Description:

The Diagnostic Automation Myeloperoxidase (MPO) ELISA test system is designed to detect IgG class antibodies to MPO in human sera. Wells of plastic microwell strips are sensitized by passive absorption with MPO antigen. The test procedure involves three incubation steps: (1) Test sera (properly diluted) are incubated in antigen coated microwells. Any antigen specific antibody in the sample will bind to the immobilized antigen. The plate is washed to remove unbound antibody and other serum components. (2) Peroxidase Conjugated goat anti-human IgG (γ chain specific) is added to the wells and the plate is incubated. The Conjugate will react with IgG antibody immobilized on the solid phase in step 1. The wells are washed to remove unreacted Conjugate. (3) The microwells containing immobilized peroxidase Conjugate are incubated with peroxidase Substrate Solution. Hydrolysis of the Substrate by peroxidase produces a color change. After a period of time the reaction is stopped and the color intensity of the solution is measured photometrically. The color intensity of the solution depends upon the antibody concentration in the original test sample.

Specimen collection in this test is done by: (1) It is recommended that specimen collection be carried out in accordance with NCCLS document M29: Protection of Laboratory Workers from Infectious Disease. (2) No known test method can offer complete assurance that human blood samples will not transmit infection. Therefore, all blood derivatives should be considered potentially infectious. (3) Only freshly drawn and properly refrigerated sera obtained by approved aseptic venipuncture procedures should be used in this assay (7,8). No anticoagulants or preservatives should be added. Avoid using hemolyzed, lipemic, or bacterially contaminated sera. (4) Store sample at room temperature for no longer than 8 hours. If testing is not performed within 8 hours, sera may be stored between 2°° and 8°C for no longer than 48 hours. If delay in testing is anticipated, store test sera at â??20°C or lower. Avoid multiple freeze/thaw cycles that may cause loss of antibody activity and give erroneous results.

Quality control must be observed in using the MPO, Myeloperoxidase (p-ANCA) test. Each time the assay is run the Calibrator must be run in triplicate. A reagent blank, negative control and positive control must also be included in each assay. Calculate the mean of the three Calibrator wells. If any of the three values differ by more than 15% from the mean, discard that value and calculate the mean using the remaining two wells. The mean OD value for the Calibrator and the OD values for the Positive and Negative Controls should fall within the following ranges:



1. Negative Control < or = 0.250

2. Calibrator > or = 0.300

3. Positive Control > or = 0.500



Where:



a. The OD of the Negative Control divided by the mean OD of the Calibrator should be < or = 0.9.

b. The OD of the Positive Control divided by the mean OD of the Calibrator should be > or = 1.25.

c. If the above conditions are not met the test should be considered invalid and should be repeated.


The Positive Control and Negative Control are intended to monitor for substantial reagent failure and will not ensure precision at the assay cut-off. Additional controls may be tested according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations.