Peptide immobilization on calcium alginate beads: applications to antibody purification and assay.
Two different procedures were developed for the non-covalent immobilization of peptide antigens on calcium alginate beads. The antigenic peptide is first synthesized in a multimeric form starting from a polydentate lysine core, and then immobilized on alginate beads (average volume 0.05 ml) by entrapment or simply by non-covalent adsorption. Coupling yields, as monitored by RP-HPLC analysis of the immobilization time course and/or by amino acid analysis of derivatized beads, were close to 1-2 mg of peptide per ml of gel. Immobilization yields were not dependent on the peptide net charge, hydrophobicity or length, but mainly on the extent of peptide multimerization. After immobilization on alginate gel, peptide antigenic properties were fully retained, as clearly demonstrated by the batchwise micropreparative purification of anti-peptide antibodies in good yields and with a high degree of purity, directly from crude sera in a single adsorption-elution step. Derivatized beads were sufficiently stable towards repeated washing-equilibration procedures, allowing very limited peptide leakage from the matrix. Peptide beads were also successfully used for the development of solid-phase immunoassays in test-tubes to characterize the corresponding antibodies, with the immobilization yield and signal-to-noise ratio being greatly enhanced in comparison with other types of conventional supports.
Palmieri
G
G
Tecnogen SCpA, Piana di Monte Verna (CE), Italy.
Cassani
G
G
Fassina
G
G
D000327:Adsorption; D000464:Alginates / Q000737:chemistry*; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007118:Immunoassay; D010455:Peptides / Q000737:chemistry*; D011817:Rabbits
D000464:Alginates; D000906:Antibodies; D006603:Hexuronic Acids; D010455:Peptides; D020723:Glucuronic Acid