Introduction

Antibody mediated rejection is the leading cause of kidney transplant failure. Not all antibodies are harmful and some may be protective. Immunoglulin Gs, of which there are four subtypes, are detected by single antigen bead testing. The aims of this study were to characterise the IgG subclass profiles for class I HLA-specific antibodies in an uncensored post-transplant population and to determine the underlying relationship between reactivity patterns and MFI cut-offs with the pan-IgG assay.

Methods

Patients were recruited to the study who were transplanted in our centre between 2009 and 2014. Prospectively stored post-transplant serum initially underwent a Labscreen Mixed assay and those positive for class I HLA-specific antibody underwent standard SAB testing, EDTA, 1 in 10 dilution and IgG subclass modifications using the Luminex platform. A total of 4947 bead reactions from 51 patients were analysed.

Results

A 1 in 10 dilution was used as a comparator pan-IgG assay for summed subclass and individual subclass linear regression analyses. Using a dilution to standard assay ratio we characterised all reactions for prozone potential i.e. how likely there is to be inhibition related to complement complex formation. We stratified samples into degrees of association and were able to determine suggested MFI thresholds of Log 5.35 for the dilution assay and Log 5.05 for the summed subclass assay when considering a Log MFI of 6.9 (1000) in the standard assay. Using individual subclass dominant reactions (>70%) we were able to determine linear relationships between the 1 in 10 dilution pan-IgG assay and the individual subclass assays (excluding prozone potential reactions for IgG1/3) enabling us to suggest Log MFI thresholds of 5.03, 3.58, 4.3 and 4.05 respectively for IgG1–4.

Discussion

We recommend a 1 in 10 dilution as the optimum pan-IgG comparator assay for a subclass analysis. We advocate the utilisation of the summed subclass assay to determine overall relationships and potential subclass failures. Following others, we recommend serum pre-treatment of the subclass assays to mitigate prozone. We suggest cut-offs for each IgG subclass which should be used with caution given the many inhibitory influences which may include competitive inhibition for bead binding, IgM and IgA interference and under-representation of specific subclasses on the bead panel.

中文翻译：

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研究I类HLA特异性免疫球蛋白G亚类，Pan-IgG单抗原珠试验和补体介导的肾移植受者血清干扰之间的关系。

介绍

抗体介导的排斥反应是肾移植失败的主要原因。并非所有抗体都是有害的，有些可能具有保护性。免疫球蛋白Gs有四个亚型，可通过单抗原珠子检测来检测。这项研究的目的是在未经审查的移植后人群中表征I类HLA特异性抗体的IgG亚类概况，并通过pan-IgG分析确定反应模式与MFI临界值之间的潜在关系。

方法

招募了参加本研究的患者，于2009年至2014年之间在我们中心进行了移植。对预先储存的移植后血清进行了Labscreen混合测定，对I类HLA特异性抗体呈阳性的患者进行了标准SAB测试（EDTA），每10份稀释一次使用Luminex平台修饰IgG和IgG亚类。分析了来自51例患者的4947例珠反应。

结果

每10个稀释液中有1个稀释液用作比较子泛IgG分析，用于汇总的亚类和单个亚类线性回归分析。使用稀释液至标准测定比例，我们表征了所有反应的前区电位，即与补体复合物形成相关的抑制作用的可能性。当将标准分析中的Log MFI考虑为6.9（1000）时，我们将样品分层为关联度，并且能够确定稀释测定的Log 5.35的建议MFI阈值和总分类法的Log 5.05的建议MFI阈值。使用单个亚类显性反应（> 70％），我们能够确定十分之一的稀释泛IgG测定与单个亚类测定（不包括IgG1 / 3的前带潜在反应）之间的线性关系，从而使我们能够提出Log MFI阈值5.03、3.58、4.3和4。

讨论区

我们建议以十分之一的稀释度作为亚类分析的最佳pan-IgG比较剂测定法。我们提倡利用汇总的子类分析来确定整体关系和潜在的子类故障。在其他方面之后，我们建议对亚类测定法进行血清预处理以减轻前带区。考虑到许多抑制性影响，包括竞争性抑制磁珠结合，IgM和IgA干扰以及磁珠组中特定亚类的代表性不足，我们建议对每个IgG亚类的临界值应谨慎使用。