Application Note Multiplex Cytokine Immunoassays using The A2®Multiplex IA Slide by Yuri Yamada Overview Cytokines are low molecular weight protein (8-30kDa) products of immune cells functioning as mediators and regulators of the immune processes. Cytokines are involved not only in regulating the immune system, causing inflammation, but also are involved in cell proliferation, cell differentiation and wound healing. The over production of cytokines can lead to respiratory obstruction and multiple organ failure. Chemokines are produced from macrophages, endothelial cells and T cells. They are members of the cytokine family of basic proteins that evolve their effect through interaction with the G protein- coupled receptor. Chemokines act specifically on the white corpuscle resulting in chemotaxis causing white corpuscles to then migrate into a cell gradient of the secreted chemotaxic factor. IL-6 is a lectin secreted from T- cell or macrophage and is involved in the control of humeral immunity. IL-6 is multifunction and involved in various physiology phenomena and pathogenic mechanisms of inflammation and autoimmune disorder. For example, the cytokine promotes B-cell differentiation into antibody-producing cells. IL-6 is responsible for the onset of G0 phase in hematopoietic stem cells, which increases blood platelet thrombocyte production. This leads to an increased population of renal mesanginal and myeloma cells. IL-6 acts upon hepatocytes, promoting acutephase protein production. The normal level of IL-6 in the body is under 4 pg/mL. Over production of IL-6 occurs in autoimmune disorders such as rheumatoid arthritis. 1|Page FGF-9 is member of the FGF Family and is a heparin-binding growth factor. The protein consists of 208 amino acid residues and contains a potential Nlinked glycosylation site enabling binding of glycan heparin sulfate. FGF-9 affects glial cells and astrocyte through FGF receptor. FGF-9 promotes osteoblast differentiation, as well as, the multiplication and activation of epithelial cell and endothelial cells. EGF protein has 53 amino acid residues and three intramolecular disulfide bonds. EGF acts upon the EGF receptor (EGFR) that in turn propagates enhanced expression of three major pathways: Ras/MAPK, PI3K/Akt and Jak/STAT pathways. The Ras/MAPK pathway is the main signaling pathway involved in cell proliferation and differential regulation. GDP-linked inactive RAS becomes GTP-linked active RAS by EGF stimulation. Active RAS activates RAF, which in turn phosphorylates, specific serine groups of MEK1/2. MEK1/2 phosphorylates Erk. Phosphorylated Erk promotes gene expression that is 2|Page involved in proliferation and differentiation. By these cascade reactions, signals are propagated from extracellular to nucleus. PI3K/Akt pathway involves phosphatidylinositol-3 kinase, PI3K,an enzyme that phosphorylates inositol phospholipids that are structural components of cell membranes. PI3K produce phosphatidylinositol-3, 4,5-trisphosphate (PIP3). The PIP3 cells phosphorylate Akt. The Jak/STAT pathway regulates proliferation, differentiation or expression of cells. EGF also controls pathogen resistance of numerous cytokine receptor systems. EGF therefore contributes to differentiation and multiplication of epithelial cells, endothelial cells and fibroblasts. EGF also promotes IFN-γproduction from spleen. IFN-γ activates β-cells and macrophage. Fibroblast Growth Factor Basic（FGF Basic） is one of 23 known members of the FGF Family having conserved sequence of 35%~60% of their amino acids in common. FGF- basic is a non-glycosylated heparin binding growth factor that is expressed in the brain, kidney, pituitary, retina, bone, testis, adrenal