Automated determination of antibody oxidation using flow injection analysis

Area of Science:

• Bioconjugation Chemistry
• Analytical Biochemistry
• Protein Modification

Background:

• Antibody carbohydrate oxidation is key for site-specific modification.
• Monitoring oxidation is crucial for reproducible antibody-based assays.

Purpose of the Study:

• Develop a flow injection analysis (FIA) system for monitoring antibody oxidation.
• Quantify antibody and label concentrations accurately and efficiently.

Main Methods:

• Antibodies were oxidized using periodate, followed by labeling with Lucifer yellow CH (LyCH).
• An FIA system measured LyCH via absorbance (428 nm) and antibody via bicinchoninic acid assay.
• Analyzed 20 microliter samples with a 2-minute analysis time per sample.

Main Results:

• Achieved limits of detection of 1 x 10(-8) M for rabbit immunoglobulin G (IgG) and 4 x 10(-7) M for LyCH.
• Demonstrated dynamic ranges up to 2 x 10(-5) M for IgG and 7 x 10(-3) M for LyCH.
• FIA system showed higher accuracy than manual methods for high LyCH concentrations and monitored oxidation time courses.

Conclusions:

• The developed FIA system provides a rapid and accurate method for monitoring antibody oxidation.
• This technique is valuable for optimizing antibody modification protocols and ensuring assay consistency.
• The system enables precise characterization of antibody conjugation and modification processes.