Affinity purification of polyclonal antibodies using immobilized multimeric peptides.
The possibility of using multiple antigenic peptides (MAP) not only for the production and characterisation of antibodies but also for their purification by affinity chromatography, has been explored with two different tetrameric MAPs synthesised starting from a tetradentate lysine core. Recognition selectivity and specificity of the multimeric antigens were retained after immobilization on preactivated affinity supports, allowing convenient antibody purification directly from crude sera in a single chromatographic step. Since antibodies raised against MAPs recognise very frequently the N-terminal portion of the peptide antigen, results suggest that only a limited number of peptide chains remains covalently linked to the solid phase, leaving the others uncoupled and free to interact fully with the antibody. Recovery of antibody immunoreactivity from affinity purifications on MAP-columns was much higher than that obtained from columns prepared by immobilizing at the same density the corresponding linear peptide antigen. The purity of thus obtained antibodies is also far superior, as detected by SDS-PAGE analysis. Retention of the multimeric peptide recognition properties for the corresponding antibodies after immobilization on solid supports suggests that production, characterization, and even the affinity purification of anti-peptide antibodies, could be carried out simply and conveniently via the synthesis of a single multimeric antigen, without additional steps.
Verdoliva
A
A
TECNOGEN S.C. p. A., Piana di Monte Verna (CE), Italy.
Cassani
G
G
Fassina
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits
D000906:Antibodies; D007106:Immune Sera; D010455:Peptides