of Recombinant 19271755 Proteins of MPO by ELISA Highly purified recombinant MPO proteins were reconstituted to 10 mg/ml with coating buffer. A 100 ml portion of the mixture was then plated to a well of a polystyrene microtitre plate and kept overnight at 4uC. Every plate contained native MPO as a positive antigen control. The plate was washed three times with PBS containing 0.1% Tween-20 . 2% BSA diluted by PBS was used to block the non-specific binding sites. The sera of subjects were diluted to 1:100 by PBST/0.5 M NaCl and were added in duplication. Every plate contained positive, negative and blank controls. The plate was incubated at 37uC for 1 hr and then washed three times with PBST, and the binding was detected with alkaline phosphatase-conjugated goat anti-human IgG at a dilution of 1:5000. The plate was washed three times with washing buffer and the P-nitrophenyl phosphate was used in substrate buffer. The results were recorded as the net absorbance value at 405 nm and samples were considered positive if the A 405 nm exceeded mean+2 S.D. of 
the A 405 nm of the sera from 35 normal blood donors. Purification 17358052 of Insoluble Proteins with 6x His-tagged Protein Purification Kit The harvested bacterial cells were lysed by ultrasonic and the precipitant was collected. The inclusion bodies in the precipitant were suspended in buffer 1 consisting of 50 mM Tris, 1 mM EDTA and 0.5% Triton. After centrifugation at 10000 rpm for 20 min at 4uC, newly formed precipitant was collected and resuspended in buffer 2 consisting of 50 mM Tris, 1 mM EDTA and 1.0%Triton. Similarly, buffer 3, buffer 4 and buffer 5 were sequentially used to wash the insoluble target proteins in inclusion bodies. Then, the inclusion bodies were dissolved in 8 M urea buffer and the supernatant was collected by centrifugation at 10000 rpm for 20 min at 4uC. Saracatinib Finally, the target protein fragments were purified by 6x Histagged protein purification kit. 4 Epitopes of MPO-ANCA Inhibition ELISA Cross-reaction between antibodies against the recombinant proteins and native myeloperoxidase was investigated using inhibition ELISA. In brief, polystyrene microtiter plates were coated with myeloperoxidase. The diluted sera were preincubated with either myeloperoxidase or recombinants, at increasing concentrations from 0.5 to 50 mg/ml at 37uC for 2 hr. The mixtures were then transferred to the myeloperoxidase-coated microtiter plates and the bound autoantibodies were detected with alkaline phosphatase-conjugated secondary antibodies, as described above. The absorbance value of the sera without the inhibitor was defined as 1, and the absorbance value of the sera preincubated with inhibitor was expressed relative to that. Similarly, the crossreaction between antibodies against the H1 fragment and human a3NC1 was also performed using inhibition ELISA with a polystyrene microtiter plate coated with human a3NC1. Statistical Analysis Differences in quantitative parameters were assessed using t test or nonparametric test. Differences in qualitative data were compared using x2 tests. The difference was considered significant if a P value was,0.05. Analysis was performed with SPSS statistical software package. The Reactivity of ANCA against the Linear Peptides of MPO The cutoff values of positive reactivity, built up by normal sera testing, for recombinants P, L, H1, H2, H3 and H4 were 0.14, 0.14, 0.23, 0.20, 0.14 and 0.23, respectively. The MPO-ANCA positive sera were demonstrated to recognize all the si