Myeloperoxidase (MPO) is a glycoprotein with an alpha2beta2 heteromultimer expressed in all cells of the myeloid linage. MPO is abundantly present in azurophilic granules of polymorphonuclear neutrophils. It is an important enzyme used during phagocytic lysis of engulfed foreign particles which takes part in the defense of the organism through production of hypochlorous acid (HOCl), a potent oxidant. MPO is rapidly released by activated polymorphonuclear neutrophils. MPO is released in the extra cellular medium where hypochlorous acid leads to chlorination of proteins leading to products as 3-chlorotyrosine. Furthermore it leads to oxidation of low density lipoprotein (LDL) and apolipoprotein A-I, the primary protein of HDL resulting in the disruption of HDL functions as cholesterol efflux. Involvement of MPO has been described in numerous diseases such as atherosclerosis, lung cancer, Alzheimer’s disease and multiple sclerosis. Autoimmune antibodies to MPO are involved in Wegener’s disease. Since the discovery of MPO deficiency, initially regarded as rare and restricted to patients suffering from severe infections, MPO has attracted more clinical attention. The classical MPO assay is an enzymatic assay for activity of MPO. This classical MPO assay is hampered by the presence of inhibitory compounds in tissue homogenates and plasma. In this type of assays spiking often gives unreliable results. The rat MPO elisa is not influenced by inhibitors of the enzyme activity.
The rat MPO ELISA kit is to be used for the in vitro quantitative determination of rat MPO in plasma, tissue homogenate and cell culture supernatant samples.
The rat MPO ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are captured by a solid bound specific antibody.
Biotinylated tracer antibody will bind to captured rat MPO.
Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of citric acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the rat MPO standards (log).
The rat MPO concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The linearity of the assay was determined by serially diluting a serum sample containing 50 ng/ml rat MPO. The diluted samples were measured in the assay. The line obtained a slope of 0.90 and a correlation coefficient of 0.999.
Normal rat blood samples (plasma), were spiked with rat MPO in concentrations of 100 ng/ml. Samples with and without rat MPO were incubated for 1 hour at room temperature. Samples were measured using the ELISA. Recovery values for rat MPO ranged between 108% and 120% (mean 114 %).