Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies.
The interaction between the humanised A33 monoclonal antibody and the corresponding F(ab)'2 or Fab' fragments with the colonic epithelial A33 antigen, purified by micropreparative HPLC from membrane extracts of the colonic carcinoma cell line LIM 1215, has been studied with the BIAcore 2000 biosensor using surface plasmon resonance detection. The surface orientation of immobilised antibody and the Fab' fragment onto the biosensor surface was controlled using alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared with the conventional N-hydroxysuccinimide (NHS)/N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC) chemistry. This increase in signal resulted in a concomitant increase in sensitivity of detection, which facilitates analysis of low levels of A33 antigen. The apparent association rate (ka) and dissociation rate (kd) constants obtained with the different immobilisation chemistries were determined. These analyses showed that the kinetic constants obtained for the IgG were not significantly affected by the method of immobilisation. F(ab)'2 and Fab' fragments immobilised using NHS/EDC chemistry showed significantly lower apparent affinity. By contrast the use of the thiol coupling chemistry with the Fab' fragment gave a five fold increase in observed KA, resulting in a similar affinity to that observed with the intact IgG molecule.
Catimel
B
B
Ludwig Institute for Cancer Research. PO Royal Melbourne Hospital, Parkville, Victoria, Australia.
Nerrie
M
M
Lee
F T
FT
Scott
A M
AM
Ritter
G
G
Welt
S
S
Old
L J
LJ
Burgess
A W
AW
Nice
E C
EC
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000941:Antigens / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D015374:Biosensing Techniques; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003106:Colon / Q000276:immunology*; D003432:Cross-Linking Reagents; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007413:Intestinal Mucosa / Q000276:immunology; D007700:Kinetics; D013205:Staphylococcal Protein A / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D000941:Antigens; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D013438:Sulfhydryl Compounds