Antibody immobilisation on fibre optic TIRF sensors.
A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab')-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10(-8) M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen-antibody complex and hence to regenerate the immunosensor surface was also evaluated.
Tedeschi
Lorena
L
Istituto di Fisiologia Clinica del C.N.R., Pisa e Centro Interdipartimentale di Ricerca E. Piaggio, Università di Pisa, Italy.
Domenici
Claudio
C
Ahluwalia
Arti
A
Baldini
Francesco
F
Mencaglia
Andrea
A
D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000032:analysis / Q000276:immunology; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000737:chemistry; D004566:Electrodes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005336:Fiber Optic Technology / Q000295:instrumentation*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D012709:Serum Albumin / Q000032:analysis* / Q000276:immunology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods
D000906:Antibodies; D000936:Antigen-Antibody Complex; D020099:Coated Materials, Biocompatible; D007074:Immunoglobulin G; D012709:Serum Albumin