gland liver, monocyte, epithelial cells and endothelial cells. The FGF family plays a central role in prenatal development, postnatal growth and regeneration of a variety of tissues by promoting cellular differentiation and proliferation. FGFbasic induces differentiation and regeneration of nerve. FGF−Basic stimulates cells from mesoderm, neuroectodemal, ectoderm and endoderm. In vivo, FGF-Basic participates in the process of vascularization, healing of wounds, as well as, embryogenesis/differentiation. Heparin Binding-Epidermal Growth Factor (HB-EGF) is a member of the EGF family and is synthesized as a membrane-anchored mitogenic and chemotactic glycoprotein. HB-EGF has 3 forms: trans membrane HB-EGF (pro HB-EGF), soluble HB-EGF(s HB-EGF) that is released by protease cleavage at the surface, and the residual C-terminal fragment resulting from the cleavage (HB-EGF-CTF). These forms each have different bioactives: pro HB-EGF performs as an intracellular signaling protein between cells. sHB-EGF interacts with EGF Receptors such as ErbB1 or ErbB4 . These accelerate cell motility and proliferation of many cells such as vascular smooth muscle cell, fibroblast and epithelial cells. HB-EGF is expressed by many kinds of cell types such as epithelial cells, myocardial cell, vascular endothelial cell smooth muscle cell and macrophage. HB-EGF-CTF moves from the cellular membrane into the nucleus where it is involved in the control of gene expression of intracraine, a second messenger and transcription factor. HB-EGF when secreted into blood is involved in cell proliferation, differentiation and inflammatory reactions. It 3|Page is also associated with oncogenesis leading to an increase in carcinoma cell proliferation. Product Description Quantiscientifics offers the A2® Multiplex IA slide (cat.no.A21018) which is based upon the use of oligonucleotides (oligos) to link affinity ligands such as antibodies to the surface of a glass substrate. Capture oligos are first printed onto substrate in a 7X6 array format consisting of 14 unique sequences printed in triplicate. Each slide contains 8 sub arrays allowing the performance of an 8 point immunoassay. The slide can be mount in a micro well chamber such as the “press to seal” silicon isolator systems (Grace Bio). Data A2 Multiplex IA Slide ELISA 2500 QS1 IL6 MFI (PBXL) 2000 QS5 FGF-9 QS9 EGF QS7 FGF-basic 1500 QS17 HB-EGF 1000 500 0 1 10 100 Analyte [pg/mL] 1000 10000 Protocol for Slide Immunoassay Following blocking of the slide with a protein blocker, oligo antibody conjugates and reference biotin-oligonucleotide are mixed together. 60 μL of the mixture is dispensed to each well (2x4 wells on slide). The slide is incubated for 1hour 4|Page under hybridization conditions. The slide is then rinse 3 times using 200µL TBST (Tris Buffered-saline with 1% Tween 20) with a 5 minute soak on the final rinse. After hybridization, a mixture of antigens is serially diluted and applied to the wells and incubated for 1 hour. The slide is processed as before. A mixture of the biotin secondary antibodies, provided at 60 μL per each well is incubated for 30 minutes. Following rinse, 60 μL of SA-PBXL (Streptavidin Surelight P1, Columbia Biosciences) is added to each well and incubated for 1 hour. The rinsed slide is read and analyzed on the A2 MicroArray System. QS1 IL-6 ELISA Slide Assay 2000 MFI (PBXL) 1600 1200 800 R² = 0.9785 400 0 1 10 100 IL-6 (pg/mL) 1000 10000 Comparison of Slide vs Plate immunoassay The oligo-antibody conjugate and reference biotin–oligonucleotide were mixed together as previously described and applied to wells of the A2 Plate (cat. no. A21002), as well as, the A2 Multiplex IA slide (cat. no. A21018) and each incubated for 1 hour under hybridization conditions. All other conditions are as previously described. The rinsed plate and slide were then read and analyzed on the A2 MicroArray System (cat. no. A21001). 5|Page QS1 IL-6 ELISA: Slide vs Plate 1200 R² = 0.9736 1000 Plate 800 600 400 200 0 0 500 1000 1500 Slide 2000 2500 The above graph shows the correlation (R2=0.97) for human IL-6 micro ELISA performed using the A2 multiplex IA slide vs the A2 plate. Table 1. Assay Range (pg/mL) for Human Cytokines Analyte Experimental 1 Low2 High2 IL-6 9.3 – 1200 3.12 300 FGF-9 46.8 - 6000 62.5 4000 EGF 8.6 - 1100 3.9 250 FGF-basic 44 - 1200 10 640 HB-EFG 13.9 - 1783 31.2 2000 Range: slide assays reported here 1 Source2: http://www.rndsystems.com References http://www.rndsystems.com/Products/d6050/RelatedInformation http://www.copewithcytokines.de/cope.cgi?key=TNF-beta http://www.okayama-u.ac.jp/user/hos/kensa/hotai/ir-2r.htm http://www.srl.info/srlinfo/kensa_ref_CD/KENSA/SRL8208.htm http://www.rndsystems.com/Products/DEG00 6|Page http://www.geocities.jp/norihisamikami/GF.html http://www.ncbi.nlm.nih.gov/gene/2254 http://www.rndsystems.com/product_results.aspx?m=1436 http://mh.rgr.jp/memo/mz0144.htm http://ja.wikipedia.org/wiki/ http://www.peprotech.com/en-US/Pages/Product/Recombinant_Human_FGF-basic_(154_a.a.)/10018B http://kusuri-jouhou.com/immunity/cytokine.html http://www.pharm.or.jp/dictionary/wiki.cgi http://www.cstj.co.jp/reference/pathway/Jak_Stat_IL_6.php http://www.cstj.co.jp/reference/pathway/Akt_PKB.php http://www.medgen.med.tohoku.ac.jp/rasmapk_j/pathway/ http://tykerb.jp/info/04.php http://cell-biology.biken.osaka-u.ac.jp/MekadaLabHP/HB-EGF.htmlhttp://cell-biology.biken.osakau.ac.jp/MekadaLabHP/HB-EGF.html http://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100009017 http://cell-biology.biken.osaka-u.ac.jp/MekadaLabHP/Iwamoto/entori/2010/6/9_4_HBEGFha_zeng_zhi_yin_zika.html http://lifesciencedb.jp/dbsearch/Literature/get_pne_cgpdf.php?year=2009&number=5413&file=CPLUS gOThVWZN9b1STkxKXgXg http://cell-biology.biken.osaka-u.ac.jp/MekadaLabHP/HB-EGFandCancer.html http://tykerb.jp/info/04.php http://www.weblio.jp/content/インターフェロン-γ http://bsd.neuroinf.jp/wiki/シグナル伝達兼転写活性化因子 3 About the Author Yuri Yamada QS Marketing Intern, 2014 Yuri was born in Hokkaido, Japan. She received a bachelor’s degree in applied chemistry & biochemistry from Gunma University (Tokyo) in 2013. Her studies included the synthesis of a peptide potentially useful as a vaccine for Malaria. Yuri is currently enrolled in the Marketing Professional Certificate Program at the University of California, Irvine. 7|Page Product Information The A2®Multiplex IA Slide (Cat. No. A21018) comprises a glass microarray substrate with 8 sub-arrays. Each subarray contains a covalently linked set of 14 capture oligos represented in triplicate (42 spots) in a 7x6 pattern. How it works The slide is intended for use with complementary oligoantibody conjugates created using QuantiScientifics’ kits to tether up to 14 different capture antibodies per sub-array. A mixture of the prepared conjugates of the user’s choice is applied to the sub-arrays under hybridization conditions to create a multiplex immunoassay (IA) platform. Conventional immunoassays such as ELISA may then be performed and the slide subsequently read using a microarray scanner. Cat. No. Description A21018 A2 Multiplex IA Slide A21004 A2 Conjugation Kit A21005 A2 Activated Oligos A21014 A2 Direct Label Assay Kit Please visit our website for more information regarding our products and protocols. QuantiScientifics, LLC 2852 Alton Parkway, Irvine, CA 92606 www.quantiscientifics.com 8|Page