The MPO BioAssay(TM) ELISA Kit, is a sandwich ELISA for quantitative detection of human MPO in cell culture supernates, cell lysates, serum, plasma (heparin, EDTA), saliva and urine. Myeloperoxidase (MPO) is a mammalian phagocyte hemoprotein thought to primarily mediate host defense reactions. It is abundantly expressed in neutrophils and secreted during their activation. Myeloperoxidase is part of the host defense system of human polymorphonuclear leukocytes, responsible for microbicidal activity against a wide range of organisms. It is located in the nucleus as well as in the cytoplasm. Intranuclear MPO may help to protect DNA against damage resulting from oxygen radicals produced during myeloid cell maturation and function. The standard product used in this kit is the product of gene recombination, consisting of 697(A49-S745) amino acids with the molecular mass of 80kD. Assay Range: 312-20,000pg/ml Sensitivity: <10pg/ml Specificity: Natural and recombinant human MPO. Crossreactivity: There is no detectable cross-reactivity with other relevant proteins. Test Principle: This human MPO BioAssay(TM) ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for MPO has been precoated onto 96-well plates. Standards (NS0, Immunogen Sequence A49-S745) and test samples are added to the wells, a biotinylated detection polyclonal antibody from goat specific for MPO is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the humanMPO amount of sample captured in plate. Kit Components: 143986A: Microtiter Strips, 1x96 wells *143986B: MPO Standard, 2x20ng 143986C: Anti-human MPO (Biotin), 1x130ul (1:100 dilution) 143696: Avidin-Biotin-Peroxidase Complex (ABC), 1x130ul (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 Storage and Stability: Store *143779B 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 min. Do not wash. 2. Add 100ul biotinylated antibody and incubate the plate at 37C for 60 min. Wash plate 3 times with 0.01M PBS or TBS. 3. Add ABC working solution and incubate the plate at 37C for 30 minutes. Wash plate 5 times with 0.01M PBS or TBS. 4. Add TMB color developing agent and incubate the plate at 37C in dark for 15-20 minutes. 5. Add 100ul TMB stop solution and read.
