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475919 Myeloperoxidase ELISA Kit

Myeloperoxidase ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
475919
  
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Overview

Replacement Information

Key Spec Table

Species Reactivity
H
Description
Overview

This product has been discontinued.



A standard sandwich ELISA intended for the quantitation of human myeloperoxidase in serum and plasma.
Catalogue Number475919
Brand Family Calbiochem®
Application Data
Materials Required but Not Delivered• Distilled or deionized water
• Precision pipettes: 5 µl, 10 µl, 100 µl and 1.0 ml
• Disposable pipette tips
• Plate reader that can read absorbance at 450 nm
• EIA plate shaker capable of shaking 96-well plates at 750 rpm
• Vortex mixer or equivalent
• Absorbent paper
• Graph paper
References
ReferencesNicholls, S. and Hazen, S. 2005. Arterioscler. Thromb. Vasc. Biol. 25, 1102.
Nicholls, S. and Hazen, S. 2004. Jpn. J. Infect. Dis. 57, S21.
Baldus, S., et. al. 2003. Circulation 108, 1440.
Brennan, M., et al. 2003. New Engl. J. Med. 349, 1595.
Olsen, R.L. and Little, C., 1983. Biochem. J. 209, 781.
Engall, E. 1980. Methods Enzymol. 70, 419.
Product Information
Form96 Tests
Format96-well plate
Kit containsMPO Antibody-Coated 96-Well Plate, MPO Standard, Standard Diluent Buffer, Sample Diluent Buffer, Anti-Mouse IgG-HRP , Enzyme Conjugate Diluent, Wash Buffer Concentrate (20X), TMB, Stop Solution, and a User Protocol.
Quality LevelMQ100
Applications
Biological Information
Assay range2.5-40 ng/ml
Assay time4.5 h
Species Reactivity
  • Human
Physicochemical Information
Sensitivity0.25 ng/ml
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 20/21/22-36/37/38

Harmful by inhalation, in contact with skin and if swallowed.
Irritating to eyes, respiratory system and skin.
S PhraseS: 24/25-26-36/37/39-45-61-A09

Avoid contact with skin and eyes.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing, gloves and eye/face protection.
In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Avoid release to the environment. Refer to special instructions/safety data sheet.
Product Usage Statements
Intended useThe Calbiochem® Myeloperoxidase ELISA Kit is intended for the quantitative determination of myeloperoxidase (MPO) in human serum and heparin plasma.
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Harmful
Storage +2°C to +8°C
Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsMPO Antibody-Coated 96-Well Plate, MPO Standard, Standard Diluent Buffer, Sample Diluent Buffer, Anti-Mouse IgG-HRP , Enzyme Conjugate Diluent, Wash Buffer Concentrate (20X), TMB, Stop Solution, and a User Protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
475919 0

Documentation

Myeloperoxidase ELISA Kit Certificates of Analysis

TitleLot Number
475919

References

Reference overview
Nicholls, S. and Hazen, S. 2005. Arterioscler. Thromb. Vasc. Biol. 25, 1102.
Nicholls, S. and Hazen, S. 2004. Jpn. J. Infect. Dis. 57, S21.
Baldus, S., et. al. 2003. Circulation 108, 1440.
Brennan, M., et al. 2003. New Engl. J. Med. 349, 1595.
Olsen, R.L. and Little, C., 1983. Biochem. J. 209, 781.
Engall, E. 1980. Methods Enzymol. 70, 419.

