Antibodies purification methods

Ayyar BV, Arora S, Murphy C, O Kennedy R. Affinity chromatography as a tool for antibody purification. Methods 56 116-129, 2012. 

The endothelin B receptor is an example of characterization of a homogeneous, affinity purified protein (Roos et al., 1998). Significant progress has been made in the development of techniques for more high-throughput identification of phosphorlyation events. Analysis of large sets of phosphorylated proteins is facilitated by the availability of affinity purification methods such as anti-phosphotyrosine or anti-phosphoserine antibodies or metal affinity chromatography (Neubauer and Mann, 1999 Soskic et al., 1999). These methods are not specific to a particular protein but rather are used to fractionate all proteins that are phosphorylated. 

Should the antibody purification nevertheless be desirable, one can use the Ab-Select Purification Kit (also from Innova Biosciences, http //www.innovabio-sciences.com/products/abselect.php) that quickly removes the contaminants, such as BSA, glycine, tris or azide. The AbSelect method involves capture of the antibody on protein A resin and the removal of unwanted substances by a simple wash procedure. The purified product is then eluted and neutralized. 

The advent of recombinant DNA technology led to the development of antibodies and fragments that are tailored for optimal behaviour in vivo [7,8]. Humanized and chimeric antibodies can be constructed to circumvent the human anti-mouse antibody response elicited by mouse antibody treatment of patients, which severely hampers the application of these powerful molecules. The treatment of rheumatoid arthritis patients with doses of as high as 10 mg kg cA2 chimeric antibody specific for TNFa [9], emphasizes that at present the production and purification methods for these proteins have been optimized to such extent that clinical studies can be considerably intensified. 

Grodzki AC and Berenstein E. Antibody Purification Ion-Exchange Chromatography. Immunocytochemical Methods and Protocols. Methods in Molecular Biology 2010 588 27-32. 

Sun, L., Ghosh, I., Xu, M.Q. (2003). Generation of an affinity column for antibody purification by intern-mediated protein ligation. J. Immunol. Methods, 282(1-2), 45-52. 

Aithal, H. N., Knigge, K. M., Kartha, S., Czyewski, E. A., and Toback, F. G. (1988) An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies,/. Immunol. Methods 112, 63—70. 

Boschetti, E. (2001) The use of thiophilic chromatography for antibody purification a review. J. Biochem. Biophys. Methods. 49, 361-389. 

Intact antibodies with biologically active glycosylation profiles, crucial for the effector functions, require eukaryotic expression (in vitro or in vivo). These circumstances have inspired many scientists to find effective methods for production, as well as methods for the selection of the best extraction-purification methods. 

This method of separation has been applied to monoclonal antibody purification (A. Schwarz, personal communication). The selectivity for the antibodies is played by the choice of the ligand while the HCIC effect is still the one described above. 

Finally, gel filtration is most often considered an appropriate polishing method when the target antibody is already pure and the only impurities are foreign protein traces or fragments or aggregates that must be eliminated. In this context, separation processes for antibody purification are logical orthogonal combinations of methods. 

TABLE 15 Possible Combinations of Methods for Antibody Purification... 

Roggenbuck, D., Marx, U., Kiessig, S. T., Schoenherr, G., Jahn, S., and Porstmann, T. (1994). Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody.. Immunol. Methods 167, 207-218. 

Recently, an immunoaffinity purification method using antimicrocystin-LR monoclonal antibodies (named M8H5) has been developed.This purification method was found to be remarkably effective in the removal of coexisting substances and in the enrichment of microcystins in samples.This work will focus on the immunoaffinity purification methods for microcystins in lake ° and tap water samples, and the analysis methods for microcystins and their metabolites in mouse and rat livers.It will also cover the reuse of the immunoaffinity column. 

Interferring compounds from the sample matrix can become a major problem in the analysis of cytokinins that normally occur at trace levels. Immunoaffinity purification methods that are based on polyclonal or monoclonal antibodies enable a selective single-step clean-up and concentration of a certain group of cytokinins. These methods are usually combined with various techniques of final analysis such as HPLC-UV, HPLC-ELTSA, HPLC-MS [277-279]. However, the antibodies prepared so far do not show suitable affinity to certain metabolites (O-glucosides, N -glucosides, nucleotides). 

Brown R, Kertiles L, Kleinmann R. Choice of immunoglobin G purification method in assays for antibodies to the thyrotropin receptor. Clin Chem 1986 32 2034-9. 

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