Protein A mimetic peptide ligand for affinity purification of antibodies.
A peptide mimicking protein A for its ability to recognize the Fc immunoglobulin portion has been identified through screening of a synthetic multimeric peptide library. Screening of the multimeric library, composed of randomized synthetic tripeptide tetramers, has been carried out using a very simple assay, measuring the library ability to interfere with the interaction between protein A and biotinylated immunoglobulins, monitored on solid phase using an enzyme-linked immunosorbent assay format. The tetrameric tripeptide identified after three screening cycles was produced in larger amounts and then immobilized in high yield on preactivated solid support for the preparation of affinity columns, which proved useful for a very convenient one-step purification of antibodies directly from crude sera. Antibody purity after affinity purification was close to 95 per cent, as determined by densitometric scanning of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of purified fractions, and up to 2 mg of antibody could be purified from 1 ml of peptide-derivatized affinity support. The ligand was stable to treatment with a vast array of sanitation agents, such as ethanol and 0.1 M sodium hydroxide, and to repeated use, thus making the ligand applicability extremely attractive for the purification of monoclonal antibodies for therapeutic use. Column binding selectivity was similar to that of protein A-affinity columns, since immunoglobulin G from several sources (rabbit, goat, sheep, mouse) was conveniently purified, with no detection of leaked ligand fragments in the purified preparations.
Fassina
G
G
TECNOGEN S.C.p.A., Piana di Monte Verna (CE), Italy.
Verdoliva
A
A
Odierna
M R
MR
Ruvo
M
M
Cassini
G
G
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D006041:Goats; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification; D008024:Ligands; D051379:Mice; D018716:Molecular Mimicry; D010455:Peptides / Q000737:chemistry*; D011817:Rabbits; D012756:Sheep; D013205:Staphylococcal Protein A / Q000737:chemistry*
D000906:Antibodies; D007141:Immunoglobulin Fc Fragments; D008024:Ligands; D010455:Peptides; D013205:Staphylococcal Protein A