Luminex single antigen flow beads (SAFB) assays are used to monitor anti‐HLA antibodies, which are associated with allograft loss. The primary antibody binding level is evaluated by the mean fluorescence intensity (MFI) provided by the tracing secondary antibody. The aim of this study was to compare the MFI obtained with two secondary antibodies and to evaluate their sensitivity to detect newly developed de novo anti‐HLA donor‐specific antibodies (dnDSA). A total of 23 and 46 different sera from 20 HLA‐sensitized kidney transplant recipients were tested with class I and II SAFB, respectively, as well as sera from 17 patients with dnDSA. The conventional secondary antibody (IgHPolyFab) was the phycoerythrin (PE)‐conjugated goat anti‐human IgG constant heavy chain (HC)‐binding polyclonal‐F(ab')2. The alternate secondary antibody was a PE‐conjugated anti‐human IgG Fc‐specific monoclonal IgG (FcMonoIgG). MFI of negative control sera were drastically lower with the FcMonoIgG than with IgHPolyFab, requesting a unique positivity threshold to be defined for each secondary antibody. MFI obtained with positive control sera were also lower with FcMonoIgG. In kidney transplant recipient's sera, the number of antigenic specificities detected by each secondary antibody was comparable, for dnDSA as well as for nonDSA. In conclusion, decision thresholds must be refined when changing the secondary tracing reagent in SAFB assays. FcMonoIgG did not demonstrate any superiority over IgHPolyFab for monitoring HLA antibodies.

中文翻译：

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使用单抗原流珠测定法检测抗HLA IgG的单克隆二级试剂不胜过多克隆二级试剂。

Luminex单抗原流珠（SAFB）分析用于监测与同种异体移植物丢失相关的抗HLA抗体。一抗结合水平通过追踪二抗提供的平均荧光强度（MFI）进行评估。这项研究的目的是比较两种二级抗体获得的MFI，并评估其检测新开发的从头抗HLA供体特异性抗体（dnDSA）的敏感性。I类和II类SAFB分别测试了来自20位HLA致敏的肾移植受者的23种和46种不同的血清，以及17例dnDSA患者的血清。常规的二抗（IgHPolyFab）是藻红蛋白（PE）偶联的山羊抗人IgG恒定重链（HC）结合多克隆F（ab'）2。备用第二抗体是PE缀合的抗人IgG Fc特异性单克隆IgG（FcMonoIgG）。阴性对照血清的MFI与FcMonoIgG相比大大低于IgHPolyFab，要求为每个二抗定义一个独特的阳性阈值。FcMonoIgG阳性对照血清获得的MFI也较低。在肾脏移植受者的血清中，对于dnDSA和非DSA，每种二抗检测到的抗原特异性数量都是可比的。总之，在SAFB分析中更改次级示踪剂时，必须完善决策阈值。FcMonoIgG在监测HLA抗体方面没有表现出优于IgHPolyFab的优势。阴性对照血清的MFI与FcMonoIgG相比大大低于IgHPolyFab，要求为每种二抗定义一个独特的阳性阈值。FcMonoIgG的阳性对照血清MFI也较低。在肾脏移植受者的血清中，对于dnDSA和非DSA，每种二抗检测到的抗原特异性数量都是可比的。总之，在SAFB分析中更改次级示踪剂时，必须完善决策阈值。FcMonoIgG在监测HLA抗体方面没有表现出优于IgHPolyFab的优势。阴性对照血清的MFI与FcMonoIgG相比大大低于IgHPolyFab，要求为每种二抗定义一个独特的阳性阈值。FcMonoIgG阳性对照血清获得的MFI也较低。在肾脏移植受者的血清中，对于dnDSA和非DSA，每种二抗检测到的抗原特异性数量都是可比的。总之，在SAFB分析中更改次级示踪剂时，必须完善决策阈值。FcMonoIgG在监测HLA抗体方面没有表现出优于IgHPolyFab的优势。对于dnDSA和非DSA，每种二抗检测到的抗原特异性数量是可比的。总之，在SAFB分析中更改次级示踪剂时，必须完善决策阈值。FcMonoIgG在监测HLA抗体方面没有表现出优于IgHPolyFab的优势。对于dnDSA和非DSA，每种二抗检测到的抗原特异性数量是可比的。总之，在SAFB分析中更改次级示踪剂时，必须完善决策阈值。FcMonoIgG在监测HLA抗体方面没有表现出优于IgHPolyFab的优势。