Myeloperoxidase, also known as MPO, is a heme peroxidase that is highly expressed in neutrophils and monocytes. As such, one of the primary physiological roles of MPO is as an essential component of the innate immune system. Once activated by its co-substrate, hydrogen peroxide, MPO is able to oxidize a range of substrates, most notably however is its capacity to react with halides, principally chloride, to generate hypochlorous acid. The highly reactive hypochlorous acid rapidly oxidatively damages various cellular components of invading microbes, leading to cell death. However, the same reactivity that make hypochlorous acid such a potent antimicrobial agent also allow it to damage components of the host. As a result, MPO activity has been implicated in the tissue damage of various inflammatory diseases.

The Myeloperoxidase Activity Assay Kit provides a rapid, sensitive and reproducible tool for determining MPO activity in various sample types. In this assay, peroxide and chloride ions are supplied at a low pH of 6.0, enhancing MPO hypochlorous acid generation. The generated hypochlorous acid then readily reacts with taurine to generate the more stable oxidant taurine chloramine. Once the reaction is quenched with catalase to remove the excess peroxide, freshly prepared 2-nitro-5-thiobenzoate (TNB) is added to react with the taurine chloramine. As taurine chloramine oxidizes TNB, the yellow color associated with TNB fades as it is converted to the colorless 5,5′-dithiobis-(2-nitrobenzoic acid) DTNB. The amount of TNB oxidized in reactions quenched at set time points can thus be used to reliably determine the MPO activity of a sample.

• Microplate reader capable of measuring absorbance at 410 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• Tubes to prepare sample dilutions
• 15 mL conical tubes
• Ultrapure water

1. Prepare the positive control and sample wells by adding the appropriate volumes of positive control and assay buffer.
2. Add the stop reagent to the background wells to establish the zero-minute time point.
3. Initiate the reaction by adding the substrate mix to the sample and positive control wells.
4. Halt the reaction at designated time points by adding the stop reagent and mix thoroughly.
5. Add the detector reagent to the wells that received the stop solution and mix thoroughly.
6. Add the prepared standards to the standard wells.
7. Measure the absorbance of all the wells using a plate reader.