Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes
Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes
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DOI：
10.1016/s0022-1759(97)00203-2
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发表时间：
1998-02-01
影响因子：
2.2
通讯作者：
Specks, U
中科院分区：
文献类型：
--
作者：
Sun, JY;Fass, DN;Specks, U
Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils (polymorphonuclear cells, PMNs), is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener's granulomatosis (WG). We have recently developed an expression system for recombinant PR3 (rPR3) that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies (moABs) and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3-ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay (ELISA) for clinical PR3-ANCA testing. Both purified PMN-PR3 and crude human mast cell line (HMC-1)/PR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 (83.5%) and 90 (82.6%) were PR3-ANCA positive by capture ELISA when PMN-PR3 and HMC-1/PR3-S176A cell lysates were used as antigen, respectively. When HMC-1/PR3 and HMC-1/PR3-S176A cells were used as indirect immunofluorescence (IIF) substrate, 88 (80.7%) and 92 (84.4%) were PR3-ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen (both negative by PR3-ANCA specific IIF). The capture ELISA can also be used to detect of PR3-ANCA immunecomplexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids. (C) 1998 Elsevier Science B.V.
