Data_Sheet_1_Characterization of the C1q-Binding Ability and the IgG1-4 Subclass Profile of Preformed Anti-HLA Antibodies by Solid-Phase Assays.docx

Humoral alloimmunity, particularly that triggered by preformed antibodies against human leukocyte antigens (HLA), is associated with an increased prevalence of rejection and reduced transplant survival. The high sensitivity of solid phase assays, based on microbeads coated with single antigens (SAB), consolidated them as the gold-standard method to characterize anti-HLA antibodies, ensuring a successful allograft allocation. Mean fluorescence intensity (MFI) provided by SAB is regularly used to stratify the immunological risk, assuming it as a reliable estimation of the antibody-level, but it is often limited by artifacts. Beyond MFI, other properties, such as the complement-binding ability or the IgG1-4 subclass profile have been examined to more accurately define the clinical relevance of antibodies and clarify their functional properties. However, there are still unresolved issues. Neat serum-samples from 20 highly-sensitized patients were analyzed by SAB-panIgG, SAB-IgG1-4 subclass and SAB-C1q assays. All 1:16 diluted serum-samples were additionally analyzed by SAB-panIgG and SAB-IgG1-4 subclass assays. A total of 1,285 anti-HLA antibodies were identified as positive, 473 (36.8%) of which were C1q-binding. As expected, serum-dilution enhanced the correlation between the C1q-binding ability and the antibody-strength, measured as the MFI (rneat = 0.248 vs. rdiluted = 0.817). SAB-subclass assay revealed at least one IgG1-4 subclass in 1,012 (78.8%) positive antibody-specificities. Among them, strong complement-binding subclasses, mainly IgG1, were particularly frequent (98.9%) and no differences were found between C1q- and non-C1q-binding antibodies regarding their presence (99.4 vs. 98.5%; p = 0.193). In contrast, weak or non-C1q-binding subclasses (IgG2/IgG4) were more commonly detected in C1q-binding antibodies (78.9 vs. 38.6%; p < 0.001). Interestingly, a strong association was found between the C1q-binding ability and the IgG1 strength (rIgG1dil = 0.796). Though lower, the correlation between the IgG2 strength and the C1q-binding ability was also strong (rIgG2dil = 0.758), being both subclasses closely related (rIgG1−IgG2 = 0.817). We did not find any correlation with the C1q-binding ability considering the remaining subclasses. In conclusion, we demonstrate that a particular profile of IgG subclasses (IgG1/IgG3) itself does not determine at all the ability to bind complement of anti-HLA antibodies assessed by SAB-C1q assay. It is the IgG subclass strength, mainly of IgG1, which usually appears in combination with IgG2, that best correlates with it.

体液同种免疫，尤其是由预先形成的针对人类白细胞抗原（HLA）的抗体所触发的免疫，与排斥反应的增多和移植存活率的降低密切相关。基于单抗原（SAB）包被微珠的固相检测的高灵敏度，使得SAB成为表征抗-HLA抗体的金标准方法，确保了同种异体移植的合理分配。由SAB提供的平均荧光强度（MFI）通常被用于分层免疫风险，并将其视为抗体水平的可靠估计，但常受到伪影的限制。除了MFI之外，其他特性，如补体结合能力或IgG1-4亚类谱，也被检验以更准确地定义抗体的临床相关性并阐明其功能特性。然而，仍存在一些未解决的问题。20例高度敏感的血清样本通过SAB-panIgG、SAB-IgG1-4亚类和SAB-C1q检测进行了分析。所有1:16稀释的血清样本还通过SAB-panIgG和SAB-IgG1-4亚类检测进行了分析。共鉴定出1,285个抗-HLA抗体为阳性，其中473个（36.8%）与C1q结合。正如预期的那样，血清稀释增强了C1q结合能力与抗体强度（以MFI衡量）之间的相关性（未稀释为0.248，稀释为0.817）。SAB亚类检测在1,012个（78.8%）阳性抗体特异性中发现至少一种IgG1-4亚类。其中，强补体结合亚类，主要是IgG1，尤其常见（98.9%），且C1q结合抗体与非C1q结合抗体在存在上无差异（99.4%与98.5%；p = 0.193）。相反，弱或非C1q结合亚类（IgG2/IgG4）在C1q结合抗体中更为常见（78.9%与38.6%；p < 0.001）。有趣的是，C1q结合能力与IgG1强度之间存在强烈的关联（rIgG1dil = 0.796）。尽管较低，但IgG2强度与C1q结合能力之间的相关性也很强（rIgG2dil = 0.758），这两个亚类密切相关（rIgG1−IgG2 = 0.817）。我们没有发现与C1q结合能力相关的其他亚类。总之，我们证明了特定的IgG亚类谱（IgG1/IgG3）本身并不能完全决定通过SAB-C1q检测评估的抗-HLA抗体的补体结合能力。通常与IgG2结合出现的IgG亚类强度，尤其是IgG1，与补体结合能力最佳相关。