Isolation and characterization of monoclonal antibodies to (Na+ + K+)-ATPase.
Isolation and characterization of monoclonal antibodies to (Na+ + K+)-ATPase.
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(Na K )-ATPase 单克隆抗体的分离和表征。
DOI：
10.1016/0304-4165(82)90228-8
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Lessard,JL
中科院分区：
文献类型：
--
作者：
Ball,WJ;Schwartz,A;Lessard,JL
用小鼠骨髓瘤细胞与免疫小鼠脾细胞融合，获得4株稳定的杂交瘤细胞株，可分泌抗羊肾髓质膜（Na ~++ K ~+）依赖性ATP酶的抗体。这些细胞系产生IgG γ 1重链和κ轻链抗体，这些抗体针对（Na++ K+）-ATP酶的催化或α亚基。结合研究，使用抗体，产生的杂交瘤在体内生长和纯化的亲和柱层析，表明这些抗体的亲和力为分离的α-亚基比为“天然”全酶。此外，这些单克隆抗体与Lamb酶的糖蛋白（β）亚基或大鼠肾脏（哇巴因不敏感器官）的（Na++ K+）-ATP酶均无反应性。共滴定结合实验表明，来自最初独立地从融合细胞的相同培养板孔群分离的两个细胞系的抗体结合至相同的决定簇位点，并且可能是相同的抗体。与其他两种抗体的共滴定和竞争结合研究揭示了另外两个不同的抗体结合位点，其似乎与第一个位点几乎没有重叠。分离的三种不同抗体中的一种引起（Na++ K+）-ATP酶活性的部分抑制。该抗体似乎针对α亚基的特定功能重要位点，是ATP结合的竞争性抑制剂。在ATP酶活性的最佳条件下，这种抑制作用不会因其他两种抗体的存在而改变。
Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na++ K+)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ1heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na++ K+)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na++ K+)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na++ K+)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.