Sample
Cell culture supernatants, cell lysates, serum, platelet-poor plasma, saliva, urine
Intended Use
For the quantitative determination of human Myeloperoxidase (MPO) concentrations in cell culture supernates, cell lysates, serum, platelet-poor plasma, saliva, and urine
Contents of Kit
1. Human MPO Microplate: 1 plate, 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody specific for human MPO.
Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at ≤ -20°C*
2. Human MPO Standard: 2 vials, Natural human MPO in a buffered protein base with preservatives; lyophilized.
Refer to the vial label for reconstitution volume.
Use a new standard for each assay Discard after use.
3. Human MPO Conjugate: 2 vials, 11 mL/vial of a polyclonal antibody specifc for human MPO conjugated to horseradish peroxidase with preservatives.
May be stored for up to 1 month at ≤ -20°C*
4. Assay Diluent: 2 vials, 6 mL/vial of a buffered protein base with preservatives.
May be stored for up to 1 month at ≤ -20°C*
5. Calibrator Diluent CD5K: 2 vials, 11 mL/vial of a buffered protein base with preservatives.For cellculture supernate/cell lysate/saliva samples.
May be stored for up to 1 month at ≤ -20°C*
6. Calibrator Diluent CD6-58: 2 vials, 11 mL/vial of a buffered protein base with preservatives. For serum/plasma/urine samples.
May be stored for up to 1 month at ≤ -20°C*
7. Wash Buffer Concentrate: 2 vials, 11 mL/vial of a 25-fold concentrated solution of buffered surfactant with preservatives. May turn yellow over time.
May be stored for up to 1 month at ≤ -20°C*
Color Reagent A: 1 vial, 12 mL of stabilized hydrogen peroxide.
May be stored for up to 1 month at ≤ -20°C*
8. Color Reagent B: 1 vial, 12 mL of stabilized chromogen (tetramethylbenzidine).
May be stored for up to 1 month at ≤ -20°C*
9. Stop Solution: 1 vial, 6 mL of 2N sulfuric add.
May be stored for up to 1 month at ≤ -20°C*
10. Plate Sealers: 4 adhesive strips.
*Provided this is within the expiration date of the kit.
Performance Characteristics
Intra-Assay Precision (Precision within an assay)Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays)Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
Serum/Plasma/Saliva/Urine - Samples from apparently healthy volunteers were evaluated for the presence of human MPO in this assay. No medical histories were available for the donors used in this study.
Cell Culture Supernates:Human monocytes were cultured in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Cells were cultured unstimulated or stimulated with 1 μg/mL LPS for 1 and 6 days. Aliquots of the cell culture supernates were removed and assayed for human MPO.
HL-60 human acute promyelocytic leukemia cells were cultured in RPMI supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. An aliquot of the cell culture supernate was removed, assayed for human MPO, and measured 18.2 ng/mL.
An aliquot of the same HL-60 cells was removed, and the cells were lysed at 5 x 10
6 cells/mL in diluted Cell Lysis Buffer. The lysate was assayed for human MPO and measured 2673 ng/mL.
General Description
Myeloperoxidase (MPO) is a heme-containing enzyme belonging to the XPO subfamily of peroxidases. It is an abundant neutrophil and monocyte glycoprotein that catalyzes the hydrogen peroxide dependent formation of hypochlorus acid (HOCl) and other reactive species (1, 2). Reaction of these compounds with macromolecules results in the nitrosylation, chlorination, and oxidation of tyrosine residues, lipids, and cholesterol, and the intermolecular crosslinking of proteins and DNA (1, 3-8). MPO is synthesized as a preproprotein that is proteolytically processed to remove a 48 amino acid (aa) signal peptide, a 116 aa propeptide, the C-terminal serine, and a 6 aa internal peptide which generates separate 60 kDa heavy and 12 kDa light chains (9). Other post-translational modifications of MPO include insertion of a heme moiety, glycosylation, and phosphorylation of mannose residues (10, 11). Enzymatically active MPO is a disulfide-linked tetramer that contains two heme groups and two copies each of the heavy and light chains (10, 11). Alternate splicing results in two additional isoforms of MPO, one with a 32 aa insertion in the light chain, and another with a deletion of the signal sequence and 47 aa of the propeptide (9). Mature human MPO shares 87-88% aa sequence identity with canine, mouse, and rat MPO. It shares 71%, 56%, and 47% aa sequence identity with comparable regions of human eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively. MPO binds albumin, the macrophage mannose receptor, cytokeratin 1 on vascular endothelial cells, high molecular weight kininogen, and the integrin CD11b/CD18 on neutrophils (12-15). These interactions promote MPO clearance, a reduction of nitric oxide and bradykinin levels, reduced vasodilation, and continued neutrophil activation (12-16). MPO gene expression is under the control of thyroid hormone, retinoic acid, estrogen, and PPARγ receptors (17, 18). A polymorphism within the MPO gene promoter confers increased steroid responsiveness and is associated with the development or severity of a variety of diseases (1, 17, 18).
Neutrophil MPO is stored in cytoplasmic azurophilic granules (19, 20). Upon cellular activation and degranulation, MPO is delivered into phagosomes where it is required for the killing of phagocytosed bacteria (10, 21). Activated neutrophils also release granule contents extracellularly. Elevated plasma MPO levels have been associated with a variety of clinical conditions including systemic inflammation, eclampsia, risk of cardiovascular events, vascular endothelial dysfunction, severity of multiple sclerosis, and prospective mortality and oxidative stress during hemodialysis (22-30). MPO levels are also elevated in arthritic synovial fluid and gingivitis crevicular fluid (31, 32). MPO is incorporated into atherosclerotic plaques and renal stones and serves as an indicator of neutrophil infiltration of inflamed or injured tissues (33-35).
The Human MPO Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human MPO in cell culture supernates, cell lysates, serum, platelet-poor plasma, saliva, and urine.
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