colorimetric sandwich ELISA kit datasheet For the quantitative detection of mouse Interleukin 6 (IL6) concentrations in serum, plasma and cell culture supernatants. general information Catalogue Number Product Name Species cross-reactivity Range (calibration Range) Tested applications KE10007 IL6 ELISA Kit Mouse IL6 15.6-1000 pg/ml Quantification ELISA database links Entrez Gene SwissProt 16193 (Mouse) P08505 (Mouse) kit components & storage Microplate - antibody coated 96-well Microplate (8 well 12 strips) 1 plate Store at 2-8 C for six months Standard - 2000 pg/bottle; lyophilized* 2 bottles Store at 2-8 C for six months Detection antibody, biotinylated (100X) - 120 µl/vial 1 vial Store at 2-8 C for six months Streptavidin-HRP (100X) - 120 µl/vial 1 vial Store at 2-8 C for six months Sample Diluent PT 1-ec - 30 ml/bottle. For serum and plasma. 1 bottle Store at 2-8 C for six months Sample Diluent PT 1-ef - 30 ml/bottle. For cell culture supernatants. 1 bottle Store at 2-8 C for six months Detection Diluent - 30 ml/bottle 1 bottle Store at 2-8 C for six months Wash Buffer Concentrate (20X) - 30 ml/bottle 1 bottle Store at 2-8 C for six months Tetramethylbenzidine Substrate (TMB) - 12 ml/bottle 1 bottle Store at 2-8 C for six months Stop Solution - 12 ml/bottle 1 bottle Store at 2-8 C for six months Plate Cover Seals 3 pieces NB: Do not use the kit after the expiration date. Sample Diluent PT 1-ec is for standard, serum and plasma. Sample Diluent PT 1-ef is for standard and cell culture supernatants. Detection Diluent is for Detection antibody and Streptavidin-HRP. *Add 2 ml Sample Diluent PT 1-ec or PT 1-ef in standard. This reconstitution gives a stock solution of 1000 pg/ml. UK & Europe: Europe@
Add # µl of Standard diluted in the previous step # µl of Sample Diluent PT 1-ec or PT 1-ef 500 µl 500 µl 500 µl 500 µl 500 µl 500 µl 2000 µl 500 µl 500 µl 500 µl 500 µl 500 µl 500 µl "sd7" "sd6" "sd5" "sd4" "sd3" "sd2" "sd1" product description KE10007 is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (Sandwich ELISA). The Mouse IL6 ELISA kit is to be used to detect and quantify protein levels of endogenous mouse IL6. The assay recognizes mouse IL6. A monoclonal antibody specific for mouse IL6 has been pre-coated onto the microwells. The mouse IL6 protein in samples is captured by the coated antibody after incubation. Following extensive washing, a polyclonal antibody of biotinylated specific for mouse IL6 is added to detect the captured mouse IL6 protein. For signal development, Streptavidin-HRP is added, followed by Tetramethyl-benzidine (TMB) reagent. Solution containing sulfuric acid is used to stop color development and the color intensity which is proportional to the quantity of bound protein is measurable at 450nm. background Interleukin-6 (Il-6) is an interleukin that acts as both a pro-inflammatory and anti-inflammatory cytokine. Il-6 protein is secreted by a variety of cell types including T cells and macrophages as phosphorylated and variably glycosylated molecule. Il-6 Plays an essential role in the final differentiation of B-cells into Ig-secreting cells involved in lymphocyte and monocyte differentiation. It induces myeloma and plasmacytoma growth and induces nerve cells differentiation Acts on B-cells, T-cells, hepatocytes, hematopoietic progenitor cells and cells of the CNS. Il-6 is also considered a myokine, a cytokine produced from muscle, and is elevated in response to muscle contraction. Il-6 has been shown to interact with interleukin-6 receptor and glycoprotein 130. Additionally, Il-6 is involved in hematopoiesis, bone metabolism, and cancer progression, and has been defined an essential role in directing transition from innate to acquired immunity. sample preparation The serum, plasma or cell culture supernatants samples may require proper dilution to fall within the range of the assay. A range of dilutions like 1:2, 1:4 is suggested according to the individual samples. 2 UK & Europe: Europe@
