Capillary electrophoresis immunoassay (CEIA) is shown to be substantially more sensitive to the antibody (Ab) reagent quality than are immunosorbent methods such as enzyme-linked immunosorbent assays (ELISA). Cyanine 5 (Cy5)-labeled monoclonal anti-ovalbumin (mAb*) was inactive for CEIA of ovalbumin (Ov), yet was functional in ELISA for Ov. ELISA showed the mAb* was at least ten times less active, accounting for the poor CEIA performance. Labeled polyclonal Ab was inactive for a dye to protein ratio greater than 1.6. An affinity protection chromatography procedure (APC) was developed for Ab labeling, which avoided degradation of the Ab binding site. Ov was covalently bound to cyanogen bromide activated cellulose gel in a column, and used to capture the Ab. The coupling efficiency for Ov to the gel was 74-97%, Ab could then be bound with 95-100% efficiency, and Ab* was recovered in 50% yield following labeling on the column. This procedure was performed successfully in three different laboratories, indicating the robustness of the optimized APC synthetic method. No inactive Ab* could be detected in the APC product. The CEIA detection limit for ovalbumin using APC labeled mAb was 173 nM, when [Ab*] was fixed at 163 nM. The association constants of mAb and mAb* were determined by CEIA.
A microsphere-based suspension array (SA) system was used for the development and characterization of an immunoassay for the toxin simulant ovalbumin. Results obtained by SA immunoassay were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) using the same immunoreagents. The limit of detection (LOD) for the SA ovalbumin assay was 4.9 ng/mL, compared to a LOD of 0.01 ng/mL for the ovalbumin ELISA. Although the ELISA LOD exceeded that of the SA assay, the SA assay was simple and rapid to perform, with assays being completed in half the time of the traditional ELISA. The well-to-well reproducibility (coefficient of variation (CV)) of the ELISA and the SA assay was 4.9% and 5.1%, respectively. The ELISA and SA assay plate-to-plate reproducibility was 14.8% and 6.1%, respectively. The protocols used to develop the SA assay for ovalbumin may be used as a template for development of other SA assays for toxins, bacteria, and viruses.
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