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Development of an ELISA for myeloperoxidase on microplate: normal reference values and effect of temperature on
Pi-Yueh Chang1, Tsu-Lan Wu, Chung-Chih Hung
1Department of Pathology, Chang Gung Memorial Hospital, Taipei, Taiwan.
Background:
Myeloperoxidase (MPO), a leukocyte enzyme, is implicated in both the pathogenesis and the progression of atherosclerosis.
Methods:
We developed a sandwich ELISA on microplate using commercial antibodies for the measurement of plasma MPO.
Results:
The in-house ELISA has a sensitivity of 15 ng/ml. Both within-day and between-day imprecision were <10%. The in-house assay was well correlated with the commercial kit from Oxis (gamma=0.96). We have established normal reference range for MPO for apparently healthy Chinese. No statistical difference was found between males and females and the various age groups. Because the coating antibodies used by two different kits are different in their affinities for MPO, the analysis by the in-house ELISA that was approximately three times that of the Oxis kit when testing the same specimens. We found that it is necessary to keep the heparinized whole blood on ice before centrifugation in order to prevent further release of MPO from the leukocytes at room temperature. For the same reason, serum is not recommended for MPO measurement. We also found that either pooled human plasma or serum containing MPO can be used as calibrators.
Conclusions:
We believe that this ELISA for MPO is useful to assess risk for inflammation, oxidative stress, nitrosative stress, and for predicting cardiac events.
Insights
A new enzyme assay accurately measures myeloperoxidase (MPO), an inflammation marker. This assay helps predict cardiac events and assess risks associated with oxidative stress.
Area of Science:
- Biochemistry
- Cardiovascular Research
- Biomarker Discovery
Background:
- Myeloperoxidase (MPO) is a leukocyte enzyme linked to atherosclerosis development and progression.
- Understanding MPO's role is crucial for cardiovascular disease research.
Purpose of the Study:
- To develop and validate a sensitive enzyme-linked immunosorbent assay (ELISA) for quantifying plasma MPO.
- To establish reference ranges for MPO in a healthy Chinese population.
Main Methods:
- A sandwich ELISA was developed using commercial antibodies for plasma MPO measurement.
- Assay validation included sensitivity, imprecision, and correlation with a commercial kit.
- Optimal sample handling procedures were determined to ensure MPO stability.
Main Results:
- The in-house ELISA demonstrated high sensitivity (15 ng/ml) and low imprecision (<10%).
- The assay showed excellent correlation with a commercial kit (gamma=0.96).
- Normal MPO reference ranges were established for apparently healthy Chinese individuals, with no significant differences across sex or age groups.
Conclusions:
- The developed ELISA is a reliable tool for MPO measurement.
- This assay can aid in assessing risks for inflammation, oxidative stress, and nitrosative stress.
- It holds potential for predicting cardiac events.
