S100A8 ELISA Kit (Bovine)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level S100 Calcium Binding Protein A8 (S100A8) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level S100 Calcium Binding Protein A8 (S100A8) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

S100A8 is a calcium- and zinc-binding protein which plays a prominent role in the regulation of inflammatory processes and immune response. It can induce neutrophil chemotaxis and adhesion. Predominantly found as calprotectin (S100A8/A9) which has a wide plethora of intra- and extracellular functions. The intracellular functions include: facilitating leukocyte arachidonic acid trafficking and metabolism, modulation of the tubulin-dependent cytoskeleton during migration of phagocytes and activation of the neutrophilic NADPH-oxidase. Activates NADPH-oxidase by facilitating the enzyme complex assembly at the cell membrane, transferring arachidonic acid, an essential cofactor, to the enzyme complex and S100A8 contributes to the enzyme assembly by directly binding to NCF2/P67PHOX. The extracellular functions involve proinflammatory, antimicrobial, oxidant-scavenging and apoptosis-inducing activities. Its proinflammatory activity includes recruitment of leukocytes, promotion of cytokine and chemokine production, and regulation of leukocyte adhesion and migration. Acts as an alarmin or a danger associated molecular pattern (DAMP) molecule and stimulates innate immune cells via binding to pattern recognition receptors such as Toll-like receptor 4 (TLR4) and receptor for advanced glycation endproducts (AGER) . Binding to TLR4 and AGER activates the MAP-kinase and NF-kappa-B signaling pathways resulting in the amplification of the proinflammatory cascade. Has antimicrobial activity towards bacteria and fungi and exerts its antimicrobial activity probably via chelation of Zn (2+) which is essential for microbial growth. Can induce cell death via autophagy and apoptosis and this occurs through the cross-talk of mitochondria and lysosomes via reactive oxygen species (ROS) and the process involves BNIP3. Can regulate neutrophil number and apoptosis by an anti-apoptotic effect; regulates cell survival via ITGAM/ITGB and TLR4 and a signaling mechanism involving MEK-ERK. Its role as an oxidant scavenger has a protective role in preventing exaggerated tissue damage by scavenging oxidants (By similarity) . The iNOS-S100A8/A9 transnitrosylase complex is proposed to direct selective inflammatory stimulus-dependent S-nitrosylation of multiple targets such as GAPDH, ANXA5, EZR, MSN and VIM by recognizing a [IL]-x-C-x-x-[DE] motif; S100A8 seems to contribute to S-nitrosylation site selectivity (By similarity) .

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to S100 Calcium Binding Protein A8 (S100A8) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to S100 Calcium Binding Protein A8 (S100A8) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain S100 Calcium Binding Protein A8 (S100A8), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of S100 Calcium Binding Protein A8 (S100A8) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery range (%) Average (%) Serum (n=5) 81-92 84 EDTA Plasma (n=5) 85-95 92 Heparin Plasma (n=5) 98-105 102 Matrix Recovery range (%) Average (%) Matrix Recovery range (%) Average (%) Serum (n=5) 81-92 84 Serum (n=5) 81-92 84 EDTA Plasma (n=5) 85-95 92 EDTA Plasma (n=5) 85-95 92 Heparin Plasma (n=5) 98-105 102 Heparin Plasma (n=5) 98-105 102

Component Amount Anti-S100A8 Microplate 96 Wells (12 x 8 Well strips) S100A8 Lyophilized Standard 2 100X Biotinylated S100A8 Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL Component Amount Component Amount Anti-S100A8 Microplate 96 Wells (12 x 8 Well strips) Anti-S100A8 Microplate 96 Wells (12 x 8 Well strips) S100A8 Lyophilized Standard 2 S100A8 Lyophilized Standard 2 100X Biotinylated S100A8 Detector Antibody 1 x 120 uL 100X Biotinylated S100A8 Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL

This assay has high sensitivity and excellent specificity for detection of S100 Calcium Binding Protein A8 (S100A8) . No significant cross-reactivity or interference between S100 Calcium Binding Protein A8 (S100A8) and analogues was observed.