Optical Density safety notes This product is sold for lab research and development use ONLY and not for use in humans or animals. Avoid any skin and eye contact with Stop Solution and TMB. In case of contact, wash thoroughly with water. assay procedure summary Step Reagent Volume Incubation Wash Notes 1 Standard and Samples 100 µl 120 min 4 times Cover Wells 2 Diluent Antibody Solution 100 µl 60 min 4 times Cover Wells 3 Diluent HRP Solution 100 µl 40 min 4 times Cover Wells 4 TMB Substrate 100 µl 15-20 min Do not wash Incubate in the dark at 37 C 5 Stop Solution 100 µl 0 min Do not wash - 6 Read plate at 450 nm and 630 nm immediately after adding Stop solution. DO NOT exceed 5 minutes. typical data These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed. 10 1 0.1 Sample Diluent PT 1-ec Four Parameter Logistic (4-PL) Curve Fit 0.01 1 10 100 1000 Mouse IL6 Concentration (pg/ml) R 2 =0.999 (pg/ml) O.D Average Corrected 0 0.037 0.039 0.038 15.6 0.078 0.078 0.078 0.040 31.25 0.11 0.129 0.120 0.082 62.5 0.189 0.216 0.203 0.165 125 0.298 0.368 0.333 0.295 250 0.635 0.649 0.642 0.604 500 1.029 1.126 1.078 1.040 1000 1.769 1.902 1.834 1.796 3 UK & Europe: Europe@
Optical Density 10 1 0.1 Sample Diluent PT 1-ef Four Parameter Logistic (4-PL) Curve Fit 0.01 1 10 100 1000 Mouse IL6 Concentration (pg/ml) R 2 =0.999 (pg/ml) O.D Average Corrected 0 0.046 0.046 0.046 15.6 0.105 0.105 0.105 0.059 31.25 0.172 0.167 0.170 0.124 62.5 0.282 0.296 0.289 0.243 125 0.532 0.556 0.544 0.498 250 0.974 1.01 0.992 0.946 500 1.679 1.715 1.697 1.651 1000 2.632 2.674 2.653 2.607 precision Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision. Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 24 24 24 Mean (pg/ml) 133.3 204.3 778.1 142.3 223.0 827.3 SD 6.1 7.6 52.7 11.6 18.8 81.2 CV% 4.5 3.7 6.8 8.2 8.4 9.8 recovery The recovery of IL6 spiked to three different levels in four samples throughout the range of the assay in various matrices was evaluated. Sample Type Average% of Expected Range (%) Mouse serum 1:2 100 94-107 1:4 98 75-119 Cell culture supernatants 1:2 105 96-118 1:4 110 99-123 4 UK & Europe: Europe@
sample values Twenty individual mouse serum samples were evaluated for the presence of mouse IL6 in this assay. Nineteen samples measured less than the lowest standard, 15.6 pg/ml, one sample measured 15.8 pg/ml. A mouse was injected with 15 μg of LPS and was bled 2 hours after injection. The serum was tested for mouse IL6 and measured 12,000 pg/ml. sensitivity The minimum detectable dose of mouse IL6 is 3.8 pg/ml. This was determined by adding two standard deviations to the concentration corresponding to the mean O.D. of 20 zero standard replicates. linearity To assess the linearity of the assay, three samples were spiked with high concentrations of mouse IL6 in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. 1:2 1:4 1:8 1:16 Mouse serum (Sample Diluent PT 1-ec) Cell culture supernatants (Sample Diluent PT 1-ef) Average% of Expected 93 86 Range (%) 89-96 84-88 Average% of Expected 101 87 Range (%) 96-106 85-89 Average% of Expected 101 85 Range (%) 100-102 83-87 Average% of Expected 111 83 Range (%) 96-126 82-85 references 1. Scheller J. et al. (1988). Behring Inst Mitt. Behring Inst Mitt. (83):40-7. 2. Heinrich PC. et al. (2003). Biochem J. 374: 1-20. 3. Rose-John S. et al. (2007). Expert Opin Ther Targets. 11: 613-24. 4. Ming JE. et al. (1989). J Mol Cell Immunol. 4: 203-11. 5. Febbraio MA. et al. (2005). Exerc Sport Sci Rev. 33:114-9. 6. J Appl Physiol. et al. (2005). J Appl Physiol. 98:1154-62. 5 UK & Europe: Europe@