| Tryptases are serine proteases implicated in asthma and are highly expressed in human mast cells. They are derived from at least 4 nonallelic genes clustered on chromosome 16p13.3: TPSAB1, which represents the alpha and beta-I tryptase alleles, TPSB2, which represents the beta-II and beta-III tryptase alleles, TPSG1, and TPSD1. Elevated levels of serum tryptase occur in both anaphylactic and anaphylactoid reactions, but a negative test does not exclude anaphylaxis. Tryptase is less likely to be elevated in food allergy reactions as opposed to other causes of anaphylaxis. The Tryptase/TPSAB1, B2 BioAssay(TM) ELISA Kit is a sandwich ELISA for quantitative detection of human Tryptase/TPSAB1, B2 in cell culture supernates, cell lysates, serum, plasma (Heparin or EDTA). Assay Range: 156pg/ml-10,000pg/ml Sensitivity: <15pg/ml Specificity: Recognizes natural and recombinant Human Tryptase/TPSAB1,B2. Test Principle: The Tryptase/TPSAB1, B2 BioAssay(TM) ELISA Kit is a solid-phase immunoassay specially designed to measure Human Tryptase/TPSAB1,B2 with a 96-well strip plate that is pre-coated with antibody specific for Tryptase/TPSAB1,B2. The detection antibody is a biotinylated antibody specific for Tryptase/TPSAB1,B2. The kit includes Human Tryptase/TPSAB1,B2 protein as standards. The kit contains recombinant human Tryptase/TPSAB1,B2 with immunogen: Expression system for standard: NS0, Immunogen sequence: Met1-Pro275. To measure Human Tryptase/TPSAB1,B2, 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 Human Tryptase/TPSAB1,B2 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 Human Tryptase/TPSAB1,B2 in the sample. Kit Components: 396814A: Microtiter Strips, 1x96 wells 396814B: Tryptase/TPSAB1, B2 Standard, 2x10ng 396814C: Anti-human Tryptase/TPSAB1, B2 (Biotin), 1x100ul 143696: Avidin-Biotin-Peroxidase Complex (ABC), 1x100ul 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 *396814B 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. |
