FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
goat anti –Dog Myeloperoxidase
Collect Sample – serum or blood plasma
Catalogue No.: QRCT –560018EIA \ UTL
Package size: 96 determinations
Storage: 2 - 8 °C
I. INTRODUCTION
Myeloperoxidase (MPO) is a hemoprotein that is abundantly expressed in polymorphonuclear leukocytes (neutrophils) and secreted during
their activation. The presence of a peroxidase in the cytoplasmic granules of leukocytes was suggested at the beginning of 20th century but it was the
early 1940s that it was purified for the first time. Native MPO is a covalently bound tetrameric complex of two glycosylated alpha chains (MW 59 – 64
kDa) and two unglycosylated beta chains (MW 14 kDa) with total MW about 150 kDa and theoretical pI 9.2.MPO plays an important role in neutrophil
microbicidal action through catalyzing chloride ion oxidation to hypochlorous acid, which is a potent antimicrobial agent. On the other hand, it was
demonstrated that MPO causes oxidative modification of low density lipoprotein (LDL) to a high uptake form that is considered to be a key event in the
promotion of atherogenesis. That’s why MPO is believed to participate in the initiation and progression of cardiovascular diseases. MPO possesses
potent proinflammatory properties and may contribute directly to tissue injury. In addition,MPO is shown to be involved in pathogenesis of lung cancer ,
Alzheimer’s disease and multiple sclerosis .Now MPO is believed to be one of the most promising cardiac markers. Recently it was demonstrated that
an increased MPO level in patient’s blood serves as a risk marker for atherosclerosis and coronary artery disease.It predicts the early risk of myocardial
infarction,as well as the risk of other major adverse cardiac events in patients with chest pain in the ensuing 30-day and 6-month periods . The value of
MPO as a marker is in that MPO predicts these outcomes independently of other known laboratory tested risk factors, including troponins, creatine
kinase MB isoform (CK-MB)，C-reactive protein (CRP) and lipid profile.Moreover, unlike troponins I and T, CK-MB, and CRP, MPO makes it possible
to identify patients at risk for cardiac events in the absence of myocardial necrosis. All these factors make MPO measurements in patients an
indispensable procedure to reveal patients with chest pain that are at increased risk of cardiovascular complications.
.
II. REAGENTS
Materials provided with the kits :
.
1、Coated Microtitration Strips 96 wells.
2、Standard 2.5, 5.0, 10, 20, 40uIU/ml, 1 set.
3、Enzyme Conjugate Solution, 12 ml.
4、Substrate
A, 6 ml.
5、Substrate
B, 6 ml.
6、Stopping Solution (1N HCl), 6 ml.
7、Rinsing buffer,60 ml( x 20).
8、dilution,15ml(x5)
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FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
goat anti –Dog Myeloperoxidase
Collect Sample – serum or blood plasma
Catalogue No.: QRCT –560018EIA \ UTL
Package size: 96 determinations
Storage: 2 - 8 °C
Materials required but not provided :
�
Precision pipettes: 0.10, 0.20, and 1.0 ml.
�
Disposable pipette tips.
�
Distilled water.
� Vortex mixer or equivalent.
� Absorbent paper or paper towels.
� Microtiter plate shaker
� Graph paper.
� A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450 nm .
III. STORAGE OF TEST KIT
Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize
exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described above. A microtiter plate
reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is acceptable for use in absorbance
measurement. All reagents should be allowed to reach room temperature before use.
IV. ASSAY PROCEDURE
STEP1
Prepare the ringing buffer by diluting the ringing buffer 20×(example: 60ml+1140ml H2O), Prepare the Sample Diluent by diluting the
Diluent 5×(example: 15ml+60ml H2O)
STEP2
Dilute samples 1:100 distribing 5ul of sample into 500 ul of dilluent .
STEP3
Place 100ul of diluted sample /reagent blank/standard/controls to the wells of the strips. Incubate for 30 min at 37℃。Wash 5 times。
STEP4
Add 100ul of
STEP5
Add 50ul Substrate A and Substrate B to each well。Incubate for 15 min at 37℃。
STEP6
Add 50ul of stop solution。read absorbance at 450nm within 15 min
Enzyme Conjugate to each well。 Incubate for 30 min at 37℃。Wash 5 times。
V. Important Notes
The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. It is recommended
that if manual pipetting is used, no more than 32 wells be used for each assay run, since pipetting of all standards, specimens and controls should be
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FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
goat anti –Dog Myeloperoxidase
Collect Sample – serum or blood plasma
Catalogue No.: QRCT –560018EIA \ UTL
Package size: 96 determinations
Storage: 2 - 8 °C
completed within 3 minutes. A full plate of 96 wells may be used if automated pipetting is available. Duplication of all standards and specimens,
although not required, is recommended.
VI. CALCULATION OF RESULTS
Calculate the average absorbance values (A
) for each set of reference standards, control, and samples. Construct a standard curve by plotting
450
the mean absorbance obtained for each reference standard against its concentration on linear graph paper, with absorbance on the vertical (y) axis and
concentration on the horizontal (x) axis.Using the mean absorbance value for each sample, determine the corresponding concentration of in from the
standard curve.
VII. EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450nm shown in the Y axis against concentrations shown in the X axis. This
standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and
standard curve.
VIII. CALCULATION OF RESULTS
The minimum detectable concentration of MPO in this assay is estimated to be 0.1μIU/ml.
IX. LIMITATIONS OF THE PROCEDURE
Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert
instructions and with adherence to good laboratory practice. The wash procedure is critical. Insufficient washing will result in poor precision and falsely
elevated absorbance readings. The results obtained from the use of this kit should be used only as an adjunct to other diagnostic procedures and
information available to the physician.
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