MPO Serum Plasma ELISA

Additional Information

Assay Background

Myeloperoxidase (MPO) is a specific polymorphonuclear enzyme that is most abundantly expressed in neutrophil granulocytes. It functions in the oxygen-dependent killing of microorganisms and was released from primary granules of neutrophils during acute inflammation. MPO is the product of a single gene, which is about 11 kb in size, composed of 11 introns and 12 exons, and located in the long arm of chromosome 17 in segment q12-24. The mature 150 kDa MPO protein is a dimer consisting of two 15 kDa light chains and two heavy chains of variable degrees of glycosylation. MPO is related to both inflammation and oxidative stress. It is a sensitive predicator for myocardial infarction in presenting with chest pain. Studies have indicated that MPO is causally linked to atherosclerosis. Moreover, a combination test of MPO and CRP (C-reactive protein) provides added benefit for risk prediction of cardiovascular mortality than just measuring CRP alone. This assay utilizes a specific monoclonal antibody to capture MPO in test samples to ensure that only myeloperoxidase is detected.

Assay Procedure

1. Add 100 µl of Standards, Controls and diluted samples (diluted beforehand 1:5 with assay buffer) into the designated microwells.
2. Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 1.5 hr. ± 5 minutes at 400 to 450 rpm.
3. Just prior to the end of the incubation time, dilute the proper amount of Tracer Antibody for the assay.
4. Wash each well five times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
5. Add 100 µL of above Tracer Antibody to each well.
6. Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 45 minutes ± 5 minutes at 400 to 450 rpm.
7. Wash each well five times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
8. Add 100 µL of ELISA HRP Substrate into each of the wells.
9. Cover the plate with aluminum foil or other material to avoid exposure to light. Incubate plate static, at room temperature for 20 minutes.
10. Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
11. Read the absorbance at 405 nm with reference filter at 620 nm or 650 nm.

Typical Standard Curve

Expected Values

Plasma samples from normal healthy adults ages 20 – 60 were collected and measured with this ELISA. Because each of the serum and plasma samples were initially diluted 1:5, the measured value of each sample has to be multiplied by 5 for the true result. The recommended normal cut – off for serum myeloperoxidase concentration by using this ELISA is 220 ng/mL, and the normal cut off for EDTA – plasma myeloperoxidase concentration is 40 ng/mL. We strongly recommend for each clinical laboratory to establish its own normal cut – off level by measuring normal serum or EDTA – plasma samples with this ELISA.