Supplementary Material for: Immunoglobulin Class Profiles of ABO Antibodies in Saliva and Serum of Healthy Individuals

<b><i>Introduction:</i></b> The coronavirus disease (COVID-19) pandemic gave rise to studies investigating the association of ABO blood group with COVID-19 susceptibility. It is hypothesized that ABO antibodies might play a role in neutralizing SARS-CoV-2. However, ABO antibodies were exclusively analyzed in blood samples. Investigation of ABO antibodies in saliva, an easy-to-obtain surrogate for respiratory secretions, may provide novel insights into mucosal immunity crucial in early defense against respiratory pathogens. <b><i>Methods:</i></b> In this study, saliva and serum samples from healthy individuals with known blood groups were investigated using a flow cytometric method for separate anti-A/anti-B IgA, IgM, and IgG class antibody detection. Saliva samples were additionally tested using hemagglutination-based neutral and indirect anti-human globulin test gel cards. This method comparison was complemented by dilution experiments with a high-titer anti-A/anti-B WHO standard. <b><i>Results:</i></b> In saliva, IgA was the most abundant ABO antibody class, followed by IgM; IgG was detected only in low levels in all non-AB blood types. In serum, IgM was the predominant ABO antibody class in all non-AB blood types, followed by IgA and IgG, the latter mainly detected in group O individuals. Saliva and serum samples of group O individuals yielded the highest variability of ABO-specific antibody levels. Regardless of sample material and blood type, major interindividual differences in ABO antibody reactivities were recorded. Antibody levels correlated moderately between these two body fluids. There were no significant sex and age-group differences in ABO antibody levels in both serum and saliva. WHO standard dilution experiments yielded technique-specific limits of detection, illustrating the inherent differences of immunofluorescence versus agglutination. <b><i>Conclusion:</i></b> For the first time, salivary ABO antibodies were investigated by separate detection of the three most relevant antibody classes IgA, IgM, and IgG in a healthy cohort. This study opens new perspectives regarding mucosal ABO antibody class profiles and their potential influence on respiratory infections.

<b><i>研究背景：</i></b> 新型冠状病毒肺炎（COVID-19）大流行催生了诸多探讨ABO血型（ABO blood group）与COVID-19易感性关联的研究。本研究提出假说，ABO抗体（ABO antibodies）可能在中和新型冠状病毒（SARS-CoV-2）的过程中发挥作用。然而此前相关研究仅在血液样本中对ABO抗体进行了分析。唾液作为一种易于获取的呼吸道分泌物替代样本，对其ABO抗体展开研究，或可为理解呼吸道病原体早期防御至关重要的黏膜免疫提供新视角。<b><i>研究方法：</i></b> 本研究针对已知血型的健康志愿者，采用流式细胞术方法（flow cytometric method）分别检测抗A/抗B的IgA、IgM与IgG类抗体，对唾液与血清样本进行分析。此外，本研究还通过基于血凝反应的中性及间接抗人球蛋白试验凝胶卡对唾液样本进行检测，并使用高滴度抗A/抗B世界卫生组织（WHO）标准品开展稀释实验以补充该方法学对比。<b><i>研究结果：</i></b> 唾液中IgA是含量最高的ABO抗体类别，其次为IgM；IgG仅在所有非AB型血个体的样本中以低水平被检出。血清中，IgM是所有非AB型血个体中主要的ABO抗体类别，其次为IgA与IgG，其中IgG主要在O型血个体中检出。O型血个体的唾液与血清样本的ABO特异性抗体水平变异度最高。无论样本类型与血型如何，均观察到ABO抗体反应性存在显著的个体间差异。两种体液中的抗体水平呈中度相关。血清与唾液中的ABO抗体水平均未表现出显著的性别与年龄组差异。世界卫生组织（WHO）标准品稀释实验得到了不同检测技术对应的检测限，阐明了免疫荧光法与凝集法的固有差异。<b><i>研究结论：</i></b> 本研究首次在健康队列中通过分别检测三类最相关的抗体类别——IgA、IgM与IgG，对唾液中的ABO抗体进行了研究。本研究为黏膜ABO抗体类别谱及其对呼吸道感染的潜在影响提供了新的研究视角。