colorimetric sandwich ELISA kit datasheet

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1 colorimetric sandwich ELISA kit datasheet For the quantitative detection of human TGF-beta1 in serum, plasma, cell culture supernatants and Urine. general information Catalogue Number Product Name Species cross-reactivity Range (calibration Range) Tested applications KE00002 TGF-beta1 ELISA Kit Human TGF-beta pg/ml Quantification ELISA database links Entrez Gene SwissProt 7040 (Human) P01137 (Human) kit components & storage Microplate - antibody coated 96-well Microplate (8 well 12 strips) 1 plate Store at 2-8 C for six months Standard pg/bottle; lyophilized* 2 bottles Store at 2-8 C for six months Detection antibody, biotinylated (100X) µl/vial 1 vial Store at 2-8 C for six months Streptavidin-HRP (100X) µl/vial 1 vial Store at 2-8 C for six months Sample Diluent PT 1-ec - 30 ml/bottle 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 and samples. Detection Diluent is for Detection antibody and Streptavidin-HRP antibody. *Add 2 ml Sample Diluent PT 1-ec in standard. This reconstitution gives a stock solution of 1000 pg/ml.

2 Add # µl of Standard diluted in the previous step # µl of Sample Diluent PT 1-ec 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 KE00002 is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (Sandwich ELISA). The TGF-beta1 ELISA kit is to be used to detect and quantify protein levels of endogenous TGF-beta1. The assay recognizes human TGF-beta1. A monoclonal antibody specific for TGF-beta1 has been pre-coated onto the microwells. The TGF-beta1 protein in samples is captured by the coated antibody after incubation. Following extensive washing, a polyclonal antibody of biotinylated specific for TGF-beta1 is added to detect the captured TGF-beta1 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 TGF-beta is a member of the transforming growth factor beta (TGFB) family of cytokines, which are multifunctional peptides that regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. TGF-beta is produced by a number of cell types including regulatory T cells, fibroblasts, epithelial cells, and endothelial cells. TGF-beta acts synergistically with TGFA in inducing transformation. It also acts as a negative autocrine growth factor. TGF-beta plays an important role in bone remodeling as it is a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts. TGF-beta appears to promote late stage progression and metastasis in some cancers. sample preparation The serum or plasma 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. To activate latent TGF-beta1 to the immunoreactive form, prepare the following solutions for acid activation and neutralization. The solutions may be stored in polypropylene bottles at room temperature for up to one month. 2

3 1 N HCl (100 ml) - To ml of deionized water, slowly add 8.33 ml of 12 N HCl. 1.2 N NaOH/0.5 M HEPES (100 ml) - To 75 ml of deionized water, slowly add 12 ml of 10 N NaOH. Add 11.9 g of HEPES. Bring fnal volume to 100 ml with deionized water. For each new lot of acidifcation and neutralization reagents, measure the ph of several representative samples after neutralization to ensure that it is within ph Adjust the volume and corresponding dilution factor of the neutralization reagent as needed. NB: Do not activate the kit standards. The kit standards contain active recombinant TGF-beta1. Cell lture supernatants/urine To 100 μl of cell culture supernate, add 20 μl of 1 N HCI. Mix ell. Incubate 10 minutes at room temperature. Neutralize the acidifed sample by adding 20 μl of 1.2 N NaOH/0.5 M HEPES. Assay immediately. The concentration read of the standard curve must be multiplied by the dilution factor, 1.4. Serum/Plasma To 40 μl serum/plasma, add 20 μl of 1 N HCl. Incubate 10 minutes at room temperure. Neutralize the acidifed sample by adding 20 μl of 1.2 N NaOH/0.5 M HEPES. Prior to the assay, dilute the activated sample with Sample Diluen PT 1-ec. See the following datas for suggested dilutions. The concentration read of the standard curve must be multiplied by the appropriate dilution factors. Activated serum and plasma samples may be stored for up to 24 hours at 2-8 C before use. Activated cell culture supernates/urine samples must be assayed immediately after activation. Do not freeze activated samples. 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 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. 3

4 Optical Density typical data These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed. precision Four Parameter Logistic (4-PL) Curve Fit R 2 = Human TGF-β1 Concentration (pg/ml) (pg/ml) O.D Average Corrected 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 n Mean (pg/ml) SD CV% recovery The recovery of TGF-beta1 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 (%) Citrate plasma 1: : Cell culture supernatants 1: : Urine 1: :

5 sample value Sample1 Sample2 Sample3 Sample4 Human plasma pg/ml 7748 pg/ml 8038 pg/ml pg/ml sensitivity The minimum detectable dose of human TGF-beta1 is 3.3 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 TGF-beta1 in various matrices and diluted with the appropriate Sample Diluent PT 1-ec to produce samples with values within the dynamic range of the assay. (The serum and plasma samples were initially diluted 1:50. The cell culture supernatants samples were initially diluted 1:1) 1:2 1:4 1:8 1:16 Citrate plasma Cell culture supernatants Urine Average% of Expected Range (%) Average% of Expected Range (%) Average% of Expected Range (%) Average% of Expected Range (%) references 1. Siegel, P.M.et al. (2003) Nat Rev Cancer 3: Bierie, B. et al. (2006) Nat Rev Cancer 6:, Tian, M. et al. (2009) Future Oncol 5: Priyadarshi S. et al.(2013) 28: