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For optimal results, the immunosensor should be cleaned after each assay. The antibodies should be removed by washing the surface with a 10 mM TRIS-HCl buffer at pH 7.4. To further improve the recovery of the antibodies, the sensor can be incubated in a 10 mM TRIS-HCl solution at pH 7.4, and for an additional 20 min at 50 °C. The antibody-BSA conjugate is soluble in the TRIS buffer and can be removed by washing with it. In addition, the TRIS buffer can be used to remove nonspecific binding, to determine the isoelectric point of the antibodies on the inner surface, and to study the antigen-antibody interaction. The procedure described above is as follows: (a) To immobilize the antibodies, a 10 mM TRIS-HCl buffer at pH 7.4 is added to the inner capillary surface at a flow rate of 1.6 mL/min. The solution is allowed to pass through the inner surface for 30 min at room temperature. The capillary is subsequently flushed with the buffer for an additional 30 min and is dried at 50 °C for 30 min.
In an attempt to enhance the affinity of ZON-mAb for ZON-BSA, ZON-BSA was conjugated with the synthetic peptide, ZON(29-41), as explained in section 2.2.2. The binding capacity of the ZON(29-41)-BSA conjugate to ZON-mAb was checked by competitive ELISA. The results are shown in Table 2. The data are expressed as the percentage of ZON-mAb bound, which is the antibody in percentage bound to ZON-(29-41) that is bound to ZON-mAb. The conjugate and the ZON-mAb complex were applied to a microtiter plate, and the amount of free antibody was estimated. The conjugate was found to bind more than 100 % of the antibody. As expected, the ZON(29-41)-BSA conjugate binds less than 100 % of ZON-mAb. As a consequence, ZON(29-41)-BSA conjugate immobilized on the microtiter plate would decrease the amount of free ZON-mAb available for binding to ZON. This decrease in affinity affects the performance of the microtiter plate-based assays. However, it is still possible to measure ZON using ZON(29-41)-BSA conjugate immobilized on the sensor surface.
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One of the drawbacks to this approach was that we needed to recompile every time we made even a small change to the code. This slowed everything else we were doing down and because we couldn't always have someone waiting for the application to finish building. 827ec27edc