Studies of the optimization and control of antibody oxidation for labeling or immobilization

The modification of antibodies for labeling or immobilization is an important aspect of many analytical and biochemical methods. One way to modify antibodies is to oxidize their carbohydrate chains with periodate to form reactive aldehyde moieties. The aldehydes can then be reacted with an amine- or hydrazide-containing label or support. In this work, the periodate oxidation of the antibody rabbit immunoglobulin G (IgG) was studied. The formation of aldehydes on the antibody carbohydrate chains was followed by labeling the aldehydes with Lucifer yellow CH (LyCH). The LyCH labeling protocol was optimized and a method was developed for purifying the oxidized antibodies. To determine the extent of LyCH labeling, and therefore the amount of oxidation that occurred, a flow injection analysis (FIA) system was developed. The FIA system monitored LyCH by absorbance measurements performed at 428 nm. The amount of protein was determined using an on-line bicinchoninic acid (BCA) protein assay. The analysis time was 2 minutes per 20 ml sample injection. The limits of detection for the IgG and LyCH were 1 $\times$ 10$\sp{-8}$ and 4 $\times$ 10$\sp{-7}$ M, and the dynamic ranges were 2 $\times$ 10$\sp{-5}$ and 7 $\times$ 10$\sp{-3}$ M, respectively. The within-run precision was $\pm$5% or less for both analytes. The antibody oxidation reaction was studied by adjusting the pH, temperature, periodate concentration and the time of the reaction. Conditions were determined that were suitable for either antibody labeling or immobilization while retaining adequate antibody activity. The long term stability of the oxidized antibodies was also considered. The kinetics of this oxidation process were studied using the FIA system. It was found that the overall rate reaction was a sum of two pseudo-first order reactions. This was due to the presence of different types of aldehyde-forming sites--a fast reacting site and a slow reacting site. The faster site is proposed to represent the exocyclic diols of sialic acids at the ends of the carbohydrate chains; the slower sites are believed to be due to cyclic diols of sugars within the carbohydrate chains