Antibody Purification: Affinity Chromatography – Protein A and Protein G Sepharose

Abstract

Affinity chromatography relies on the reversible interaction between a protein and a specific ligand immobilized in a chromatographic matrix. The sample is applied under conditions that favor specific binding to the ligand as the result of electrostatic and hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. After washing away the unbound material the bound protein is recovered by changing the buffer conditions to those that favor desorption. The technique has been used not only to isolate antigen-specific antibodies but also to remove specific contaminants from biological samples. Methods are described for the purification of immunoglobulins, namely IgG, IgG fragments and subclasses, using the high affinity of protein A and protein G coupled to agarose. In the Subheading 3 there are also protocols for affinity purification using a specific ligand coupled to commercial matrices like CNBr- Sepharose 4-B and Affigel.

A monoclonal antibody to an abrin chimera recognizing a unique epitope on abrin A chain confers protection from abrin-induced lethality

“…16,28 Monoclonal antibodies were purified from the corresponding hybridoma culture supernatants using Protein A-Sepharose affinity chromatography 29 and the concentration of the purified proteins estimated by Lowry's method. Indirect ELISA ELISA plates were coated with proteins (500 ng/100 ml per well) in phosphate buffer, pH 7.2 containing 150 mM NaCl (PBS) overnight at RT.…”

Statistical and Bioanalytical Considerations for Establishing a Depletion Criterion for Specificity Testing During Immunogenicity Assessment of a Biotherapeutic

“…The specificity step usually involves the depletion of the antibody signal by adding an excess amount of biotherapeutic (5)(6)(7)(8)(9)13,18). The analytical considerations around such assays and statistical methods needed to establish a DCP require further evaluation.…”

“…Purification of the rabbit positive control antibodies specific to the idiotypic region of the biotherapeutic was performed by using a column with biotherapeutic covalently bound to cyanogen bromide Sepharose 4B gel (GE Healthcare). Subsequent to the affinity column purification, immunoadsorption against human immunoglobulin bound to Sepharose 4B was performed (13). This enables generation of a positive control antibody that is predominantly reactive to the F (ab) region of the biotherapeutic and helps eliminate Fc reactivity.…”

Affinity Chromatography: Principles and Applications

“…Sepharose CL-4B; agarose crosslinked with 2,3-dibromopropanol and desulphated by alkaline hydrolysis under reductive conditions), polyacrylamide, and magnetic beads (Grodzki & Berenstein, 2010;Hober et al, 2007;Katoh et al, 2007;Tyutyulkova & Paul, 1995 …”