Zhao M H, Lockwood C M
Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, UK.
J Immunol Methods. 1996 Oct 16;197(1-2):121-30. doi: 10.1016/0022-1759(96)00123-8.
Indirect immunofluorescence (IIF) techniques have shown that anti-neutrophil cytoplasm autoantibodies (ANCA) are useful serological markers for certain small vessel vasculitides and the non-vasculitic inflammatory disorders. ELISA procedures, using purified molecules as solid phase ligands, helped to identify proteinase 3 (PR3) and myeloperoxidase (MPO) as two major ANCA antigens; and recently we characterised bactericidal/permeability-increasing protein (BPI) as another important ANCA antigen. ANCA against these three antigens are associated with different clinical disorders. Therefore purified antigens are needed to determine these different autoantibody specificities in order to help diagnosis and guide treatment. Here we describe a method using Orange-A dye ligand chromatography and cation exchange chromatography for the sequential purification of PR3, MPO and BPI, from the same starting material, an acid extract of normal human neutrophil granules. After separation the three antigens were free of contamination by each other and no traces were found of other known minor ANCA antigens.
间接免疫荧光（IIF）技术已表明，抗中性粒细胞胞浆自身抗体（ANCA）是某些小血管炎和非血管炎性疾病有用的血清学标志物。使用纯化分子作为固相配体的酶联免疫吸附测定（ELISA）程序，有助于将蛋白酶3（PR3）和髓过氧化物酶（MPO）鉴定为两种主要的ANCA抗原；最近，我们将杀菌/通透性增加蛋白（BPI）鉴定为另一种重要的ANCA抗原。针对这三种抗原的ANCA与不同的临床疾病相关。因此，需要纯化抗原以确定这些不同的自身抗体特异性，从而有助于诊断和指导治疗。在此，我们描述了一种使用橙黄-A染料配体色谱法和阳离子交换色谱法，从相同起始材料（正常人中性粒细胞颗粒的酸性提取物）中依次纯化PR3、MPO和BPI的方法。分离后，这三种抗原彼此无污染，也未发现其他已知次要ANCA抗原的痕迹。
