ELIZEN MPO. Immunoenzymometric assay For the quantitative determination of MYELOPEROXIDASE in biological fluids. Code number: E-HB-96

Transcription

1 ELIZEN MPO Immunoenzymometric assay For the quantitative determination of MYELOPEROXIDASE in biological fluids ELISA assay (96 determinations) Code number: E-HB-96 TECHNICAL AND CLINICAL CHARACTERISTICS ELIZEN MPO / E-HB-96/ rev 0 Page 1 of 14

2 TABLE OF CONTENTS 1. Introduction 1.1 Structure and function 1.2 Clinical applications 2. Technical characteristics 2.1. Assay principle 2.2. Reagents supplied 2.3. Reagents preparation & conservation 2.4. Specimen collection and preparation 2.5. Assay procedure 2.6. Typical calibration curve 2.7. Assay characteristics Sensitivity Precision Accuracy Limitation Troubleshooting 2.8. Reference intervals 3. Correlation between Zentech and competition 4. Features 5. Literature ELIZEN MPO / E-HB-96/ rev 0 Page 2 of 14

3 1.INTRODUCTION 1.1. Structure and function Myeloperoxidase (MPO) is a hemoprotein with a molecular weight of 140 kda, composed of two glycosylated alfa chains and two unglycosylated beta chains. It is a major constituent of cytoplasmic primary granules of neutrophilic myeloid cells. Being an actor of the defense mechanism of the polymorphonuclear leukocytes, the functional role of myeloperoxidase and other enzymes contained in primary granules is destruction of microorganisms and debris within the phagocytic vacuole of the cell. When MPO is released into the phagosome and the extracellular medium, it catalyzes the oxidation of substances into powerful oxidative agents, like the chloride anion (Cl-) into hypochlorous acid (HOCl) thanks to hydrogen peroxide (H ). The reactive species are used in the defense against pathogens but they can inflict oxidative damage on biological targets, including lipids and proteins Clinical applications In many inflammation situations, MPO is released into extracellular medium where its measurement can be used as an index of neutrophil activation and a marker of oxidative stress. Furthermore, involvement of MPO has been described in numerous diseases such as atherosclerosis, lung cancer, Alzheimer s disease and multiple sclerosis and it has been shown that MPO might be involved in several articular diseases including rheumatoid arthritis and osteoarthritis. ELIZEN MPO / E-HB-96/ rev 0 Page 3 of 14

4 2.TECHNICAL CHARACTERISTICS 2.1. Assay principle ELIZEN MPO is a sensitive solid phase two-site enzyme linked immunosorbent assay. The microtiter plates are coated with a polyclonal antibody to human MPO. Patient sample prior diluted is added into the wells and incubated. After carefull washing of the microplate wells, an enzyme coupled to an antibody to human MPO is added in the wells and incubated. After washing away any unbound material, the enzyme activity is measured by adding a chromogen substrate. The reaction is stopped and the intensity of colour development proportional to the concentration of MPO in the patient sample is measured at 405 and 450nm with a spectrophotometer Reagents supplied Each assay contains reagents for 96 wells, sufficient to measure 40 patients samples in duplicate. Reagents Quantity Physical state Stability after reconstitution Microtiterplate 1 x 96 Ready for use - Plate sealers Calibrators x 1.5 ml Lyophilized Stable two months at 2-8 C Control plasma 2 x 1.5 ml Lyophilized Stable two months at 2-8 C HRP conjugate 1 x 15 ml Ready for use - Sample diluent 1 x 53 ml Ready for use - Washing Solution 1 x 100 ml Concentrated 10 x Stable one month at 2-8 C Chromogenic substrate 1 x 15 ml Ready for use - Blocking Reagent 1 x 15 ml Ready for use - ELIZEN MPO / E-HB-96/ rev 0 Page 4 of 14

5 2.3. Reagents preparation and conservation Before opening or reconstitution, all the components are stable until the expiry date shown on the vial label, if kept at 2 to 8 C. A. Calibrators: 6 vials of lyophilized calibrators. Reconstitute with 1.5mL of distilled water. For the exact value, refer to the data sheet included. Store lyophilized at 2 8 C and after reconstitution, store at 2-8 C for 2 months. B. Control plasma: 2 vials of lyophilized controls. Reconstitute with 1.5mL of distilled water. For the exact value, refer to the data sheet included. Store lyophilized at 2 8 C and after reconstitution, store at 2-8 C for 2 months. C. Washing solution: 1 vial of 100mL of phosphate buffer. Bring the vial content to 1000mL (final volume) with distilled water. The diluted washing solution is stable for 1 month at 2-8 C. If undissolved crystals are detected, put back into solution by placing the vial at 37 C for few minutes Specimen collection and preparation It is recommend to use EDTA PLASMA for correct determination in order to avoid any Polymorpho- nuclear degranulation and a release of circulating MPO. Avoid using hemolized, lipemic or bacterially contaminated samples. Thoroughly mix thawed specimens before assay and avoid repeated freeze/thawing cycles which may cause loss of antibody activity and give erroneous results. Samples may be stored at 2-8 C for up to 24 hours. For long term storage, samples should be stored frozen at -20 C or lower. ELIZEN MPO / E-HB-96/ rev 0 Page 5 of 14

