colorimetric sandwich ELISA kit datasheet

Transcription

1 colorimetric sandwich ELISA kit datasheet For the quantitative detection of human HER2 in serum, plasma, cell culture supernatants and urine. general information Catalogue Number Product Name Species cross-reactivity Range (calibration Range) Tested applications KE00053 HER2 ELISA Kit Human HER ng/ml Quantification ELISA database links Entrez Gene SwissProt 2064 (Human) P04626 (Human) kit components & storage Microplate - antibody coated 96-well Microplate (8 wells 12 strips) 1 plate Store at 2-8 C for six months Standard - 20 ng/bottle; lyophilized* 2 bottles Store at 2-8 C for six months Detection antibody (0 ) µl/vial 1 vial Store at 2-8 C for six months HRP-conjugated antibody (0 ) µl/vial 1 vial Store at 2-8 C for six months Sample Diluent PT 1-a - 30 ml/bottle; For serum and plasma samples 1 bottle Store at 2-8 C for six months Sample Diluent PT 1-ec - 30 ml/bottle; For cell culture supernatants and urine 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 (20 ) - 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-a is for standard, serum and plasma samples. Sample Diluent PT 1-ec is for standard, cell culture supernatants and urine. Detection Diluent is for Detection antibody and HRP-conjugated antibody. *Add 2 ml Sample Diluent PT 1-a or PT 1-ec in standard. This reconstitution gives a stock solution of ng/ml. USA: Proteintech@

2 Add # µl of Standard diluted in the previous step # µl of Sample Diluent PT 1-a or 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 KE00053 is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (Sandwich ELISA). The HER2 ELISA kit is to be used to detect and quantify protein levels of endogenous HER2. The assay recognizes human HER2. A polyclonal antibody specific for HER2 has been pre-coated onto the microwells. The HER2 protein in samples is captured by the coated antibody after incubation. Following extensive washing, a monoclonal antibody specific for HER2 is added to detect the captured HER2 protein. For signal development, horseradish peroxidase (HRP)-conjugated antibody 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 HER2, also known as ErbB2 and Neu, is a member of the epidermal growth factor (EGF) receptor family. Overexpression of HER2 have been reported in numerous cancers, including breast, ovarian, lung, gastric, and oral cancers. It is a therapeutic target for the treatment of breast cancer and other carcinomas. HER2 is a 185-kDa transmembrane glycoprotein, the extracellular domain (ECD) of which can be cleaved from the cell surface by matrix metalloproteases and then released into blood. Elevated serum HER2 levels are reported in 23% to 80% of patients with metastatic breast cancer. Raising serum HER2 levels have been correlated with indicators of poor prognosis and a poor response to chemotherapy. 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. 2 USA: Proteintech@

3 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 0 µl 60 min 4 times Cover Wells 2 Diluent Antibody Solution 0 µl 60 min 4 times Cover Wells 3 Diluent HRP Solution 0 µl 40 min 4 times Cover Wells 4 TMB Substrate 0 µl min Do not wash Incubate in the dark at 37 C 5 Stop Solution 0 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 Assay Diluent PT 1-ec Four Parameter Logistic (4-PL) Curve Fit R 2 = Human HER2 Concentration (ng/ml) (ng/ml) O.D Average Corrected USA: Proteintech@

4 Optical Density precision Assay Diluent PT 1-a Four Parameter Logistic (4-PL) Curve Fit R 2 = Human HER2 Concentration (ng/ml) (ng/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) 7, , , , SD CV% recovery The recovery of HER2 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 (%) Human plasma 1: : Cell culture supernatants 1: : Urine 1: : USA: Proteintech@

5 sample value Twenty-eight serum samples from healthy volunteers were evaluated for HER2 in this assay. No medical histories were available for the donors used in this study. Sample Type The Detection Rate Mean of Detectable (ng/ml) Range (ng/ml) serum (n=28) 37% sensitivity The minimum detectable dose of human HER2 is 0.02 ng/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 HER2 in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The plasma samples were initially diluted 1:2) 1:2 1:4 1:8 1:16 Human plasma (Sample Diluent PT 1-a) Cell culture supernatants (Sample Diluent PT 1-ec) Urine (Sample Diluent PT 1-ec) Average% of Expected Range (%) Average% of Expected Range (%) Average% of Expected Range (%) Average% of Expected Range (%) references 1. Hung MC, et al. Basic science of HER-2/neu: a review. Semin Oncol. 26(4 Suppl 12):51-9 (1999). 2. Slamon DJ, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 344(11): (2001). 3. Pupa SM, et al. The extracellular domain of the c-erbb-2 oncoprotein is released from tumor cells by proteolytic cleavage. Oncogene. 8(11): (1993). 4. Carney WP, et al. Potential clinical utility of serum HER-2/neu oncoprotein concentrations in patients with breast cancer. Clin Chem. 49(): (2003). 5. Colomer R, et al. Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer. Clin Cancer Res. 6(6): (2000). 5 USA: Proteintech@

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