Characterisation of monoclonal antibodies to haemocyte subpopulations of tiger shrimp (Penaeus monodon): immunochemical differentiation of three major haemocyte types
Characterisation of monoclonal antibodies to haemocyte subpopulations of tiger shrimp (Penaeus monodon): immunochemical differentiation of three major haemocyte types
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DOI：
10.1006/fsim.1998.0185
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发表时间：
1999-04
影响因子：
4.7
通讯作者：
H. Sung;P. Wu;Yen-ling Song
中科院分区：
文献类型：
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作者：
H. Sung;P. Wu;Yen-ling Song
摘要为了研究斑节对虾的免疫细胞效应因子，从血细胞中制备单克隆抗体（mAb）可能成为分离和鉴定血细胞亚群的潜在工具。本研究采用刀豆球蛋白（concanavalin，conA）连接的磁珠对虎虾颗粒细胞（granular cell，GC）进行负选择，制备了4株单克隆抗体Z5 E10（IgG 2a）、Z6 A5（IgG 1）、Z6 A6（IgG 1）和Z6 H8（IgG 2b）。免疫酶染色法测定血细胞反应性，Western印迹法分析抗原分子量。Z5 E10 mAb显示与GC和半颗粒细胞（SGCs）的反应性，并识别29 kDa抗原。Z6 A6特异性结合SGCs，同时识别163 kDa抗原。另外两种mAb（Z6 A5和Z6 H8）分别与SGCs反应，并且能够在非还原条件下识别两种大于205 kDa的抗原，但在还原条件下不能识别它们。高碘酸盐氧化试验的数据显示，Z6 A6抗体对一种糖蛋白具有特异性，而其他三种mAb识别的抗原由蛋白质分子组成。根据这些结果，可以得出结论，Z5 E10抗原和Z6 A6抗原不仅彼此不同，而且与其他两种mAb识别的抗原也不同。此外，加和性测试结果显示mAb对Z6 H8-Z6 A6的加和性指数值（29%）高于Z6 A5-A6 A6的加和性指数值（0%）。提示Z6 A5和Z6 H8单克隆抗体可对不相同的表位具有特异性。在用mAb连接的珠进行阴性选择后，观察到血细胞悬浮液中透明细胞密度增加，这表明由四种mAb识别的表位位于血细胞表面上，并且当这些mAb用于标记和分离血细胞亚群以进一步研究血细胞功能时，这些mAb可能是有用的。
Abstract In order to study immune cellular effectors in tiger shrimp ( Penaeus monodon ), monoclonal antibodies (mAbs) raised from haemocytes could be a potential tool for separating and identifying haemocyte subpopulations. In this study, four mAbs – Z5E10 (IgG2a), Z6A5 (IgG1), Z6A6 (IgG1), and Z6H8 (IgG2b) – were produced by immunising balb/c mouse with a high-density granular cell (GC) suspension prepared from tiger shrimp through negative selection with concanavalin A-linked beads. Haemocyte reactivities were determined by means of immunoenzyme staining, and the molecular masses of antigens were analysed by Western blotting. Z5E10 mAb showed reactivity with both GCs and semigranular cells (SGCs) and recognised a 29 kDa antigen. Z6A6 specifically bound to SGCs while recognising a 163 kDa antigen. The other two mAbs (Z6A5 and Z6H8) separately reacted with SGCs and were able to recognise two antigens which were larger than 205 kDa under non-reducing conditions but could not recognise them under reducing conditions. Data from a periodate oxidation assay revealed that Z6A6 antibody was specific for one glycoprotein, while the antigens recognised by the other three mAbs consisted of protein molecules. According to these results, it was concluded that the Z5E10 antigen and the Z6A6 antigen were different not only from each other, but also from those recognised by the other two mAbs. Furthermore, additivity test results showed that the additivity index value for the mAb pairs Z6H8-Z6A6 (29%) was higher than that of Z6A5-A6A6 (0%). It is suggested that Z6A5 and Z6H8 mAbs could be specific for non-identical epitopes. Following the negative selection with mAb-linked beads, an increase in hyaline cell density in haemocyte suspension was observed which suggests that the epitopes recognised by the four mAbs were located on the surface of haemocytes and that these mAbs may be useful when they are employed to label and separate haemocyte subpopulations for further study of haemocyte functions.