Purification and Characterisation of Immunoglobulins from the Australian Black Flying Fox (Pteropus alecto) Using Anti-Fab Affinity Chromatography Reveals the Low Abundance of IgA

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.现有大量证据表明，蝙蝠可传播一系列新发及再现病毒病原体，此类病原体常会导致动物与人类患病乃至死亡。尽管如此，目前关于蝙蝠与高致病性病毒共生的免疫学机制，相关研究资料仍较为匮乏。免疫球蛋白（Immunoglobulin）是适应性免疫系统的核心组成部分。早期研究显示，与常规实验动物相比，蝙蝠对模式抗原的抗体应答在数量上相对偏低。为进一步解析蝙蝠的抗体应答机制，本研究从健康黑狐蝠（Pteropus alecto）体内纯化并鉴定了主要免疫球蛋白类别。本研究采用全新策略：先纯化免疫球蛋白G（IgG），并以此制备抗Fab特异性抗体；随后将固定化的抗Fab特异性抗体用于从IgG耗竭血清中捕获其他免疫球蛋白。尽管成功从血清中分离出大量免疫球蛋白M（IgM），但未能分离到免疫球蛋白A（IgA）；通过质谱分析法仅能在血清中检测到极微量的IgA。针对IgA的固定化配体（Jacalin、肽M及葡萄球菌超抗原样蛋白）同样未能从血清中捕获黑狐蝠的IgA。免疫球蛋白M（IgM）是血清中仅次于IgG的第二丰富抗体类别。对黏膜分泌物的调查显示，黏膜中的优势抗体类别为IgG，而非IgA。本研究证实，健康黑狐蝠的血清IgA水平远低于预期；黏膜分泌物中较高水平的IgG，或可弥补IgA丰度过低或缺乏的缺陷。本研究开发的相关方法与试剂，未来可用于解析这一重要病毒宿主的类别特异性抗体应答。