Schultz H, Csernok E, Johnston T W, Lockwood C M, Gross W L
Department of Rheumatology, University of Lübeck, Rheumaklinik Bad Bramstedt GmbH, Germany.
J Immunol Methods. 1997 Jul 14;205(2):127-33. doi: 10.1016/s0022-1759(97)00067-7.
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.
抗天然杀菌/通透性增加蛋白（nBPI）的抗中性粒细胞胞浆抗体（ANCA）在炎症性肠病和囊性纤维化中的诊断意义日益增加。然而，常规检测BPI-ANCA需要大量的纯抗原。由于nBPI难以分离且极易被蛋白水解切割并导致随后的表位丢失，本研究的目的是确定重组BPI（rBPI）是否可作为nBPI的替代品，用作诊断程序中ANCA的靶抗原。因此，使用nBPI、rBPI、未糖基化的rBPI和rBPI的21 kDa氨基末端片段，在不同的酶联免疫吸附测定（ELISA）中对93份BPI-ELISA阳性血清和对照进行了比较。ELISA结果通过免疫印迹得到证实，所有血清均采用间接免疫荧光法（IFT）检测。在两种ELISA系统中，ANCA对nBPI和rBPI的识别一致性为88%（82/93），然而，以相对单位定量的BPI-ANCA结果不太理想，仅具有45%的相关性（p < 0.01）。大多数血清对nBPI、rBPI和未糖基化的rBPI的识别相同，这表明糖基化对抗原识别没有影响。只有两份血清对21 kDa的nBPI呈阳性，这表明ANCA的结合位点要么是构象表位，和/或主要位于BPI分子的羧基末端部分。大多数BPI-ELISA阳性血清在IFT中呈阴性（43%），但也观察到核周（pANCA，30%）、胞浆（cANCA，10%）或非典型ANCA（aANCA，2%）染色模式，以及仅在甲醛固定粒细胞上的胞浆模式（13%）。总体而言，IFT中未观察到BPI-ELISA阳性血清的特征性模式。综上所述，这些数据表明，rBPI为广泛的BPI-ANCA ELISA提供了一种优于nBPI的极佳替代品，在进一步研究BPI-ANCA相互作用中将具有重要价值。
