Characterization of a Monoclonal Antibody Directed against <i>Mytilus spp</i> Larvae Reveals an Antigen Involved in Shell Biomineralization

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.本研究针对靶向地中海贻贝（Mytilus galloprovincialis）幼虫及后幼虫阶段表达抗原所开发的M22.8单克隆抗体（monoclonal antibody, mAb），在成体样本中展开了相关研究。通过蛋白质印迹（Western blot）开展的抗原表征实验显示，抗原MSP22.8的分布具有组织局限性，在成体样本中仅涵盖外套膜边缘组织、外套腔液、外套腔液血细胞及贝壳有机基质。其余组织如中央外套膜、生殖腺组织、消化腺、唇瓣、足以及足丝收缩肌均不表达该抗原。免疫组织化学（immunohistochemistry）实验检测发现，MSP22.8存在于外套膜边缘外褶上皮直至套膜线的细胞中。流式细胞术（flow cytometry）分析结果表明，外套腔液来源的血细胞胞内也含有该抗原。此外，当暴露于无细胞外套腔液溶液时，血淋巴来源的血细胞能够内化该抗原。本研究结果提示，血细胞可能在生物矿化过程中发挥关键作用，因此该抗原被纳入贻贝贝壳形成模型。本研究首次针对地中海贻贝（M. galloprovincialis）报道了外套膜边缘分泌至外套腔的蛋白质，及其如何成为贝壳基质框架组成部分的相关机制。