Abstract: The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an online bicinchoninic acid protein assay. The analysis time was 2 min per 20 µL sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10^-8 and 4 x 10^-7 M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10^-5 and 7 x 10^-3 M. The within-run precision was±5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.

Abstract: Sample (100 µL) was injected into a phosphate-buffered saline binding buffer stream (0.7 mL min-1) and passed through a Protein A affinity column (prepared as described by Ngo, Biotechnol., 1986, 4, 134). The absorbance of the effluent was monitored at 280 nm. When the absorbance peak returned to baseline any bound immunoglobulin on the column was eluted with 0.1 M sodium acetate of pH 3 (1.5 mL min-1) and the eluate was monitored at 280 nm. Calibration graphs were rectilinear up to at least 4 and 10 mg mL-1 of IgG in human and bovine standards, respectively. Recoveries were 95.8 to 100.6%.

Abstract: The effects of incubation time, IgG subclass affiliation and immunoassay type on microtiter plate ELISA and FIIA were investigated. The advantages and limitations of the sandwich and the competitive assay for mouse IgG are discussed.