Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin (CD62P)
Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin (CD62P)
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DOI：
10.1034/j.1399-0039.2000.560202.x
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发表时间：
2000-08-01
期刊：
影响因子：
--
通讯作者：
Pizcueta, P
中科院分区：
文献类型：
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作者：
Massaguer, A;Engel, P;Pizcueta, P
P-选择素（CD 62 P）是在活化的内皮和活化的血小板上表达的粘附分子，其参与白细胞对发炎的血管内皮的初始附着。阻断性单克隆抗体（mAb）和P-选择素缺陷小鼠已经表明P-选择素是抗炎治疗中的潜在靶点。大多数针对P-选择素的mAb不结合保守表位，包括配体结合区，因为来自哺乳动物物种的P-选择素具有高氨基酸序列同源性。本研究的目的是产生一个新的面板的抗P-选择素单克隆抗体的保守表位存在于几种动物物种。为了产生这些mAb，P-选择素缺陷小鼠用转染有人P-选择素cDNA的前B细胞系免疫。获得了12个识别人P-选择素的小鼠mAb。通过流式细胞术、免疫组织化学、粘附试验和免疫沉淀法对与人、大鼠、小鼠、兔和猪活化血小板结合的单个mAb进行表征。这些mAb中的四种（P-sel.KO. 2.3，P-sel. KO.2.4、P-sel.KO.2.7和P-sel.KO.2.12）与人、大鼠和小鼠P-选择素交叉反应。另外三种单克隆抗体（P-sel.KO.2.2，P-sel.KO.2.11和P-sel.KO.2.12）阻断HL 60细胞与P-选择素转染的COS细胞的粘附，证明这些单克隆抗体抑制P-选择素介导的粘附。MBb交叉阻断实验表明，这三种mAb结合非常接近和重叠的表位。设计使用mAb P-sel.KO.2.3和P-sel.KO.2.12的ELISA测定法来测量可溶性大鼠、小鼠和人P-选择素。这些抗P-选择素单克隆抗体是独特的，因为它们识别在哺乳动物进化过程中保守的共同表位，并且它们可能用于研究不同物种中炎症模型中的P-选择素功能。
P-selectin (CD62P) is an adhesion molecule expressed on the activated endothelium and activated platelets that is involved in the initial attachment of leukocytes to inflamed vascular endothelium. Blocking monoclonal antibodies (mAbs) and P-selectin-deficient mice have shown that P-selectin is a potential target in anti-inflammatory therapy. Most mAbs against P-selectin do not bind to conserved epitopes, including the ligand-binding region, since P-selectin from mammalian species shares high amino acid sequence homology. The aim of this study was to generate a novel panel of anti-P-selectin mAbs against the conserved epitopes present in several animal species. To produce these mAbs, P-selectin-deficient mice were immunized with a pre-B-cell line transfected with human P-selectin cDNA. Twelve mouse mAbs that recognize human P-selectin were obtained. Individual mAbs that bound to human, rat, mouse, rabbit and pig activated platelets were characterized by flow-cytometry, immunohistochemistry, adhesion assays and immunoprecipitation. Four of these mAbs (P-sel.KO.2.3, P-sel.-KO.2.4, P-sel.KO.2.7 and P-sel.KO.2.12) cross-reacted with human, rat and mouse P-selectin. Another three mAbs (P-sel.KO.2.2, P-sel.KO.2.11 and P-sel.KO.2.12) blocked the attachment of HL60 cells to P-selectin-transfected COS cells, demonstrating that these mAbs inhibit P-selectin-mediated adhesion. MBb cross-blocking experiments showed that these three mAbs bind to very close and overlapping epitopes. An ELISA assay using mAbs P-sel.KO.2.3 and P-sel.KO.2.12 was designed to measure soluble rat, mouse and human P-selectin. These anti-P-selectin mAbs are unique since they recognize common epitopes conserved during mammalian evolution and they may be useful for studying P-selectin function in inflammatory models in various species.