Stabilization of antibodies by haptens

A new concept for the stabilization of antibodies in regenerable immunochemical sensors is described. The influence of a chemiluminescence (CL) substrate on the activity of different immobilized monoclonal antibodies (MAbs) directed to triazines has been investigated. One MAb (4A54) showed a decrease in activity of more than 50% upon incubation of the CL substrate for 2 h. It could be shown that MAb 4A54 can be stabilized by saturation with a suitable hapten. Total preservation of the antibody activity was observed by adding atrazine or simazine as stabilizing agents to the CL substrate at concentration levels of 10 μg/L and 100 μg/L. Additional amounts of hydrogen peroxide in concentration levels of 0.004% and 0.04% added to the test solutions did not increase the observed loss of activity of the antibody and also did not alter the stabilizing effect of atrazine and simazine on MAb 4A54. Loss of activity caused by low pH could not be prevented by hapten saturation under the employed experimental conditions. For practical use of this stabilization concept in immunochemical sensors, complete dissociation of the hapten used as stabilizing agent must be achieved prior to the use of the antibody in a successive sensor cycle. Therefore, the dissociation of the bound triazines was investigated. For simazine, a dissociation rate constant of 0.0052 s^-1 in neutral washing buffer was determined. After a dissociation time of 10 min, no bound simazine could be detected any more. As a control experiment, ELISA calibration curves were determined for MAb 4A54 after incubating the antibody with the CL substrate. The CL substrate caused significant reduction of the ELISA signal, which could be prevented by saturation of the antibody with a hapten. Evidence was given for the full dissociation of the hapten after the stabilization step. This indicates that the proposed stabilization concept is suitable for practical applications in immunochemical sensors.