Evaluation of antibody immobilization technqiues for fiber optic-based fluoroimmunosensing

Fiber optic chemical sensors have been developed that employ immunochemicals to perform fluoroimmunoassays. A regenerable fiber optic sensor was developed with a capillary delivery system to perform repetitive assays using antibodies immobilized to beads, “immunobeads”. The sensitivity of these sensors is directly proportional to the amount of antibody present. For the immunobeads, the amount immobilized (loaded) and the ability of the immobilized antibody to maintain antibody recognition (activity) are two criteria which will affect the sensitivity of the sensors. Four reagents, 1,1′-carbonyldiimidazole (CDI), Protein A, glycidoxypropyltrimethoxysilane (GOPS) and 2-fluoro-1-methylpyridium toluene-4-sulfonate (FMP)], were evaluated using these two criteria. Two antibody—antigen systems were employed to investigate the four procedures. The first combination is a polyclonal rabbit anti-human IgG with a F(ab)^′₂ fragment human IgG as the antigen. The second combination is a monoclonal mouse anti-benzoa]pyrene tetraol (BPT) IgG with BPT as the hapten. In the case of the BPT, CDI demonstrated superior performance, combining high loading and high retention of antibody activity. In the case of the large antigen, CDI again immobilized the most antibody but suffered activity losses of about 40%. The large amount of inactive antibody may make this procedure less attractive than the Protein A beads, which maintained the activity of the anti-human IgG but exhibited less loading than the CDI. However, in terms of active antibody there are similar amounts. FMP yielded similar results to CDI for the large antigen case but the sample preparation is more labor intensive. GOPS yielded losses of up to 70% of the antibody activity, which makes it unattractive as a reagent. The two systems tested give an indication of antibody—antigen interactions but may not be representative of all cases. The ideal immobilization reagent and conditions will probably change from system to system.