van de Braak C B, Taverne N, Botterblom M H, van der Knaap W P, Rombout J H
Fish Culture and Fisheries Group, Wageningen, The Netherlands.
Fish Shellfish Immunol. 2000 Aug;10(6):515-30. doi: 10.1006/fsim.2000.0269.
Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.
用对虾血细胞的膜裂解物免疫小鼠，制备了对斑节对虾血细胞具有特异性的单克隆抗体（mabs）。使用流式细胞术、光学显微镜、激光扫描显微镜、电子显微镜和免疫沉淀法对四种单克隆抗体（WSH 6、WSH 7、WSH 8和WSH 16）进行了表征。WSH 6识别85 kDa分子上的碳水化合物决定簇。WSH 7、WSH 8和WSH 16分别识别50 kDa、35 kDa和115 kDa的分子。对于所有单克隆抗体，当血细胞立即固定在含有阿氏液（AS）的2%甲醛中时，与保存在AS（可减少血细胞激活）或L15细胞培养基中的未固定血细胞相比，发现标记量和强度存在差异。WSH 6与所有固定血细胞的细胞膜发生反应，而WSH 7和WSH 16与超过80%的固定血细胞的细胞膜发生反应。当细胞保存在L15培养基中时，膜标记似乎减少。WSH 8不与血细胞细胞膜发生反应。当血细胞立即固定时，所有单克隆抗体都与一些颗粒发生反应，这些颗粒主要存在于透明细胞中。当未固定的细胞保存在AS和L15培养基中时，在半颗粒和颗粒血细胞以及第四种细胞类型的最大颗粒中也观察到阳性颗粒，第四种细胞类型含有许多大小和电子密度不同的颗粒。在L15培养基中的细胞中可观察到免疫反应性细胞外线状物质。WSH 8的染色模式变化最大，WSH 6和WSH 7的变化较小，WSH 16的变化最小。双重标记显示，所有单克隆抗体在细胞膜和颗粒上均呈现不同的染色模式。WSH 16在组织切片的细胞质小泡以及血淋巴血浆中也显示出标记。提出的假设是，这些单克隆抗体识别的免疫反应性分子与血细胞激活因子有关。
