Immobilization of ultra-thin layer of monoclonal antibody on glass surface.
When preparing an affinity column and a biosensor, it is desirable to immobilize a unimolecular layer of pure protein on a matrix. In this work, we tried to immobilize a monoclonal antibody on a surface of a glass test-tube as a model, to confirm the stability of this ultra-thin layer by an enzyme immunoassay, and to estimate the thickness of the layer on a slide glass by Fourier transform infrared reflection spectrometry. A new test-tube was washed and dried. The tube was filled with 5% 3-aminopropyltriethoxysilane. The 3-aminopropylsilylated surface was treated with glutaraldehyde and 5.6.10(-2) mg/ml solution of a normal mouse monoclonal antibody. The Schiff base between glutaraldehyde and the antibody was further reduced with 7.9.10(-3)% NaBH4. The tube was washed with 0.05% Tween 20 to block non-specific binding. The antibody immobilized on the surface was measured by an enzyme immunoassay based on a reaction of anti-mouse immunoglobulin G labelled with alkaline phosphatase, with which p-nitrophenol was produced from p-nitrophenylphosphate as a substrate. Meanwhile, various amounts of the antibody were immobilized on slide glasses in the same manner. The antibody on each surface was measured by Fourier transform infrared reflection spectrometry. The antibody immobilized under the final conditions was detectable by the enzyme immunoassay, and stable at 4 degrees C for ten days. The antibody on the slide glass was a unimolecular layer, as judged from the Fourier transform infrared spectra referred to -CONH- band semiquantitatively. Thus, we found the optimal conditions for immobilizing an ultra-thin layer of the monoclonal antibody on the glass surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoshioka
M
M
Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Mukai
Y
Y
Matsui
T
T
Udagawa
A
A
Funakubo
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation; D005583:Fourier Analysis; D005898:Glass; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D013053:Spectrophotometry / Q000379:methods; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G