Automated determination of antibody oxidation using flow injection analysis.
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 on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters 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.
Wolfe
C A
CA
Department of Chemistry, University of Nebraska, Lincoln 68588.
Hage
D S
DS
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D001331:Automation; D005456:Fluorescent Dyes; D007074:Immunoglobulin G / Q000737:chemistry*; D007546:Isoquinolines; D010084:Oxidation-Reduction; D010504:Periodic Acid; D011817:Rabbits; D013053:Spectrophotometry / Q000379:methods
D000906:Antibodies; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D007546:Isoquinolines; D010504:Periodic Acid; C017475:lucifer yellow