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Most EIA for demonstrating and quantifying PR3-ANCA still rely on the use of human PR3 purified from normal neutrophils, directly coated on the solid phase. A detailed protocol for isolating azurophil granules from nitrogen bomb cavitated normal neutro-phils has been given elsewhere (6). Procedures for purifying PR3 (7) and MPO (8) from these granules have also been described elsewhere and will not be mentioned here. Puri-fied PR3 and MPO can also be purchased from several commercial suppliers, but the pu-rity of a given preparation as well as its conformational intactness should be tested before use, since both contamination and protein degradation may affect serologic results.
PR3 at a concentration of about 0.2-0.5 mg/ml in 0.05 M sodium carbonate buffer, pH 9.6, with 0.01% Triton X-100 is used for coating high-binding microtiter plates overnight at 40 C, 100 ml/well. Plates are then washed three x in 0.15 M NaCl containing 0.05 % Tween 20. Negative control serum, positive calibrator serum, high, intermediate and low positive control sera and patient sera diluted 1:50 in incubation buffer (Tris 0.05 M, NaCl 0.15 M, Tween 20 0.05 % and bovine serum albumin 0.2 %) are added to duplicate wells (100 ml/well) and incubated for one hour at room temperature, followed by 3 x washing as above. Alkaline phosphatase-labelled goat anti-human IgG (Sigma) diluted 4000 x in incubation buffer is added to each well and incubated at room temperature for 20 min. The plate is washed three x as above and 100 ml paranitrophenyl phosphate sub-strate in diethanolamine 1 M, pH 9.8, is incubated for 30 min. at room temperature, after which OD 405 nm values are read in an ELISA reader. Values in arbitrary units/ml are calculated from the calibration curve.
EIA for MPO-ANCA
High-binding microtiter plates are coated at a concentration of 1 mg/ml of human purified MPO in 0.05 M sodium carbonate buffer, pH 9.6, 100 ml/well, overnight at 40 C. The procedure used for the rest of the assay closely follows the one described above using negative, high, intermediate and low positive control sera and calibrator serum as well as patient sera diluted 1:50.
To set cut-off values of significant clinical utility for the two EIA techniques it is advised to calculate them in vasculitis patients towards inflammatory disease controls (and not only towards healthy donors) using ROC curves (4) aiming at setting a diagnostic speci-ficity of 90-95%. In addition, it is advised to run all samples also on uncoated wells to avoid false positive results due to "sticky" sera.
Other EIA techniques used for ANCA determanation.
Some laboratories use a capture principle for binding PR3 and MPO to the microtiter plates utilizing a non-competing mouse monoclonal antibody on the solid phase to cap-ture the respective antigen. Preliminary investigations indicate that this principle may augment the sensitivity of PR3-ANCA detection without deminishing the diagnostic specificity (9). Preliminary data indicate that the capture EIA technique for MPO-ANCA does not give a similar increase in sensitivity.