CANVAX HUMAN IL-6 ELISA Manual for cat. nº : Upon Receipt Store Kits at +4ºC PRODUCT MANUAL Version 3.0 Last updated: July 2012 www.canvaxbiotech.com 1 Canvax Citokine ELISA: Better Tools for the Best Science 3. 4. 5. 6. 7. 8. 9. 10. ....................................................................................................................... 3 PROTEIN STRUCTURE ........................................................................................................................... 4 GENE ................................................................................................................................................ 4 CLINICAL SIGNIFICANCE......................................................................................................................... 4 STANDARD RANGE ............................................................................................................................... 4 SENSITIVITY ........................................................................................................................................ 4 ACCURACY ......................................................................................................................................... 4 SPIKE RECOVERY.................................................................................................................................. 4 PRECISION ......................................................................................................................................... 5 2. MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION DATE ...........................................5 11. 12. OTHER SUPPLIES REQUIRED ................................................................................................................... 5 STORAGE TEMPERATURE AND PREPARATION OF WORKING SOLUTIONS ........................................................... 6 3. DETAILED PROTOCOL .......................................................................................................8 4. PLATE LAYOUT ...............................................................................................................10 5. PRODUCT USE LIMITATION............................................................................................. 11 Table of contents TABLE OF CONTENTS............................................................................................................2 1. INTRODUCTION ...............................................................................................................3 1. 2. PRINCIPLE .......................................................................................................................................... 3 HUMAN INTERLEUKIN- 2 Canvax Citokine ELISA: Better Tools for the Best Science - 1. INTRODUCTION proliferation of thymocytes. It is responsible the production of neutrophils in the bone marrow. IL-6 plays an essential role in the final differentiation of B-cells into Ig-secreting cells and induces the in people with autoimmune diseases or infections. addition, the encoded protein has been shown to be an endogenous pyrogen capable of inducing fever secreted into the serum and acts both as a pro-inflammatory and anti-inflammatory cytokine. In primarily produced at sites of acute and chronic inflammation, during an infection or after a trauma. It is osteoblasts, mast cells, glial cells, and keratinocytes also produce IL6 after stimulation. The protein is Macrophages, T-cells and B-lympho-cytes, granulocytes, smooth muscle cells, eosinophils, chondrocytes, The main IL6 cell producers in vivo are stimulated monocytes, fibroblasts, and endothelial cells. physiological mediators of acute phase reaction. influencing antigen-specific immune responses and inflammatory reactions. It is one of the major Hybridoma growth factor (HPGF), CTL differentiation factor (CDF). IL6 is a pleiotropic cytokine has been described: B cell differentiation factor (BCDF), HSF, MGI-2, B-cell stimulatory factor 2 (BSF-2), The plethora of biological activities of IL-6 is exemplified by the many different acronyms under which it 1. Principle CvxBio Human IL-6 ELISA kit is a sandwich ELISA for the quantitative measurement of human IL-6 in cell culture supernatant, serum, plasma and urine. This assay employs an antibody against human IL-6 coated on a 96-well plate. Once IL-6 or standards are bound to the immobilized antibody, the wells are washed and biotinylated anti-human IL-6 antibody is added. The plates are revealed by the reaction of the peroxidase of the HRP-streptavidin conjugated. 2. 3 Canvax Citokine ELISA: Better Tools for the Best Science 3. Protein Structure IL6 is a protein of 185 amino acids glycosylated at positions 73 and 172. It is synthesized as a precursor protein of 212 amino acids. Monocytes express at least five different molecular forms of IL6 with molecular masses of 21,5-28 kDa. They mainly differ by post-translational alterations such as glycosylation and phosphorylation. IL6 isolated from various cell types shows some microheterogeneity in its N-terminus. 4. Gene IL6 gene has a length of approximately 5 kb and contains five exons. It is on the p-arm of chromosome 7 (7p21) between the markers D7S135 and D7S370. The IL6 gene promoter contains many different regulatory elements allowing the induction of expression by various stimuli, including glucocorticoids and cAMP. The NF-kappa-B binding site is responsible in non-lymphoid cells for the induction of the IL6 gene expression by IL1 or TNF. In lymphoid cells, a factor related to the rel oncogene functions as a repressor that prevents the interaction of transcription factors with the IL6-kappa-B binding site. 5. Clinical significance • IL-6 is implicated in a wide variety of inflammation-associated disease states, including prostate cancer, systemic lupus erythematosus, rheumatoid arthritis, depression, diabetes mellitus, atherosclerosis, Alzhei-mer´s disease and systemic juvenile rheumatoid arthritis. • Determination of IL6 serum levels may be useful to monitor the activity of myelomas and to calculate tumour cell masses. • Inhibitors of IL-6 (including estrogen) are used to treat postmenopausal osteoporosis. 