6 2.5. Assay Procedure Procedural notes In order to obtain reproducible results, the following rules must be observed: - Do not mix reagents of different lots. - Do not use reagents beyond their expiry date. - Use thoroughly clean glassware. - Use distilled water, stored in clean containers. - Avoid any contamination among samples; for this purpose, disposable tips should be used for each sample and reagent. - Allow reagents and samples to warm up at room temperature. - Mix samples by inversion before use. Sample pre-dilution Before testing, samples must be diluted 1:21 as follows : 25µl of sample + 500µl of sample diluent. Procedure 1. Allocate the wells of microtiterplate for calibrators, controls and samples 2. Pipette 100µl of each calibrator, control serum and diluted sample into the corresponding wells. 3. Incubate for 60 minutes at 37 C (NB. The incubation time begins after the last sample addition) 4. Wash the wells 4 times with 300µl of diluted washing solution. Aspirate all liquid from the wells. 5. Add 100µl of HRP conjugate into the wells. 6. Incubate for 60 minutes at 37 C. 7. Wash the wells 4 times with 300µl of diluted washing solution. Aspirate all liquid from the wells. 8. Pipette 100µl of substrate-chromogen solution into the wells 9. Incubate 15 minutes at 37 C, avoid direct light exposure (NB. The incubation time begins after the first TMB addition) 10. Pipette 100µl of blocking reagent into the wells 11. Read the absorbance of the wells (450 and 405nm) 620nm as reference. Reading must be completed within 20 minutes from the end of the assay. N.B. In order to obtain a better sensitivity, the present method employs spectrophotometric reading at two wavelengths (450 and 405 nm). For all O.D. overflow at 450nm, mutliply the O.D. 405nm by the correction factor calculated by the ratio between O.D. 450nm and O.D. 405nm. To establish this factor, use a sample reading at 405nm and 450nm. ELIZEN MPO / E-HB-96/ rev 0 Page 6 of 14

7 Assay Scheme Wells Blank Calibrators (0-5) Controls Samples Reagents Calibrator (0-5) µl Control plasma µl ---- Diluted samples µl - Cover the plate and incubate: 60' at 37 C - Aspirate and wash: 4 X 300 µl Conjugate µl 100 µl 100 µl - Cover the plate and incubate: 60' at 37 C - Aspirate and wash: 4 x 300 µl Chrom-Substrate 100 µl 100 µl 100 µl 100 µl - Cover the plate an d incubate: 15' at 37 C Blocking Reagent 100 µl 100 µl 100 µl 100 µl - Read : nm and 620 nm as reference Typical calibration curve Description Concentration (ng/ml) O.D Calibrator ,075 Calibrator ,263 Calibrator ,812 Calibrator ,268 Calibrator ,165 Calibrator ,807 Control plasma ,649 Control plasma ,505 ELIZEN MPO / E-HB-96/ rev 0 Page 7 of 14

8 Example of a typical calibration curve for myeloperoxidase Abs. (OD) 6,000 5,000 4,000 3,000 2,000 1,000 0,000 0,1 1,0 10,0 100,0 Conc. (ng/m l) 2.7. Assay Characteristics Analytical sensitivity The sensitivity was calculated upon the calibration curve and expressed as the minimal dose showing a significant difference from the zero calibrator (mean value + 2 S.D.). This dose is 0.21 ng/ml Precision Intra-assay Sample Mean ± S.D. C.V. Replicates value (ng/ml) (%) N ± ± ± Inter-assay Sample Mean ± S.D. C.V. Replicates value (ng/ml) (%) N ± ± ± ELIZEN MPO / E-HB-96/ rev 0 Page 8 of 14

9 Accuracy Linearity of Sample Dilution Dilution Measured Expected Recovery (ng/ml) (ng/ml) (%) 1/ / / / / % of recovery = measured MPO concentrations X 100 % expected MPO concentrations Recovery test Sample Measured Expected Recovery (ng/ml) (ng/ml) (ng/ml) (%) S S1 + Cal S1 + Cal S1 + Cal S1 + Cal S1 + Cal S1 + Cal % of recovery = measured MPO concentrations X 100 % expected MPO concentrations ELIZEN MPO / E-HB-96/ rev 0 Page 9 of 14

