New zwitterionic polymer-coated immunoaffinity beads were developed to resist nonspecific protein adsorption from undiluted human serum for diagnostic applications of exosomes. A zwitterionic sulfobetaine monomer with an amine functional group was employed for simple surface chemistry and antifouling properties. An exosomal biomarker protein, epithelial cell adhesion molecule (EpCAM), was selected as a target molecule in this work. The beads were coated with polyacrylic acids (PAA) for increasing biorecognition sites, and protein G was then conjugated with carboxylic acid groups on the surfaces for controlling EpCAM antibody orientation. The remaining free carboxylic acid groups were modified with sulfobetaine moieties, and anti-EpCAM antibody was finally introduced. The amount of anti-EpCAM on the beads was increased by 40% when compared with PAA-uncoated beads. The surfaces of the beads exhibited near-net-zero charge, and nonspecific protein adsorption was effectively suppressed by sulfobetaine moieties. EpCAM was captured from undiluted human serum with almost the same degree of efficiency as from PBS buffer solution using the newly developed immunoaffinity beads.
A sensitive immunoanalysis system was developed for the quantitation of
imazethapyr, the active
ingredient in PURSUIT herbicide. Imazethapyr
[5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid] is one of the imidazolinone class of
herbicides. The assay was based on sequential
competitive binding of imazethapyr and liposomes for a limited number
of antibody binding sites.
A capillary tube (20 cm × 0.53 mm i.d.) with immobilized
antibody was used as the immunoreactor
column. Liposomes that entrap fluorescent molecules as the
detectable label provide instantaneous,
rather than time-dependent, enhancement, common with enzyme
immunoassays. In this study,
liposomes encapsulated carboxyfluorescein dye and were made antigenic
by incorporating in the
bilayer a phospholipid that had the analyte conjugated to its polar
head group. The calibration
curve for imazethapyr in Tris-buffered saline solution had a working
range of 0.1−100 ng/mL. In
the range between 1 and 100 ng/mL, recoveries from fortified tap and
pond water samples ranged
from 93 to 114%. Filtration was the only step needed for sample
cleanup, and an assay could be
performed in <10 min.
Keywords: Imazethapyr; FILIA (flow injection liposome immunoanalysis);
capillary immunocolumn; immunoassay; herbicide; liposomes
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