Brochure

Title
Kit SourceBook - 2nd Edition EURO
User Protocol

Revision21-September-2007 JSW
Form96 Tests
Format96-well plate
Specieshuman
StorageUpon arrival store the entire contents of the kit at 4°C.
Intended useThe Calbiochem® Myeloperoxidase ELISA Kit is intended for the quantitative determination of myeloperoxidase (MPO) in human serum and heparin plasma.
BackgroundMyeloperoxidase (MPO) plays an important role in host defense systems. This 140 kDa protein, composed of two heavy chains of 53 kDa each and two light chains of 15 kDa each, was first discovered in the 1960s. The release of MPO from the granules of neutrophils and monocytes in response to the activation of leukocytes allows the conversion of hydrogen peroxide and chloride ions into hypochlorous acid (HOCl), a strong oxidizing agent. Although MPO serves an important purpose in the defense system, various studies show that MPO plays a role in several inflammatory conditions. An elevated level of MPO has been linked to coronary artery diseases. In atherosclerosis, a condition that involves hardening of artery walls, high concentrations of MPO and its oxidation products can be found in areas of atheroma. MPO has also been linked to vulnerable plaque and endothelial dysfunction. Nitric oxide (NO) signals blood vessels to relax and dilate, but when MPO uses NO as a substrate it can lead to atherosclerosis and endothelial dysfunction. MPO also affects cholesterol levels through the oxidation of LDL and HDL. Other studies show that an elevated MPO level in serum and plasma indicate a higher risk for cardiovascular events in patients hospitalized for chest pain. Patients with low CRP and TnT levels but a high MPO level are at greater cardiac risk than patients with low CRP, TnT and MPO levels. In the study by Baldus, results indicate that an elevated MPO level occurs before myocardial injury and this increase in MPO indicates unstable plaque formations, making MPO a good predictor of potential negative cardiac events.
Principles of the assayThe Calbiochem® Myeloperoxidase ELISA Kit is based on the principle of solid phase, sandwich, enzyme-linked immunosorbent assay. The assay system utilizes a unique monoclonal antibody specific for a distinct antigenic determinant on human MPO. This mouse anti-MPO monoclonal antibody is coated on the wells of a 96-well plate and is used to capture the MPO standard, as well as MPO from serum and plasma samples. MPO is then detected using a second mouse anti-MPO monoclonal antibody conjugated to horseradish peroxidase (HRP). TMB substrate is added and the plate is incubated for 20 min with shaking, until the development of a blue color occurs. The color development is stopped with the addition of Stop Solution, which changes the color from blue to yellow. The absorbance is measured spectrophotometrically at 450 nm. The amount of MPO in each sample is directly proportional to the color intensity. The concentration is measured by comparing unknown samples to a standard curve.
Materials provided• MPO Antibody-Coated 96 Well Plate (Kit Component No. KP31803): 1 plate, 96-wells
• MPO Standard (Kit Component No. KP31804): 4 vials, lyophilized at 40 ng/ml in bovine serum containing preservatives; please refer to the label for specific reconstitution volume. Note: One time use only; DO NOT REUSE after reconstitution.
• Standard Diluent Buffer (Kit Component No. KP31805): 1 vial, 13 ml, bovine serum containing preservatives
• Sample Diluent Buffer (Kit Component No. KP31806): 1 bottle, 50 ml, phosphate buffer-BSA solution containing preservatives
• Anti-Mouse IgG-HRP Concentrate (25X) (Kit Component No. KP31807): 1 vial, 600 µl, mouse anti-MPO monoclonal antibody conjugated to horseradish peroxidase
• Enzyme Conjugate Diluent (Kit Component No. KP31808): 1 vial, 13 ml, Tris-based buffer containing preservatives
• Wash Buffer (20X) (Kit Component No. KP31809): 1 bottle, 50 ml, phosphate buffer containing detergents
• TMB (Kit Component No. KP31810): 1 vial, 11 ml, ready-to-use
• Stop Solution (Kit Component No. KP31811): 1 vial, 11 ml, 1N HCl
Materials Required but not provided• Distilled or deionized water
• Precision pipettes: 5 µl, 10 µl, 100 µl and 1.0 ml
• Disposable pipette tips
• Plate reader that can read absorbance at 450 nm
• EIA plate shaker capable of shaking 96-well plates at 750 rpm