10 Limitation The autoantibodies directed against human MPO present in some patient capture the circulating enzyme and mask some of its epitopes involving an undervaluation of the quantity of MPO really present in the sample Troubleshooting Unexpected colour development: - inadequate incubation time - inadequate incubation procedure - uncontrolled water ingredients Drift: - inadequate volume of reconstitution for the calibration curve - expiration date for the reagents exceeded Poor precision: - non-homogeneous sample after freezing - turbidity, particles or high lipid content of the sample - carry over between samples/ calibrators - unequal volumes added to the wells - inadequate aspiration of fluids - incomplete washing 2.8. Normal values The normal range values determined is only indicative since various agents may affect them. It is recommended that each laboratory establish its own range of normal MPO levels. n Mean +/- SD (ng/ml) Plasma EDTA /- 6.0 ELIZEN MPO / E-HB-96/ rev 0 Page 10 of 14

11 3. CORRELATION WITH COMPETITORS The ELIZEN MPO assay was compared to one commercial ELISA immunoassay Comparison of methods: ELIZEN MPO ZENTECH Commercial ELISA immunoassay Washing step 5 x 250 µl Calibrators & Controls 100µL 100 µl Samples 100µl 100 µl Incubation 60' at 37 C 60' R.T. 400 rpm Washing step 4 x 300µl 5 x 250 µl Conjugate-HRP 100µl 100µl Incubation 60' at 37 C 60 ' R.T. 400rpm Washing step 4 x 300µl 5 x 250 µl Substrate-chromogen solution 100µl 100µl Incubation 15' at 37 C 20-30' R.T. Stop 100µl 50µl Read nm (réf. 620nm) nm (réf. 690nm) Total Timing 2h15 2h30 Material: 31 human plasma samples were analyzed ELIZEN MPO / E-HB-96/ rev 0 Page 11 of 14

12 Correlation graph: Correlation (Zentech/Commercial ELISA immunoassay) 180,0 Commercial ELISA immunoassay (ng/ml) 160,0 140,0 120,0 100,0 80,0 60,0 40,0 20,0 0,0 0,0 50,0 100,0 150,0 200,0 Zentech (ng/ml) The correlation coefficient between the two kits is r = 0,9910. The regression analysis comparison equation is y (ZENTECH)= Y = X ELIZEN MPO / E-HB-96/ rev 0 Page 12 of 14

13 4. TECHNICAL AND CLINICAL FEATURES ELIZEN MPO offers several advantages over competitor s assays: - CE marked ELISA kit - Short incubation time: results in 2 hours 15 minutes. - High stability of myeloperoxidase in calibrators and controls ; the lyophilized state guarantees a good stability of MPO concentration before use. - Convenient to use: - Microplate ready for use, so there is no pre-washing step. - All the reagents are ready for use except the calibrators, controls and wash solution. - Rehydrated curve ready for use and stable at 2-8 C during 2 months - High sensitivity: detection limit < 0.4 ng/ml. - Incubation temperature of 37 C, so there is no variation of test s conditions if the procedure is respected. - Convenient for automatization. ELIZEN MPO / E-HB-96/ rev 0 Page 13 of 14

14 5. Literature 1. Brennan M-L, Penn MS, Van Lente F, Nambi V, Shishehbor MH, Aviles RJ, Goormastic M, Pepoy ML, McErlean ES, Topol EJ, Nissen SE, Hazen SL. Prognostic value of myeloperoxidase in patients with chest pain. New England J. Med. 2003;349: Joseph A. Vita et al, (2004) Circulation. 110 : Franck T., Grulke S, Deby-Dupont G, Deby C, Duvivier H, Peters F, Serteyn D. Develoment of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood, J Vet et Diagn Invest. (2005) 17(5) : Nambi V. The use of myeloperoxidase as a risk marker for atherosclerosis (2005) Curr Atheroscler. Rep. 7(2): Anitra C. Carr, Mark R. McCall, Balz Frei, Oxidation of LDL by Myeloperoxidase and reactive species : reaction pathways and antioxidant protection, Arterioscler. Thromb. Vasc. Biol. (2000) 20: Liege Sciences Park Avenue du Pré Aily, ANGLEUR BELGIUM Tel: (0) Fax : (0) info@zentech.be ELIZEN MPO / E-HB-96/ rev 0 Page 14 of 14