection antibody pairs evaluated shown respectively: (A) CNTO328/Invitrogen 505E23C7, (B) Thermo-Pierce 5IL6/Invitrogen 505E23C7, (C) Epitomics EBI-R14-19/Invitrogen 505E23C7, (D) MSD IL-6 kit assay and (E) MSD IL-6 kit assay with the Humanzyme IL-6 standard. Solid line indicates LLOQ at 9.77 pg ml^–1 and dotted line indicates LLOQ at 1.22 pg ml^–1. IL-6, interleukin 6; HS, human serum; RA, rheumatoid arthritis; MSD, Meso Scale Discovery; LLOQ, lower limit of quantification

Figure 5

IL-6 concentrations in normal and disease sera samples as measured by panoptic IL-6, MSD assay; IL-6, interleukin 6; MSD, Meso Scale Discovery; HS, human serum; RA, rheumatoid arthritis

Figure 6

Superdex 200 gel filtration and immunologic characterization of IL-6 in serum from patients with RA (n = 7) and patients with MDS (n = 6). (A) IL-6 concentrations of each fraction assayed using commercial MSD assay and (B) IL-6 concentrations of each fraction assayed using the panoptic IL-6 assay. IL-6, interleukin 6; MDS, myelodysplastic syndrome; MSD, Meso Scale Discovery; SEC, size exclusion column, RA 1, RA 2, RA 3, RA 4, RA 5, RA 6, RA 7, MDS 1, MDS 2, MDS 3, MDS 4, MDS 5, MDS 6