Antibodies affinity purification

Fassina, G., Verdoliva, A., Palombo, G., Ruvo, M., and Cassani, G. (1988). Immunoglobulin specificity of TG19318 A novel synthetic ligand for antibody affinity purification. J. Mol. Recognition 11, 128-233. 

Several milliliters of affinity medium used repeatedly can be sufficient for the development and preliminary testing of an assay. Large-scale trials or extensive optimizations may require significant amounts of purified antibody. Affinity purification on that scale may benefit from standard chromatography equipment (columns, pumps, a UV/pH monitor and a fraction collector). 

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. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

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. Meth. 112, 63-70. 

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. 

Pitt JC, Lindemeier J, Habbes HW, Veh RW (1998) Haptenylation of antibodies during affinity purification a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling. Histochem Cell Biol 110 311 322 Polak JM, Van Noorden S (1997) Introduction to immunocytochemistry. BIOS Scientific... 

Parmley, S and Smith, G (1988) Antibody-selectable filamentous fd phage vectors affinity-purification of target genes Gene 73, 305—318. 

The multivalency of peptide dendrimers has also been used to increase the binding avidity of antigens to antibodies in various immunological assays and diagnostic tests. 27,43,47,48,55-59 Examples in the literature have been reported where the MAP format increases the binding avidity and sensitivity, in some cases >100000 fold. In addition to immunology, MAP types of dendrimers have found applications in areas such as inhibitors, 44,45,51,60,61 artificial proteins, 62 affinity purification, 63 and intracellular transportation, 41 as well as in drug discovery. 53 ... 

Affinity purification may be performed using gravity flow through a disposable column (e.g., a Bio-Rad Poly-Prep chromatography column). In this case, flow reversal is not possible and recovery of high affinity antibodies may be compromised. Nevertheless, acceptable results are often achieved. 

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