Isolation of functional human coagulation factor V by using a hybridoma antibody.
Isolation of functional human coagulation factor V by using a hybridoma antibody.
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使用杂交瘤抗体分离功能性人凝血因子 V。
DOI：
10.1073/pnas.78.1.162
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发表时间：
1981
影响因子：
11.1
通讯作者：
Mann,KG
中科院分区：
文献类型：
--
作者：
Katzmann,JA;Nesheim,ME;Hibbard,LS;Mann,KG
用部分纯化的人凝血因子V免疫小鼠，取脾细胞与NS-1小鼠骨髓瘤细胞融合，筛选出杂交瘤细胞。对培养上清液进行抗因子V活性筛选，获得抗体阳性克隆，并以小鼠腹水瘤传代。杂交瘤小鼠的腹水可以被稀释到1：10(6)，然后在抗因子V放射免疫分析中失去反应。当固定在琼脂糖凝胶上时，该单抗定量地从人血浆中去除因子V的活性。凝血因子V活性可用1.2M氯化钠在pH 6.5时洗脱。均一因子V经柠檬酸钡吸附、聚乙二醇6000沉淀血浆后经抗体柱层析、苯基琼脂糖层析分离得到。分离的凝血因子V在十二烷基硫酸钠中呈单一条带，其表观分子质量与牛凝血因子V(330,000)相当。在凝血酶暴露后，在缺乏凝血因子V的血浆中测得的凝血因子V活性增加了53倍。这种活性的增加与母体分子的离散蛋白分解有关。
Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:10(6) before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M NaCl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mr comparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.
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通讯作者：
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影响因子：
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作者：
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影响因子：
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