Purification of antibodies

HA chromatography processes large amounts of serum in one step and concentrates the antibodies. It is not necessary to dialyze the serum before performing the chromatography over a column buffer. The yield is good, and the antibodies relatively clean. HA chromatography does not completely separate monoclonal antibodies from tissue culture supernatants with fetal calf serum from albumin. 

Bukovsky, J., and Kennet, R. (1987). Simple and Rapid Purification of Monoclonal Antibodies from Cell Culture Supernatants and Ascites Fluids by Hydroxyl Apatite Chromatography on Analytical and Preparative Scales, Hybridoma 6 219-228. 

The ammonium sulfate precipitation of serum (with 45% saturation for rabbit serum) is a quick-and-dirty method, with an emphasis on dirty. The precipitation takes a few hours and afterward you still have to dialyze. 

This easy and efficient purification method has the disadvantage that not all IgG subclasses bind to the column with high affinity and thus get lost (e.g., mouse IgGi). There are species differences protein A binds rabbit antibody well and rat antibody badly. 

Protein G (MW 35 kd) binds antibody and albumin. An artificial variant of protein G binds only antibody. The advantage of protein G is its complementary specificity to protein A the antibodies that do not bind to protein A, such as many monoclonal antibodies, often bind to protein G. 

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Zopf, D.A., Smith, D.F., Drzeniek, Z., Tsai, C.-M., and Ginsburg, V. (1978a) Affinity purification of antibodies using oligosaccharide-phenethylamine derivatives coupled to Sepharose. In Methods in Enzymology, (V. Ginsburg, ed.), Vol. 50, pp. 171-175. Academic Press, New York. 

Kent, U.M. (1999). Purification of antibodies using affinity chromatography. Methods Mol. Biol., 115, 23-28. 

Metal affinity columns can be used for the purification of antibodies with a hexa-Histidine tag. Immobilized metal affinity chromatography is incompatible, in our own experience, with direct loading of antibodies in supernatants... 

Application of Affinity Chromatography to purification of antibodies Also a hnk to Exclusion Chromatography... 

The starting point for the purification of antibodies is usually serum, ascitic fluid, or tissue culture supernatant. If it is necessary to prepare serum from an immunised animal, then the following procedure can be used. 

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