Optimization of antibody immobilization for on-line or off-line immunoaffinity chromatography

被引:17
作者
Beyer, Natascha Helena [1 ,2 ]
Hansen, Morten Zoega [1 ]
Schou, Christian [1 ]
Hojrup, Peter [3 ]
Heegaard, Niels H. H. [1 ]
机构
[1] Statens Serum Inst, Dept Clin Biochem & Immunol, DK-2300 Copenhagen S, Denmark
[2] Statens Serum Inst, Dept Biosecur, DK-2300 Copenhagen S, Denmark
[3] Univ So Denmark, Dept Biochem & Mol Biol, Odense M, Denmark
关键词
Antibody; Covalent coupling; Immobilization; Immunoaffinity chromatography; LC-MS; AMYLOID P COMPONENT; AFFINITY-CHROMATOGRAPHY; MASS-SPECTROMETRY; SOLID-PHASE; COVALENT IMMOBILIZATION; SAMPLE PREPARATION; SUPPORT SURFACES; PROTEINS; PEPTIDE; PERFORMANCE;
D O I
10.1002/jssc.200800702
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
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 offline 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.
引用
收藏
页码:1592 / 1604
页数:13
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