Liquid-Phase Binding Assay of α-Fetoprotein Using DNA-Coupled Antibody and Capillary Chip Electrophoresis

Abstract

An immunoassay using DNA-coupled antibody for bound/free separation in a liquid-phase binding assay format is described. Anti-alpha-fetoprotein monoclonal antibody was conjugated with DNA, mixed with alpha-fetoprotein (AFP), and incubated, and then 1 muL of the mixture was applied to capillary electrophoresis on a microchip. The DNA molecule of the antibody-DNA conjugate and the DNA-conjugated immune complex peak were detectable fluorophotometrically using intercalator dye within 90 s, whereas the Alexa-labeled antibody was detected as a broad and slower migrating peak. The electrophoretic mobility of the immune complex could be optimized for resolution and sharpness by changing the length of the DNA coupled to the antibody. The detection limit of AFP was approximately 300 pM in a sample. This immunoassay method utilizing a liquid-phase binding assay format is simple and convenient for antigen measurements on microchips.

“Electrokinetic Analyte Transport Assay” for α‐fetoprotein immunoassay integrates mixing, reaction and separation on‐chip

“…This allows the injection of the immune complex into the separation channel as a sharp plug even though the initial sample zone is a very wide plug. In addition, DNA conjugate enables higher S/N in the CGE separation step by making the complex peak sharper [17]. The result is a dramatic increase in assay sensitivity due to sample concentration, providing sufficient number of analyte molecules for direct fluorescent detection without further amplification of signal.…”

“…In a mobility-shift immunoassay for AFP using fluorescencelabeled DNA-WA1, the AFP immune complex migrated slightly slower than unreacted DNA-WA1 [17]. In that assay format, the detection sensitivity suffered because tailing signal from the large, unreacted fluorescence-labeled DNA-WA1 peak overlapped with the small AFP immune complex peak (data not shown).…”

“…In order to concentrate molecules by ITP, the molecules should have electrophoretic mobilities that are faster than the trailing ion and slower than the leading ion, which are HEPES and chloride ion. We had reported that the mobility of DNA-WA1 could be easily controlled by changing the length of the DNA fragment attached to the antibody [17]. Therefore, we optimized the DNA length coupled to anti-AFP WA1 Fab' antibody so that the DNA-conjugated antibody as well as immune reaction complex can be concentrated by ITP.…”

“…Therefore, the competitive immunoassay format, for which the labeling target molecule can be relatively small and electrophoretically homogeneous, has been a better method for achieving high sensitivity with on-chip immunoassays. In order to realize high sensitivity with a non-competitive assay, we demonstrated the usefulness of the DNA-coupled antibody [17]. Introducing high negative charge into antibody by coupling with a DNA fragment minimized the heterogeneity of the antibody charge-to-mass ratio.…”

Studying drug–plasma protein interactions by two‐injector microchip electrophoresis frontal analysis

“…MCE has already been shown with further benefits inherent to miniaturization such as lower sample and reagent consumption, higher speed, potential for on-site use, ease of multichannel or highthroughput analysis, and convenient integration of several laboratory procedures [16][17][18][19]. MCE has been applied to characterize weak affinity interactions [20,21].…”