9607 Dr. Perry Road Suite 103. Ijamsville, MD Tel: (301) Fax: (301) Ultrasensitive Samoa Human IL-33 Immunoassay

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1 AssayGate, Inc Dr. Perry Road Suite 103. Ijamsville, MD Tel: (301) Fax: (301) Ultrasensitive Samoa Human IL-33 Immunoassay INTRODUCTION Interleukin-33 (IL-33), a cytokine belonging to the IL-1 superfamily, is a 30 kda pro-inflammatory protein that potently drives production of T helper-2 (Th2)- associated cytokines. IL-33 plays an important role in Th2-associated immune responses and cardiac pathology (1-3). IL-33 is expressed on a wide variety of cell types such as macrophages, mast cells, dendritic cells, fibroblasts, osteoblasts, endothelial cells, and epithelial cells (4). It is upregulated under inflammatory conditions (5). Full length IL-33 binds NFκB and inhibits NFκB transactivation (5-7). Mature IL-33, an kda C- terminal fragment is released from cleavage of full length IL-33 (8-9). Additional isoforms of human IL-33 are generated by alternative splicing (10-11). IL-33 is a ligand for IL33R (IL1RL1) that is highly expressed on Th2 cells, mast cells and group 2 innate lymphocytes (12). IL-33 mediates its biological effects by interacting with the receptors ST2 (also known as IL1RL1) and IL-1 Receptor Accessory Protein (IL1RAP), activating intracellular molecules in the NF-κB and MAP kinase signaling pathways that drive production of type 2 cytokines such as IL-5 and IL-13. IL-33 signaling through ST2 also triggers VE-Cadherin phosphorylation and internalization on vascular endothelial cells, which elicits increased vascular permeability, vessel sprouting and tubule formation (13). IL-33 induces the migration of helper T cells, mast cells, eosinophils, and basophils to inflammation sites and production type 2 cytokines (9, 14-16). By enhancing neutrophil sensitization to TLR and Dectin-1 signaling, phagocytic activity, and migration to sites of infection, IL-33 plays an important role in infection clearance (15, 17-18). IL-33 is also found at elevated levels in bronchoalveolar lavage from patients with idiopathic pulmonary fibrosis (19). IL-33 inhibits the development of atherosclerotic plaques and induces the production of anti-oxidized LDL antibodies (16). It can also enhance eosinophilic perimyocarditis and impair heart function (20). Clinically, serum IL-33 has been found at elevated levels in patients with heart failure, and is associated with 1

2 coronary in-stent restenosis and mortality in patients with ST elevation myocardial infarction (21-23). The AssayGate Human IL-33 Simoa Assay Kit is designed to detect and quantitate human IL-33 in serum, plasma and cell culture supernatants. PRINCIPLE OF THE ASSAY The AssayGate Human IL-33 Simoa Assay is a 2-hour ultrasensitive immunoassay using fully automated Simoa HD-1 Analyzer. A capture antibody specific for human IL-33 has been coupled to magnetic microspheres. Standards and samples are pipetted into the Simoa 96-well plate. After IL-33 is captured by the bead, a biotinylated detection antibody specific for IL-33 is introduced. Following a wash step to remove any unbound biotinylated antibody, beads are incubated with streptavidin beta galactosidase (SBG) Reagent. Again, following a wash step to remove any unbound SBG, beads are incubated with resorufin D galactopyranoside (RGP) Reagent. The mixture of incubated beads and RGP is then transferred into the sample wells on a Simoa Disc. Beads either settle into the wells, one bead per well, or rest on the surface of the array. The instrument moves the disc to the sealing oil station and the pump delivers oil into the disc inlet port. The oil seals beads into the microwells and flushes away loose beads. The instrument moves the disc to the imaging station, where a CCD camera images the sealed wells, capturing the signal emitted by fluorescing beads. The Simoa software analyzes the photographic image, determines the average enzymes/bead (AEB), generates a standard curve and calculate IL-33 concentrations in samples. ASSAY PERFORMANCE CHARACTERISTICS A representative standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed. 2

