Description
MPO Activity Assay Kit (BA0134) (BA0134)
The MPO Activity Assay Kit (SKU: BA0134) provides a sensitive fluorometric method for determining myeloperoxidase (MPO; EC 1.11.2.2) peroxidation activity in biological samples. Myeloperoxidase is a peroxidase enzyme found in neutrophils, monocytes and some soft tissue macrophages, and it uses chloride as a cosubstrate with hydrogen peroxide to generate the powerful antimicrobial agent hypochlorous acid. Excessive production of hypochlorous acid can lead to oxidative stress and tissue damage, and increased MPO levels may raise the risk of myocardial infarction and cardiovascular disease. In this assay, the MPO reaction with hydrogen peroxide oxidises the dye reagent to a highly fluorescent product measured at 530/585 nm, the intensity of which is proportional to the total peroxidation activity in the sample. A supplied MPO inhibitor is used to suppress peroxidase activity due to MPO in order to differentiate other peroxidase activities that may be present. The linear detection range is 0.0025 to 2 U/L for a 10 minute reaction at 25 degrees C.
| Product Name: | MPO Activity Assay Kit (BA0134) |
| SKU: | BA0134 |
| Detection Method: | Fluorometric |
| Detection Range: | 0.0025 to 2 U/L (20 uL sample, 10 min reaction at 25 degrees C) |
| Sample Type: | Cell lysates and tissues |
| Species Reactivity: | All |
| Assay Time: | Approximately 20 minutes plus 10 minute pre-incubation |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20 degrees C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This kit is based on the MPO enzyme reaction with hydrogen peroxide, which oxidises the dye reagent to a highly fluorescent product. The fluorescence intensity of this product, measured at 530/585 nm, is proportional to the total peroxidation activity in the sample, and the provided MPO inhibitor is used to suppress peroxidase activity due to MPO in order to differentiate other peroxidase activities.
- Fast and sensitive with a linear detection range of 0.0025 to 2 U/L (20 uL sample) for a 10 minute reaction at 25 degrees C
- Convenient and high-throughput homogeneous mix-incubate-measure type assay
- Can be readily automated on HTS liquid handling systems for processing thousands of samples per day
- MPO peroxidation activity determination in biological samples such as cell lysates and tissues
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Bring all reagents to room temperature, briefly centrifuge tubes, and prepare enough 1x MPO Inhibitor by diluting the provided 20x MPO Inhibitor 20-fold in distilled water (20 uL of 1x inhibitor is required per sample). |
| 2 | Prepare 250 uL 30 uM Resorufin Premix by mixing 15 uL Resorufin and 235 uL water, then transfer 100 uL water and 100 uL 30 uM Resorufin into two separate wells of a black flat-bottom 96-well plate. |
| 3 | For each sample prepare two parallel wells and add 20 uL of sample to each; add 20 uL of 1x MPO inhibitor to one well and 20 uL Assay Buffer to the other, then incubate at room temperature for 10 minutes. |
| 4 | Prepare 0.07% H2O2 by mixing 4.7 uL 3% H2O2 with 195.3 uL distilled water, then prepare 0.007% H2O2 by mixing 60 uL 0.07% H2O2 with 540 uL distilled water and use within one hour. |
| 5 | Prepare enough Working Reagent for all reaction wells by mixing, for each 96-well assay, 60 uL Assay Buffer, 1 uL 0.007% H2O2 and 1 uL Dye Reagent, then add 60 uL Working Reagent to all sample and inhibitor wells and tap the plate briefly to mix. |
| 6 | Read fluorescence at 530/585 nm at 0 minutes and 10 minutes at room temperature. |
MPO Activity = [(delta RSAMPLE - delta RINB) / (RRESORUFIN - RH2O)] x [Resorufin]/t x (Reaction Vol / Sample Vol) x n = [(delta RSAMPLE - delta RINB) / (RRESORUFIN - RH2O)] x 15 x n (U/L), where delta RSAMPLE = RSample,10min - RSample,0min and delta RINB = RINB,10min - RINB,0min, [Resorufin] = 30 uM, Reaction Vol = 100 uL, Sample Vol = 20 uL, reaction time t = 10 minutes and n is the sample dilution factor. One unit of enzyme will catalyse the formation of 1 umole resorufin per minute under the assay conditions.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 degrees C |
| 20x MPO Inhibitor | 120 uL | -20 degrees C |
| 3% Stabilized H2O2 | 100 uL | -20 degrees C |
| Resorufin | 1.5 mL | -20 degrees C |
| Dye Reagent | 120 uL | -20 degrees C |
