User Protocol CBA024 Rev. 23 May 2005 RFH Page 1 of 6 InnoZyme Myeloperoxidase Activity Kit Cat. No. CBA024 Table of Contents Page Storage 1 Intended Use 1 Background 1 Principle of the Assay 2 Materials Provided 2 Materials Required but not Provided 2 Reagent Preparation 2 Detailed Protocol 4 Calculation of Results 4 Assay Characteristics 5 Assay Examples 5 References 6 Related Products 6 Sample 96-Well Template 6 Storage The unopened kit should be stored at 20 0 C and should not be used beyond the expiration date stated on the label. All kit components, once opened, can be stored for up to 3 months under the following conditions: Refrigerate (4 o C): Freezer (-20 o C): TMB 96-well plate Myeloperoxidase Assay Buffer Stop Solution Hydrogen Peroxide Sample Buffer Note: Following initial use the Myeloperoxidase and the Hydrogen Peroxide should be dispensed into aliquots and stored at 20 C. Avoid freeze/thaw cycles. Intended Use The Calbiochem InnoZyme TM Myeloperoxidase Activity Kit is designed to measure human myeloperoxidase activity in cell lysates and biological samples and to screen enzyme inhibitors. Background Myeloperoxidase (MPO) (EC 1.11.1.7) catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant formed from chloride ions and hydrogen peroxide. It is stored in the azurophilic granules of neutrophils and is released as a result of inflammation and phagocytosis, making it an indicator of neutrophil activation. The products resulting from MPO activity exhibit strong microbiocidal effects. It is also a major component of myelocytic
User Protocol CBA024 Rev. 23 May 2005 RFH Page 2 of 6 precursor cells and is also used as a marker for acute leukemia. MPO is a hemoprotein that consists of two 53-kDa subunits, two 15-kDa subunits, and two prosthetic porphyrins that are an essential part of the catalytic cycle. Historically the enzyme is considered the most important component of the anti-bacterial activity of phagocytes, but recent reports implicate MPO in roles outside the host immune system. Brennen, et al. identified MPO as a new marker of inflammation that is superior to existing markers for assessing cardiac risk, as the level of enzyme was found to correlate with short-term risk of acute cardiac events among emergency care patients with chest pain. Recent findings indicate that MPO may play a role in the pathology of atherogenesis through the oxidation of apolipoprotein A 1 and formation of defective HDL and altering its ability to bind and remove free cholesterol from peripheral tissues. Finally, Vita, et al. reports that serum myeloperoxidase level is an independent indicator of endothelial cell dysfunction in humans. Principle of the Assay The Calbiochem InnoZyme TM Myeloperoxidase Activity Kit is a specific and sensitive assay for measuring active human MPO. The assay uses a polyclonal antibody specific for human MPO immobilized on a 96-well plate to specifically capture the enzyme. Activity of captured myeloperoxidase is measured using a detection reagent that includes TMB and hydrogen peroxide. Following color development, the reaction is stopped with sulfuric acid and the absorbance of the oxidized TMB is detected at 450 nm. Materials Provided Coated 96-well Plate: 96-well polystyrene plate, 12 X 8 well strips, coated with an antibody against human myeloperoxidase, 1 each Myeloperoxidase : 1 each, supplied at 100 µg/ml, 1 vial Hydrogen Peroxide: 1 each, 30% hydrogen peroxide, supplied as 100X TMB: 1 ml, 1 vial Assay Buffer: 5 ml, 1 bottle Sample Buffer, 25 ml, supplied as 20X, 1 bottle ELISA Stop Solution: 12 ml, 2.5N H 2 SO 4,1 bottle Plate Sealer: 2 each Materials Required but not Provided Distilled H 2 O Pipettors or multi-channel pipettor carefully calibrated to the target volume 37 0 C incubator Microplate reader capable of measuring absorbance at 450 nm Reagent Preparation All reagents necessary to perform the assay are supplied with the kit. Equilibrate all reagents to room temperature (15-25 0 C) just prior to use. Sample Buffer (1X): Dilute Sample Buffer 1:20 by adding 25 ml Sample Buffer to 475 ml dh 2 O.