6. Standard range 10-3160 pg/ml. The standard range is the range in which determinations of analyte concentration can be done with precision, accuracy and linearity. 7. Sensitivity The limit of detection of this assay is 1 pg/ml. It is the lowest concentration that is possible to detect but not necessarily quantify with precision and accuracy. 8. Accuracy The standard of this ELISA has been calibrated against an international standard from NIBSC*. One ng of supplied standard equals 100 units of 89/548 NIBSCstandard. Please note that the calibration is batch specific. 9. Spike recovery 4 Canvax Citokine ELISA: Better Tools for the Best Science Addition of a specified amount of standard to a serum/plasma sample gives a mid-curve recovery of 79111% in repeated experiments. 10. Precision The intraassay variation is 2.5%(CV). The interassay variation is 3.8%(CV). *National Institute of Biological Standards and Control, Potters Bar, Hertfordshire EN6 2. MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION DATE CANVAX HUMAN IL-6 ELISAs Item Quantity 96- well plate coated with coating antibody and blocked non-specific binding sites Human IL-6 Standard protein lyophilised (100µl) 1 Plate Storage 4ºC 2 vial 4ºC Streptavidin -HRP (20µl) 1 vial 4ºC Detection Antibody, biotinylated 6 ml 4ºC Wash Buffer 20X Assay Buffer Substrate/TMB Solution Stop Solution Adhesive cover slip. Reagent Reservoirs 50ml 25 ml 12 ml 12 ml 2 1 4ºC 4ºC 4ºC 4ºC Expiration date: See on the kit label. We recommend to use opened kit components within one month. 11. Other supplies required • Microplate reader capable of measuring absorbance at 450 nm. • Pipettes and pipette tips. • Deionized or distilled water. • 500 mL graduated cylinder. • Squirt bottle, manifold dispenser, or automated microplate washer. • Test tubes for dilution of standards. 5 Canvax Citokine ELISA: Better Tools for the Best Science 12. Storage temperature and preparation of working solutions Store unopened kit at 4ºC until ready to use. After opening the kit and reconstituting the reagents, storage following the guidelines below. • 96-well plate is stable at 4ºC until kit expiration date. Allow the plates to adjust to room temperature (18-25°C) before opening the bags. Store the remaining strips in the foil bag containing the desiccant at 4-8°C. • Wash Buffer is stable at 4ºC until kit expiration date. Add 50ml Wash Buffer 5x to 950ml deionized water and mix. Store at 4ºC until kit expiration date. • Assay Buffer. This solution is ready for use. Storage at 4ºC until kit expiration date. • Cytokine Standard: On arrival all components of the kit, the protein standard, should be stored at at -20°C. Avoid repeated freeze-thaw cycles of the cytokine standard. Preparation of standard curve: Prepare a serial dilution of the standard no more than 30 min prior to the experiment. Dilute the standard stock solution to create a standard curve ranging from 3.16-10000 pg/ml according to the scheme below. For the assay background control (0 pg/ml), use only ”Assay Buffer”. Figure 1. Recommended serial dilution of cytokine standard. 6 Canvax Citokine ELISA: Better Tools for the Best Science • Detection Antibody is stable at 4ºC until kit expiration date. It is ready for use. Store at –20ºC as aliquots for ? Avoid freeze-thaw cycles. • Streptavidin Peroxidase stable at 4ºC until expiration date. Centrifuge spin before opening. Dilute Streptavidin-HRP 1:1000 with Assay Buffer just before use. This dilution must be used within 30min after preparation. • Substrate solution store at 4ºC. It is ready for use. • Stop solution store at 4ºC. It is ready for use. Avoid sodium azide in Buffers because is an inhibitor of peroxidase activity. 7 Canvax Citokine ELISA: Better Tools for the Best Science 3. DETAILED PROTOCOL 1. Add 0.3ml Wash Buffer 1x per well. Empty wells with aspiration or invert the plate and tap microwell strips on absorbent paper. 2. Use the microwell plate immediately after washing. 3. Prepare serial dilutions of Standard in Assay Diluent .Dispense 0.1ml dilutions of Standard in the appropriated wells. Include a blank. 4. Prepare desired dilutions of the samples in Assay Diluent. Dispense 0.1ml dilutions of the samples in the appropriated wells. 5. Cover plate with the adhesive cover slip and incubate for 1hour at 37ºC. 6. Wash plate three times with washing Buffer 1x as is indicated, step 1. 7. Transfer 0.1ml Detection Antibody to all wells. 8. Cover plate with the adhesive cover slip and incubate for 1 hour at 37ºC. 9. Wash plate three times with washing Buffer 1x as is indicated, step 1. 10.Transfer 0.1ml diluted Streptavidin Peroxidase into all wells. 11.Cover plate with the adhesive cover slip and incubate 30 minutes at room temperature. 12.Wash plate five times in washing Buffer 1x as is indicated, step 1. 13.Transfer 0.1ml substrate solution into each well. 14.Incubate at room temperature in the dark and wait to blue colour development, between 15 and 30 minutes. 15.Add 0.1ml of stop solution to each well. In positive wells will result a yellow color. It is important that the Stop Solution is spread quickly and uniformly throughout the microwells. 16.Read absorbance of microwell within 30 minutes on a spectrophotometer at 450nm 8 Canvax Citokine ELISA: Better Tools for the Best Science Calculation of Results. 17.Create a standard curve (standard absorbance on the y-axis, against concentration standard on the x-axis). Draw a best fit curve. 18.To determine the concentration of human cytokine for each sample interpolate to the standard curve. The concentration read from the standard curve must be multiplied by the dilution factor. 9 Canvax Citokine ELISA: Better Tools for the Best Science 4. PLATE LAYOUT Use this plate layout to record standards and samples assayed. 10 Canvax Citokine ELISA: Better Tools for the Best Science 5. Product use limitation This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. Please refer to www.canvaxbiotech.com for Material Safety Data Sheet of the product. The kit should not be used beyond the expiration date on the kit label. 11 Canvax Citokine ELISA: Better Tools for the Best Science NOTES 12 Canvax Citokine ELISA: Better Tools for the Best Science