3 SENSITIVITY The analytical sensitivity or limit of detection (LOD) was determined as the value calculated from the standard curve at the point lying 2 standard deviations above the mean background (twenty zero standard replicates). The LOD of human IL-33 Simoa assay is pg/ml. LOWER LIMIT OF QUANTIFICATION The lower limit of quantification (LLOQ) in serum/plasma was determined using a 2-fold dilution series of the standards in standard diluent assayed in triplicate over three different runs, and is defined as the point at which the coefficient of variation (CV) for the measurement was 30%. The CV was calculated and plotted against concentration, and LLOQ was interpolated from this plot. The LLOQ of Human IL-33 Simoa assay is pg/ml. PRECISION 3

4 Intra-assay coefficient of variation (CV) was calculated from three EDTA plasma samples with known concentrations tested sixteen times on one plate. Inter-assay CV was calculated from three EDTA plasma serum samples with known concentrations tested in sixteen separate assays. Intra-Assay Precision Inter-Assay Precision Sample n Mean, pg/ml Standard deviation, pg/ml CV (%) RECOVERY The recovery of human IL-33 was determined by spiking four different levels of standards to appropriately diluted samples. Sample Type Average %Recovery Range Serum* (n=6) EDTA plasma* (n=6) *Samples were first diluted 1:4 in sample diluent and then spiked. LINEARITY Samples were spiked with high concentration of human IL-33 and diluted with sample diluent to produce samples with IL-33 concentrations within the dynamic range of the assay. Dilution Factor Serum EDTA plasma (n=6) 1 : 3 Average % of Expected (n=6) Range (%) : 9 Average % of Expected Range (%) : 27 Average % of Expected Range (%) : 81 Average % of Expected Range (%) : 243 Average % of Expected Range (%) SPECIFICITY The anti-human IL-33 antibody used in this kit is specific for human IL-33 protein. The following factors at 5 ng/ml were assayed and no cross-reactivity and interference were observed: 4

5 Recombinant human: pro-il-33 (aa 1-111), ST2/Fc Chimera Recombinant mouse: IL-33 REFERENCES 1. Gadina, M. and C.A. Jefferies (2007) Sci. STKE 390:pe Barksby, H.E. et al. (2007) Clin. Exp. Immunol. 149: Ohno, T. et al. (2012) Allergy 67: Mirchandani, A; Salmond, R; Liew, F (2012). Trends in Immunology 33 (8): Carriere, V. et al. (2007) Proc. Natl. Acad. Sci. USA 104: Baekkevold, E.S. et al. (2003) Am. J. Pathol. 163: Ali, S. et al. (2011) J. Immunol. 187: Talabot-Ayer, D. et al. (2009) J. Biol. Chem. 284: Schmitz, J. et al. (2005) Immunity 23: Tsuda, H. et al. (2012) J. Invest. Dermatol. 132: Hong, J. et al. (2011) J. Biol. Chem. 286: Yagami, A. et al. (2010). J. Immunol. 185 (10): Choi, Y.S. et al. (2009) Blood 114: Allakhverdi, Z. et al. (2007) J. Immunol. 179: Humphreys, N.E. et al. (2008) J. Immunol. 180: Miller, A.M. et al. (2008) J. Exp. Med. 205: Le, H.T. et al. (2012) J. Immunol. 189: Alves-Filho, J.C. et al. (2010) Nat. Med. 16: Luzina, I.G. et al. (2012) J. Immunol. 189: Abston, E.D. et al. (2012) Circ. Heart Fail. 5: Zhang, H.F., et al. (2012) J. Transl. Med. 10: Demyanets, S. et al. (2014) Cytokine. 67: Demyanets, S. et al. (2014) PLoS One. 9:e