Behaviors of the MS2 virus and related antibodies in capillary isoelectric focusing with whole-column imaging detection.
Capillary isoelectric focusing (CIEF) has potential importance for the study of viruses. CIEF with whole-column imaging detection (WCID) is a novel CIEF mode, providing the advantages of high resolution, high speed, and easy method development. To facilitate the application of CIEF-WCID to the immunoassay of viruses, a basic knowledge of related aspects is necessary. In this study, the MS2 bacteriophage was used as a virus model, and the behaviors of MS2 and related antibodies in CIEF were investigated with UV absorbance-WCID and laser-induced fluorescence (LIF)-WCID. The adsorption of the virus and antibodies on the capillary wall was found to be the critical issue in method development. Addition of salt was found to be an effective way to reduce the adsorption and to improve peak shape. The formation of an immunocomplex, which forms the basis of an immunoassay, was monitored with CIEF-WCID. In comparison with UV-WCID, LIF-WCID was advantageous due to its higher detection sensitivity and the elimination of precipitation. Utilization of the noncovalent fluorescent dye, NanoOrange, was demonstrated to be a potential approach for the fluorescent labeling of the virus model and antibody and the associated immunocomplex. The change in microheterogeneity during the immune interactions at different ratios was also observed.
Liu
Zhen
Z
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada.
Pawliszyn
Janusz
J
D000327:Adsorption; D000914:Antibodies, Viral / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D005240:Feasibility Studies; D005455:Fluorescent Antibody Technique / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods*; D007834:Lasers; D017909:Levivirus / Q000276:immunology / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000914:Antibodies, Viral; D000936:Antigen-Antibody Complex