User Protocol CBA024 Rev. 23 May 2005 RFH Page 3 of 6 Myeloperoxidase (1µg/ml): Dilute the Myeloperoxidase 1:100 by adding 5 µl Myeloperoxidase to 495 µl chilled Sample Buffer (1X) obtain a concentration of 1 µg/ml. Maintain the diluted Myeloperoxidase (1 µg/ml) on ice. Note: Following initial thaw the Myeloperoxidase should be dispensed into aliquots and stored at 20 C. Avoid freeze/thaw cycles. Prepare Myeloperoxidase dilutions in the range of 5-100 ng/ml using the following guidelines: Samples: Concentration Myeloperoxidase Sample Buffer (1X) (1 µg/ml) A 100 ng/ml 50 µl 450 µl B 80 ng/ml 40 µl 460 µl C 60 ng/ml 30 µl 470 µl D 40 ng/ml 20 µl 480 µl E 20 ng/ml 10 µl 490 µl F 10 ng/ml 5 µl 495 µl G 5 ng/ml 2.5 µl 497.5 µl H 0 ng/ml (Blank) 0 µl 500 µl Dilute samples with Sample Buffer (1X) as needed. Note: If sample dilution is not required for sensitivity reasons, prepare the sample by adding 475 µl sample to 25 µl 20X Sample Buffer to ensure appropriate ph for the first incubation. Hydrogen Peroxide (0.3%): Dilute the Hydrogen Peroxide (30%) 1:100 by adding 5 µl Hydrogen Peroxide (30%) to 495 µl chilled dh 2 O. Note: A total of 40 µl is required to cover the entire Coated 96-Well Plate. If the entire plate is not to be used at one time, prepare fresh Hydrogen Peroxide (0.3%) each time the assay is performed. Following initial thaw the Hydrogen Peroxide should be dispensed into aliquots and stored at 20 C. Avoid freeze/thaw cycles. TMB Detection Reagent: Note: The desired amount of TMB Detection Reagent should be prepared fresh, just prior to use. Prepare 10 ml TMB Detection Reagent to cover the entire 96-well plate by mixing the following components: 7.16 ml dh 2 O 2.00 ml Assay Buffer 0.80 ml TMB 40 µl H 2 O 2 (0.3%) Vortex gently 3 X 3 seconds
User Protocol CBA024 Rev. 23 May 2005 RFH Page 4 of 6 To prepare TMB Detection Reagent for a variable number of strips, use the following guidelines: Number of 8-Well dh 2 O Assay Buffer TMB Hydrogen Peroxide (0.3%) Strips 2 1.43 ml 0.40 ml 0.16 ml 8.0 µl 4 2.86 ml 0.80 ml 0.32 ml 16.0 µl 6 4.31 ml 1.21 ml 0.48 ml 24.1 µl 8 5.00 ml 1.41 ml 0.56 ml 28.2 µl 10 5.73 ml 1.60 ml 0.64 ml 32.0 µl 12 7.16 ml 2.00 ml 0.80 ml 40.0 µl Detailed Protocol It is recommended that all samples and standards be assayed in duplicate. 1. Prepare Sample Buffer (1X), Myeloperoxidase dilutions, and samples as indicated in the previous section. Remove the desired number of strips from the Coated 96-Well Plate, return the remaining strips to foil pouch, and close the re-sealable edge. 2. Add 100 µl Myeloperoxidase dilutions and samples to designated wells. The Blank should contain 100 µl Sample Buffer (1X). If inhibitor is to be included in the assay, add the desired amount of inhibitor to designated wells containing Myeloperoxidase or sample; the total volume, including MPO and inhibitor should be 100 µl. 3. Cover the plate with the plate sealer and incubate 1 hour at room temperature with gentle shaking. 4. Wash the plate four times with 400 µl Sample Buffer (1X). Following each wash discard the contents of the wells by inverting the plate over the sink; tap the plate on a paper towel to remove residual liquid. 5. Add 100 µl TMB Working Solution to each well and incubate 20 30 minutes at 37 o C. 6. Add 100 µl ELISA Stop Solution to each well to stop the color development. 7. Read the absorbance at 450 nm. Calculation of Results Several options are available for calculating the concentration of active myeloperoxidase in unknown samples. We recommend that the data be handled by a plate reader software package utilizing a linear or 4 parameter logistic curve-fitting program. If data processing software is not available, the concentration of active myeloperoxidase can be calculated as follows: 1. Correct the absorbance value of all samples and Myeloperoxidase dilutions by subtracting the value of the blank. 2. Calculate the mean absorbance value for each sample in duplicate. 3. Plot the mean absorbance values (OD) of each Myeloperoxidase on the OY-axis against the concentration of MPO in each Myeloperoxidase (ng/ml) on the OX-axis. 4. Determine the concentration of unknown samples by interpolation from the standard curve. 5. For samples that have been diluted, the active MPO value obtained from the standard curve must be multiplied by dilution factor. Assay characteristics Assay Range: 5 100 ng/ml Sensitivity: The minimum detectable activity, determined as the concentration of MPO at two standard deviations above the mean values of ten samples containing only diluent is 330 pg/ml.
User Protocol CBA024 Rev. 23 May 2005 RFH Page 5 of 6 2 MPO Std. curve OD at 450 nm 1.5 1 0.5 R 2 = 0.9949 0 0 20 40 60 80 100 120 MPO (ng/ml) Figure 1: MPO Curve Figure 2: Myeloperoxidase activity in biological samples in the absence and presence of MPO inhibitor, ABAH. MPO activity was measured in the presence of 20 µm ABAH (4-aminobenzoyl hydrazide), an irreversible inhibitor of myeloperoxidase. Cells were cultured in RPMI 1600 medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared by treating cell pellets with CytoBuster TM Protein Extraction Reagent (see related products) according to the manufacturer s recommended protocol. Normal EDTA and heparin plasma and CSF were acquired from a commercial supplier. The myeloperoxidase activity assay was performed according to the Detailed Protocol. References Weil, S.C., et al. 1988. Science 240, 790. Winterbourn, C.C., et al. 2000. Curr. Opin. Hematol. 7, 53. Brennan, M.L., et al. 2003. New Engl. J Med. 349, 1595. Zheng, L., et al. 2004. J. Biol. Chem. in press Vita, J.A., et al. 2004. Circulation, 110, 1134.
User Protocol CBA024 Rev. 23 May 2005 RFH Page 6 of 6 Related Products Myeloperoxidase Inhibitor-I (ABAH) (Cat. No. 475944) CytoBuster Protein Extraction Reagent (Cat. No. 71009) Myeloperoxidase, Human Polymorphonuclear Leukocytes (Cat. No. 475911) Hydrogen Peroxide (Cat. No. 386790) The following template may be used as a guide for setting up the 96-Well Plate: 1 2 3 4 5 6 7 8 9 10 11 12 A 100 ng/ml 100 ng/ml B 80 ng/ml 80 ng/ml C 60 ng/ml 60 ng/ml D 40 ng/ml 40 ng/ml E 20 ng/ml 20 ng/ml F 10 ng/ml 10 ng/ml G 5 ng/ml 5 ng/ml H 0 ng/ml (Blank) 0 ng/ml (Blank)