A non-chromatographic method for the purification of a bivalently active monoclonal IgG antibody from biological fluids.
A non-chromatographic method for the purification of a bivalently active monoclonal IgG antibody from biological fluids.
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DOI：
10.1021/ja9023836
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发表时间：
2009-07-08
影响因子：
15
通讯作者：
Whitesides, George M.
中科院分区：
文献类型：
--
作者：
Bilgicer, Basar;Thomas, Samuel W., III;Shaw, Bryan F.;Kaufman, George K.;Krishnamurthy, Vijay. M.;Estroff, Lara A.;Yang, Jerry;Whitesides, George M.
本文描述了一种从腹水中纯化单克隆抗体（大鼠抗 2,4-二硝基苯基 IgG：IgGDNP；和小鼠抗地高辛 IgG：IgGDgn）的方法。该程序（针对 IgGDNP）分为三个步骤： i) 用硫酸铵沉淀比免疫球蛋白重的蛋白质； ii) 通过使其与含有多个 DNP 基团的合成多价半抗原结合，形成 IgGDNP 的环状复合物； iii) 选择性沉淀目标抗体的这些二聚体、三聚体和高级寡聚体，然后再生游离抗体。该过程将目标抗体从抗体混合物以及生物液体中的其他蛋白质和球蛋白中分离出来。该方法适用于具有多种单价结合常数（0.1 μM 至 0.1 nM）的抗体。我们使用的多价配体（DNP 和地高辛的衍生物）从腹水中分离出 IgGDNP 和 IgGDgn，收率 > 80%，纯度 > 95%。与用于纯化抗体的传统色谱方法相比，该技术有两个优点：i) 与具有一个或零个活性 Fab 结合位点的抗体相比，它对具有两个活性 Fab 结合位点（两个位点都需要形成环状复合物）的抗体具有选择性； ii) 不需要色谱分离。它的缺点是半抗原的结构必须与二价和/或三价类似物的合成相容。
This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgGDNP; and mouse anti-digoxin IgG: IgGDgn) from ascites fluid. This procedure (for IgGDNP) has three steps: i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; ii) formation of cyclic complexes of IgGDNP by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 μM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgGDNP and IgGDgn from ascites fluid in yields of > 80% and with > 95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi and/or trivalent analogs.
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影响因子：
5.6
作者：
Martin, TD
通讯作者：
Martin, TD
影响因子：
3.5
作者：
MOTTI, C;NUZZO, M;MONACI, P
通讯作者：
MONACI, P
DOI：
10.1016/0019-2791(72)90096-1
发表时间：
1972-01-01
期刊：
IMMUNOCHEMISTRY
影响因子：
--
作者：
HORNICK, CL;KARUSH, F
通讯作者：
KARUSH, F
影响因子：
11.4
作者：
FOLGORI, A;TAFI, R;NICOSIA, A
通讯作者：
NICOSIA, A
影响因子：
3.4
作者：
Hlavacek, WS;Perelson, AS;Posner, RG
通讯作者：
Posner, RG