On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice.
On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice.
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DOI：
10.4049/jimmunol.131.6.2895
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发表时间：
1983-12
影响因子：
4.4
通讯作者：
P. Parham
中科院分区：
文献类型：
--
作者：
P. Parham
研究了用胃蛋白酶和其它蛋白酶从BALB/c单克隆IgG 1、IgG 2a和IgG 2b生产和纯化F（ab '）2片段的方法。对降解的总体敏感性为IgG 2b大于IgG 2a大于IgG 1。在pH 3.5至4.0下用胃蛋白酶从IgG 1可以高产率产生稳定的F（ab '）2，并且可以通过胃蛋白酶处理含有IgG 1的腹水或细胞培养上清液直接制备。IgG 2a被胃蛋白酶分两步裂解，首先裂解为F（ab '）2，然后裂解为Fab'。通过仔细选择的条件，可以以可接受的产率获得F（ab '）2。IgG 2a重链的主要裂解似乎位于重链间二硫化物的COOH末端侧，次要裂解位于NH 2末端侧。对于IgG 2b，情况正好相反，并且尚未获得有用量的F（ab '）2;然而，IgG 2b的初级切割相对于两条重链似乎是不对称的，并且可以制备具有一个Fab加Fc的Fab/c片段。用弹性蛋白酶消化导致Fab/c的最佳产量。这一发现可能提供了一种方法，用于保留针对细胞表面抗原的单克隆抗体的细胞毒性，同时消除它们的调节能力。三类IgG的切割模式根据其铰链区的结构合理化。
Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.