| Bead-based enzyme-linked sandwich immunoassays were developed for two types of simulants: Bugbead and ovalbumin. Bugbead is an artificial microorganism model made of microspheres coated with proteins (mouse and guinea pig IgGs) via biotin-avidin bonding. These proteins represent the epitopes on the real microorganisms that are recognized by antibodies. Ovalbumin is chosen as a simulant for toxin. In both cases, primary antibodies were immobilized on the streptavidin-coated magnetic beads. Secondary antibodies were alkaline phosphatase conjugated. p-aminophenyl phosphate was the phosphatase substrate and the p-aminophenol product was detected amperometrically using flow injection analysis-amperometric and rotating disk electrodeamperometric detectors.; The method for Bugbead preparation was developed. The capturing event between the capture beads and the Bugbeads was observed by optical microsocpe. The minimum detected amount of Bugbeads was 360, using RDE-amperometric system. These results demonstrate that the Bugbead could be used in the initial steps of developing microbial sensors.; The lowest detectable concentration of ovalbumin was 0.1 ng/mL, comparable to published results with the significant improvement of incubation time. The developed assays can detect ovalbumin in some biological and food media. Normal rat serum did not show any interference effect while ice cream and artificial blood showed a slight increase in assay signals as compared to those obtained from assay prepared in HEPES buffer.; Different procedures were studied to improve the performance of the bead-based immunoassay. The number of washes and the volume of buffer used for each wash affect the efficiency of the washing step and the degree of non-specific binding (NSB). Apart from the normally used BSA, other blockers such as gelatin, CHAPS and superblock™, may be used to reduce NSB. The efficiency of the capture beads with different surface modifications (neutravidin-coated bead, dendrimer-streptavidin-coated bead, dendrimer-neutravidin-coated bead, and dendrimer-lactone-neutravidincoated bead) were compared with the efficiency of commercial streptavidin-coated bead. Dendrimer used in this study was commercial PAMAM G. 5.5. Dendrimer capture beads show better capture at high analyte concentration, due to a higher amount of primary antibody that the capture bead can accommodate. At low analyte concentration, there was no significant difference between capture bead with and without dendrimer. |
