Related Experiment Videos
Use of native and recombinant bactericidal/permeability-increasing proteins (BPI) as antigens for detection of
H Schultz1, E Csernok, T W Johnston
1Department of Rheumatology, University of Lübeck, Rheumaklinik Bad Bramstedt GmbH, Germany.
Abstract:
Anti-neutrophil cytoplasmic antibodies (ANCA) against native bactericidal/permeability-increasing protein (nBPI) have gained increasing diagnostic significance in inflammatory bowel disease and cystic fibrosis. However, routine detection of BPI-ANCA requires pure antigen in large quantities. As nBPI is difficult to isolate and is very susceptible to proteolytic cleavage with subsequent epitope loss, it was the aim of this study to determine whether recombinant BPI (rBPI) can be used as an alternative to nBPI as target antigen for ANCA in diagnostic procedures. Therefore, 93 BPI-ELISA-positive sera and controls were compared in different ELISAs using nBPI, rBPI, unglycosylated rBPI and a 21-kDa amino-terminal fragment of rBPI. ELISA results were confirmed by immunoblotting and all sera were tested in indirect immunofluorescence (IFT). There was an 88% (82/93) agreement in recognition of nBPI and rBPI by ANCA in both ELISA systems, yet the quantitation of BPI-ANCA in relative units showed a less optimal result and correlated only by 45% (p < 0.01). Most sera recognized nBPI, rBPI and unglycosylated rBPI equally suggesting that glycosylation has no influence on antigen recognition. Only two sera were positive for the 21-kDa nBPI indicating that the binding sites for ANCA are either conformational epitopes and/or are located mainly on the carboxy-terminal part of the BPI molecule. Most BPI-ELISA-positive sera were negative in IFT (43%), but a perinuclear (pANCA, 30%), a cytoplasmic (cANCA,10%) or an atypical ANCA (aANCA, 2%) staining pattern, as well as a cytoplasmic pattern only on formaldehyde-fixed granulocytes (13%) were also observed. Overall, no characteristic pattern was seen for BPI-ELISA-positive sera in IFT. Taken together, these data suggest that rBPI offers an excellent alternative to nBPI for broad-based BPI-ANCA ELISA and will be of great value in further investigations of BPI-ANCA interactions.