| Fas, also known as APO-1, CD95 and TNFRSF6, is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily and mediates apoptosis.1 The nucleotide sequence of the cDNAs reveales that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40.2 The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 is previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. The APO-1 antigen is found to be a membrane glycoprotein of 48kD.3 Fas antigen is expressed and functional on papillary thyroid cancer cells and this may have potential therapeutic significance.4 Fas can play a role as an inducer of both neurite growth in vitro and accelerates recovery after nerve injury in vivo. The sFAS BioAssay(TM) ELISA Kit is a sandwich ELISA for quantitative detection of mouse sFAS in cell culture supernates, serum and plasma (Heparin, EDTA, citrate). Detection Range: 31.2pg/ml-2000pg/ml Sensitivity: <3pg/ml Specificity: Recognizes natural and recombinant mouse sFAS. Test Principle: The sFAS , BioAssay(TM) ELISA Kit is a solid-phase immunoassay specially designed to measure mouse sFAS with a 96-well strip plate that is pre-coated with antibody specific for sFAS. The detection antibody is a biotinylated antibody specific for sFAS. The capture antibody is monoclonal antibody from rat and the detection antibody is polyclonal antibody from goat. The kit includes recombinant mouse sFAS protein with immunogen: Expression system for standard: NS0, Immunogen sequence: Q22-R169. To measure mouse sFAS, add standards and samples to the wells, then add the biotinylated detection antibody. Wash the wells with PBS buffer, and add Avidin-Biotin-Peroxidase Complex (ABC-HRP). Wash away the unbounded ABC-HRP with PBS buffer and add TMB. TMB is an HRP substrate and will be catalyzed to produce a blue color product, which changes into yellow after adding the acidic stop solution. The absorbance of the yellow product at 450nm is linearly proportional to mouse uPAR in the sample. Read the absorbance of the yellow product in each well using a plate reader, and benchmark the sample wells readings against the standard curve to determine the concentration of mouse sFAS in the sample. Kit Components: 144039A: Microtiter Strips, 1x96 wells *144039B: sFAS Standard, 2x10ng 144039C: Anti-mouse sFAS (Biotin), 1x100ul (1:100 dilution) 143696: Avidin-Biotin-Peroxidase Complex (ABC), 1x100ul (1:100 dilution) 143697: Sample Diluent Buffer, 1x30ml 143698: Antibody Diluent Buffer, 1x12ml 143699: ABC Diluent Buffer, 1x12ml 143700: TMB Color Developing Agent, 1x10ml 143701: TMB Stop Solution, 2N H2SO4, 1x10ml 143702: Wash Buffer, 25X, 1x20ml Storage and Stability: Store *144039B powder at 4C. Once reconstituted store at 4C for up to 12 hours or at -20C for up to 48 hours. Store other components at 4C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Assay Summary: 1. Add 100ul of samples and standards and incubate the plate at 37C for 90 minutes or at RT for 2 hours. Do not wash. 2. Add 100ul biotinylated antibody and incubate the plate at 37C for 60 minutes or at RT for 90 minutes. Wash plate 3 times with Working Wash Buffer. 3. Add 100ul of ABC working solution and incubate the plate at 37C for 30 minutes or at RT for 40 minutes. Wash plate 5 times with Working Wash Buffer. 4. Add 90ul of TMB color developing agent and incubate the plate at 37C in dark for 15-25 minutes or at RT for 30 minutes. 5. Add 100ul TMB Stop Solution and read. |
