Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red-Cross Blood Service of Baden-Württemberg-Hessen, Germany.
Vox Sang. 2011 Aug;101(2):147-53. doi: 10.1111/j.1423-0410.2011.01470.x. Epub 2011 Apr 4.
Granulocyte-associated antibodies can cause several clinical granulocytopenic disorders. The monoclonal-antibody-specific immobilization of granulocyte antigens (MAIGA) is currently used as the standard assay to specify these antibodies. Here we describe an assay for specific analysis of granulocyte antibodies (SASGA) which is able to simultaneously detect and specify granulocyte IgG- and IgM-antibodies using flow cytometry.
Bead populations with distinct fluorescence intensities were used as solid phase for immobilization of mAb. Typed granulocytes were incubated with human sera and a mix of three distinct mouse monoclonal antibodies against specific granulocyte antigens (for example CD16, CD11a, HLA class I). After cell lysis and incubation of lysate with beads, goat antibodies against human IgG and IgM antibodies were added. Seventy-one frozen sera of donors and patients previously implicated in transfusion reactions and various underlying disorders were analysed for specific granulocyte-binding antibodies using MAIGA and SASGA.
The SASGA assay was able to simultaneously detect granulocyte-specific antibodies for different glycoproteins. Overall, the results of MAIGA and SASGA were concordant in 92·9%. 5 sera containing anti-HNA-1b (n=2) and -HLA class I (n=3) were not detected by MAIGA, but were recognized by the SASGA. In serial dilution tests with sera containing anti-HNA-1a, -1b, -2a and HLA class I, the SASGA assay detected the antibodies at higher dilutions than MAIGA.
The SASGA assay permits reliable detection of specific granulocyte antibodies. Six distinct antibodies can be simultaneously determined. This method will potentially open the way to investigations on additional specific antibodies as it facilitates laboratory diagnosis.
粒细胞相关抗体可引起多种临床粒细胞减少症。目前，单克隆抗体特异性固定粒细胞抗原（MAIGA）被用作确定这些抗体的标准检测方法。本文介绍了一种用于特异性分析粒细胞抗体（SASGA）的检测方法，该方法能够使用流式细胞术同时检测和鉴定粒细胞 IgG 和 IgM 抗体。
使用具有不同荧光强度的珠群作为固相固定 mAb。将定型的粒细胞与人类血清和针对特定粒细胞抗原（如 CD16、CD11a、HLA Ⅰ类）的三种不同鼠单克隆抗体的混合物孵育。细胞裂解后，将裂解液与珠粒孵育，然后加入针对人 IgG 和 IgM 抗体的山羊抗体。使用 MAIGA 和 SASGA 分析了 71 份先前涉及输血反应和各种潜在疾病的供体和患者的冷冻血清，以分析特异性结合粒细胞的抗体。
SASGA 检测法能够同时检测不同糖蛋白的粒细胞特异性抗体。总体而言，MAIGA 和 SASGA 的结果一致性为 92.9%。5 份含有抗-HNA-1b（n=2）和-HLA Ⅰ类（n=3）的血清不能被 MAIGA 检测到，但被 SASGA 识别。在含有抗-HNA-1a、-1b、-2a 和 HLA Ⅰ类的血清系列稀释试验中，SASGA 检测法在更高的稀释度下检测到抗体。
SASGA 检测法能够可靠地检测特异性粒细胞抗体。六种不同的抗体可以同时确定。该方法将为进一步研究其他特异性抗体开辟道路，因为它有助于实验室诊断。