• Vortex mixer or equivalent
• Absorbent paper
• Graph paper
Precautions and recommendations• This kit contains human material. The source material used for manufacture of this component tested negative for HBsAg, HIV 1/2, and HCV by FDA-approved methods. However, no method can completely assure absence of these agents. Therefore, all human blood products, including serum and plasma samples, should be considered potentially infectious. Handling should be as defined by an appropriate national biohazard safety guideline or regulation, where it exists.
• Avoid contact with the Stop Solution (1N HCl). It may cause skin irritation and burns. If contact occurs, wash with copious amounts of water and seek medical attention if irritation persists.
• Do not mix or use components from kits with different lot numbers.
• Replace caps on reagents immediately. Do not switch caps.
• Do not pipette reagents by mouth.
• Pipetting of all standards, samples, and controls should be completed within 15 min.
• All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same.
• It is recommended that the wells be read within 15 min following addition of Stop Solution.
• Good laboratory practice requires that quality control specimens (controls) be run with each calibration curve to verify assay performance. To ensure proper performance, control material should be assayed repeatedly to establish mean values and acceptable ranges.
• Avoid grossly hemolytic (bright red) samples (after centrifugation). Hemolyzed samples will give inaccurate results.
• Specimens should not be repeatedly frozen and thawed prior to testing. DO NOT store in "frost free" freezers, which may cause occasional thawing. Specimens that have been frozen, and those that are turbid and/or contain particulate matter, must be centrifuged prior to use.
• Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the user protocol and with adherence to good laboratory practice.
• The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
• Serum and plasma samples may contain human anti-mouse antibodies (HAMA) that are capable of giving falsely elevated or depressed results with assays that utilize mouse monoclonal antibodies. This assay has been designed to minimize interference from HAMA-containing specimens. Nevertheless, complete elimination of this interference from all samples cannot be guaranteed. Results should be interpreted with caution.
Preparation• Heparin Plasma Samples: Whole blood should be collected in sodium heparin tubes using standard venipuncture techniques. Invert the tube several times to adequately mix the blood with the anticoagulant. Blood tubes should be stored at room temperature for at least 2 h, but no more than 5 h before centrifuging samples at 2,500 rpm for 20 min at 4°C. Following centrifugation, remove the plasma supernatant and store at 4°C for up to 48 h. Aliquot and freeze (-20°C or below) for long term storage. • Serum Samples: Whole blood should be collected using standard venipuncture techniques. Invert the tube several times to adequately mix the blood. Blood tubes should be stored at room temperature for at least 2 h, but no more than 5 h before centrifuging samples at 2,500 rpm for 20 min at 4°C. Following centrifugation, remove the serum supernatant and store at 4°C for up to 48 h. Aliquot and freeze (-20°C or below) for long term storage. • Sample Dilution: Patient serum and heparin-plasma should be diluted 100 fold prior to use. For example mix 5 µl serum or heparin plasma with 495 µl Sample Diluent Buffer. Samples with expected MPO concentrations over 4 µg/ml should be further diluted with Sample Diluent Buffer and re-assayed. For example, mix 30 µl of the 100-fold diluted sample with 270 µl Sample Diluent Buffer.
Reagent preparationNote: Warm all reagents to room temperature (18-25°C) prior to use.
• MPO Standard: Reconstitute one vial lyophilized MPO Standard with dH2O as indicated on the label. Allow the reconstituted material to stand at room temperature for approximately 20 min and mix gently. Prepare MPO Standard dilutions using Standard Diluent Buffer as outlined in the table below. Reconstituted and diluted MPO standards should be discarded after use. A new vial of MPO Standard should be reconstituted for each assay. Four vials of MPO Standard are provided for a maximum of 4 separate test runs.

