Membranous nephropathy (MN) results from accumulation of antigen-antibody immune complexes along the subepithelial region of the glomerular basement membranes. Over the last years, 13 target antigens have been discovered and include PLA2R, THSD7A, EXT1 and EXT2, NELL1, SEMA3B, NCAM1, CNTN1, HTRA1, FAT1, PCDH7, NTNG1, PCSK6 and NDNF, accounting for 80-90% of MN antigens. MN associated with many of these antigens have distinctive clinicopathologic findings. It is important to accurately identify the antigen in MN. Immunohistochemical (IHC) and/or immunofluorescence (IF) methods are currently used to detect PLA2R, THSD7A, NELL1, SEMA3B and EXT1/EXT2. However, for the remaining antigens, IHC/IF methods do not exist and are not practical for detection. Here, we developed laser microdissection-based mass spectrometry methodology (LMD/MS) as a one-stop clinical test for the detection of MN antigens using paraffin-embedded kidney biopsy tissue. The LMD/MS test was validated in two steps. LMD/MS was used to detect the antigen in 75 cases of MN with known antigens and correctly identified the antigen in all these cases. Next, LMD/MS was used to identify the antigen in 61 MN cases where the antigen was unknown and identified one of the known antigens in 40 of 61 cases including many of the less common antigens. This lower-than-expected detection rate is explained by intentional enrichment of the cohort with PLA2R-negative MN. Overall, PLA2R was identified in 16.4%, one of the other antigens detected in 49.1%, and in the remaining 34.5% of cases, none of the above antigens was detected. Thus, LMD/MS is an extremely useful and reliable method for the detection of known MN antigens and possibly indicating an unknown MN antigen for eventual discovery.

中文翻译：

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使用激光显微切割和质谱法检测膜性肾病抗原的可靠临床检测


膜性肾病 （MN） 是由于抗原抗体免疫复合物沿肾小球基底膜的上皮下区域积累所致。在过去的几年中，已经发现了 13 种靶抗原，包括 PLA2R、THSD7A、EXT1 和 EXT2、NELL1、SEMA3B、NCAM1、CNTN1、HTRA1、FAT1、PCDH7、NTNG1、PCSK6 和 NDNF，占 MN 抗原的 80-90%。与许多这些抗原相关的 MN 具有独特的临床病理表现。准确鉴定 MN 中的抗原很重要。免疫组织化学 （IHC） 和/或免疫荧光 （IF） 方法目前用于检测 PLA2R、THSD7A、NELL1、SEMA3B 和 EXT1/EXT2。然而，对于其余抗原，IHC/IF 方法不存在，因此无法进行检测。在这里，我们开发了基于激光显微切割的质谱方法 （LMD/MS），作为使用石蜡包埋的肾活检组织检测 MN 抗原的一站式临床测试。LMD/MS 检测分两步验证。LMD/MS 用于检测 75 例具有已知抗原的 MN 病例的抗原，并正确鉴定了所有这些病例的抗原。接下来，使用 LMD/MS 鉴定抗原未知的 61 例 MN 病例中的抗原，并在 61 例病例中的 40 例中鉴定出一种已知抗原，包括许多不太常见的抗原。这种低于预期的检出率可以通过有意富集 PLA2R 阴性 MN 队列来解释。总体而言，PLA2R 在 16.4% 的病例中被鉴定出来，在 49.1% 的病例中检测到其他抗原之一，在其余 34.5% 的病例中，未检测到上述抗原。因此，LMD/MS 是一种非常有用且可靠的方法，可用于检测已知的 MN 抗原，并可能指示未知的 MN 抗原以供最终发现。