Affinity purification of polyclonal antibodies using a new all-D synthetic peptide ligand: comparison with protein A and protein G.
This study investigates the applicability of the protein A Mimetic (PAM) affinity ligand, obtained from the screening of a multimeric combinatorial peptide library, in immunoglobulin isolation from serum. To avoid protease degradation, the ligand has been substituted by its inverso form, named D-PAM, synthesized by replacing all amino acids with the corresponding D derivatives. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture IgG directly from the serum in a single chromatographic step, with a recovery yield ranging from 60% to 90%, a purity degree higher than 90%, and with a full recovery of antibody activity. Column capacity, determined by applying a large excess of purified IgG to 1 ml bed volume column, was close to 52 mg/ml for bovine IgG, 58 mg/ml for goat IgG, 66 mg/ml for horse IgG, 50 mg/ml for human IgG, 52 mg/ml for mouse IgG, 36 mg/ml for rabbit IgG and 48 mg/ml for sheep IgG. D-PAM peptide was found to be very stable to protease activity, and after prolonged incubation with mouse serum. Similarly, the corresponding derivatized matrix tested before and after various treatments, including sanitization and autoclaving procedures maintained its IgG binding properties, thus indicating a very high stability in terms of ligand leakage and degradation.
Verdoliva
Antonio
A
TECNOGEN S.C.p.A., Parco Scientifico, 81015 Piana di Monte Verna (CE), Italy. verdo@tecnogen.it
Pannone
Filomena
F
Rossi
Maria
M
Catello
Sergio
S
Manfredi
Vincenzo
V
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D040761:Biomimetic Materials / Q000138:chemical synthesis / Q000737:chemistry*; D015153:Blotting, Western; D002352:Carrier Proteins; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats; D006736:Horses; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000737:chemistry*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; C405695:Sh2d3c protein, mouse; D013205:Staphylococcal Protein A