Effect of Three Elution Buffers on the Recovery and Structure of Monoclonal Antibodies

Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography

“…Even if the extracted antibodies refold in their native conformation because of immediate neutralisation and/or dialysis following elution (Narhi et al, 1997a(Narhi et al, , 1997b we did not investigate the effect of the elution buffers on their conformation and so cannot exclude that an irreversible denaturation occurred. Nevertheless, our 280 nm absorption measurements of the column eluates indicated that those fractions which lacked anti-LPS antibodies by ELISA also lacked measurable protein content and thus were unlikely to contain significant amounts of denatured antibody.…”

“…Glycine at acidic pH is commonly used as an elution buffer (Narhi et al, 1997a(Narhi et al, , 1997b. We therefore tested 0.1 M glycine, 0.1 M NaCl at pH 3, pH 2.8, pH 2.6 and pH 2.4 (Fig.…”

“…Although this method for recovering active antibodies is potentially selective, rapid and simple, allowing antibody purification in a single chromatographic step, the recovery is typically low (Casey et al, 1995;Cuatrecasas and Anfinsen, 1971). Optimised conditions need to be determined to permit efficient purification of the desired antibodies without altering their native structure (Narhi et al, 1997a).…”

Immunoaffinity Chromatography: A Review

“…Unfortunately, there is no way to figure out a priori what will be an effective eluent from a particular immunoadsorbent; this can only be determined empirically. The elution method of choice is often the use of low pH (2.0-2.5) which disrupts both ionic and hydrogen bonds between antigen and antibody (Narhi et al, 1997). If that procedure is not effective, the next best choice may be to resort to a commercially available eluent such as Gentle Elution Buffer (Thermo Scientific), the composition of which is proprietary and is reported to destabilize the antigen-antibody complex without damaging either partner of the complex.…”

A study of the performance characteristics of immunoaffinity solid phase microextraction probes for extraction of a range of benzodiazepines

“…Fractions with higher specific binding were obtained from the oxazepam affinity column by elution with higher strength eluents (7 M urea in 50 mM sodium acetate buffer pH 4 (urea) or 6 M guanidine hydrochloride in 50 mM sodium acetate buffer pH 4 (GnHCl)) [27]. The pools from urea and GnHCl elutions were re-natured after elution [27].…”