Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity chromatography.
An enzyme-linked immunosorbent assay (ELISA)-elution assay was developed to screen a large variety of elution buffers for selection of a suitable one for purification of the fusion protein FV/TNF-alpha by affinity chromatography. Various commonly used buffer systems utilizing widely differing conditions such as extreme pH, denaturants, chaotropic ions and polarity reducing reagents were investigated. Ammonia solution (1 M, pH 11.5) proved to exert the most suitable influence on dissociation of the FV/TNF-alpha/TAG72 complex while having a minimal protein denaturing effect on FV/TNF-alpha. The total yield of purified FV/TNF-alpha using the TAG72-affinity column with this elution system was 300-fold higher than that using the common elution buffer, 0.1 M glycine, 0.5 M NaCl, pH 2.7. Our study indicates that the ELISA-elution assay will be most useful in the selection of suitable elution buffers for affinity chromatography.
Yang
J
J
Department of Oncology, Saskatoon Cancer Center, University of Saskatchewan, Canada.
Moyana
T
T
Xiang
J
J
D000230:Adenocarcinoma / Q000276:immunology; D000951:Antigens, Neoplasm / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003110:Colonic Neoplasms / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D000951:Antigens, Neoplasm; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72