Table 1: Dilution of MPO Standards

• 1X Anti-Mouse IgG-HRP: Dilute the Anti-Mouse IgG-HRP Concentrate (25X) 1:25 with the Enzyme Conjugate Diluent. To determine the volume need for the number of strips, refer to the table below. Note: Do not reuse the 1X Anti-Mouse IgG-HRP; prepare a fresh dilution before each assay.

Table 2: Preparation of 1X Anti-Mouse IgG-HRP

• 1X Wash Buffer: Dilute the Wash Buffer (20X) 1:20 with diH2O. For example, add 50 ml Wash Buffer (20X) to 950 ml diH2O. The 1X Wash Buffer is stable at 4°C for 30 days. Note: If crystals are present in the Wash Buffer (20X), they must be redissolved at room temperature before diluting.
Detailed protocol1. Remove the the desired number of strips from the 96-well plate and place them in the holder. Store the unused strips in the pouch at 4°C.
2. Add 100 µl diluted MPO standards and diluted samples to designated wells.
3. Incubate for 90 min at room temperature (18-25°C) on an orbital shaker set at ~750 rpm.
4. Empty the wells by shaking the contents into the sink or other appropriate waste container. Add 300 µl 1X Wash Buffer to each well to rinse and empty the contents of the wells as outlined above. Repeat for a total of 5 times. Following the final wash, gently tap the inverted plate on absorbent paper towels to remove all residual liquid.
5. Add 100 µl 1X Anti-Mouse IgG-HRP to each well.
6. Incubate for 90 min at room temperature (18-25°C) on an orbital shaker set at ~750 rpm.
7. Wash the wells as outlined in step 4.
8. Add 100 µl TMB to each well.
9. Incubate for 20 min at room temperature (18-25°C) on an orbital shaker set at ~750 rpm.
10. Stop the reaction by adding 100 µl Stop Solution to each well.
11. Gently mix for 30 s. It is important to make sure that all the blue color completely changes to yellow.
12. Read the absorbance at 450 nm.
Calculations1. Calculate the mean absorbance value for each set of reference standards, controls, and samples. 2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in ng/ml on graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis. 3. The corresponding concentration of MPO (ng/ml) can be determined from the standard curve using the mean absorbance value for each sample. Depending on experience and/or the availability of computer capability, other methods of data reduction may also be used. 4. The obtained values of serum and heparin-plasma patient samples should be multiplied by the dilution factor of 100 to obtain MPO results in ng/ml. Samples with MPO concentrations greater than 4 µg/ml should be further diluted as outlined in the Sample Preparation section and the final MPO values should be multiplied by 1,000 to obtain MPO results in ng/ml. Samples with MPO concentrations less than 0.25 ng/ml before multiplying by dilution factor (1:100) should be reported as "< 25 ng/ml MPO.
Standard curveResults of a typical standard curve are shown below. NOTE: This raw data and standard curve are provided only as an example and should not be used to calculate the MPO concentration in your samples. Each user must run a standard curve with each assay.

Table 3: Example Standard Curve Raw Data

Figure 1: Example Standard Curve
Example dataIt is recommended that each user establish a normal range based on the patient population from which the samples are derived. The MPO ranges obtained from apparently normal human samples using the Calbiochem® Myeloperoxidase ELISA Kit and with another commercially available MPO ELISA are shown below.

Table 4: Expected Values
Sensitivity0.25 ng/ml
Sensitivity NotesThe minimum detectable concentration of MPO as measured by 2SD from the mean of a zero standard is estimated to be 0.25 ng/ml, therefore the ELISA test has a minimum detectablilty of 25 ng/ml after multiplying by the dilution factor of 1:100.
Assay Range2.5-40 ng/ml
Precision

Table 5: Intra-Assay Precision

Intra-assay precision was determined by 20 replicate determinations of 4 different serum samples in a single assay.



Table 6: Inter-Assay Precision

Inter-assay precision was determined by 5 replicate measurements of 4 different serum samples over a series of individually calibrated assays.
RecoveryVarious patient serum samples of known MPO levels were combined and assayed in duplicate. The mean recovery was 101.9%.

Table 7: Recovery
LinearityFive serum samples were serially diluted to determine linearity. The mean recovery was 109.0%.

Table 8: Linearity
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
Interactive Pathways™ is a trademark of EMD Chemicals, Inc.