Optimization of antibody immobilization for on-line or off-line immunoaffinity chromatography.
The covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen-binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup-based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on-line IA-LC-MS was achieved using aminogroup immobilization of intact antibodies by epoxy- or aldehyde-activated POROS R20-matrices and in some cases by chemical crosslinking to Protein G-POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow conditions are not used, covalent crosslinking onto Protein G-POROS or Protein G-agarose is to be recommended.
Beyer
Natascha Helena
NH
Department of Clinical Biochemistry and Immunology, Statens Serum Institute, Copenhagen S., Denmark.
Hansen
Morten Zoega
MZ
Schou
Christian
C
Højrup
Peter
P
Heegaard
Niels H H
NH
D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology; D000941:Antigens / Q000276:immunology* / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004852:Epoxy Compounds / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000276:immunology
D055910:Antibodies, Immobilized; D000936:Antigen-Antibody Complex; D000941:Antigens; D004852:Epoxy Compounds; C029152:G-substrate; D007140:Immunoglobulin Fab Fragments; D009419:Nerve Tissue Proteins