Noncompetitive Immunoassay of Small Analytes at the Femtomolar Level by Affinity Probe Capillary Electrophoresis:  Direct Analysis of Digoxin Using a Uniform-Labeled scFv Immunoreagent

Abstract

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.

Microchip separations of protein biotoxins using an integrated hand-held device

“…In order to detect trace levels of these toxins in the environment, high detection sensitivity is required. Laser-induced fluorescence (LIF) has been demonstrated in CE applications to detect down to picomolar concentrations of fluorescently tagged protein without preconcentration [13,14]. In order to achieve the sensitivities required for biosensing applications, we developed a compact LIF detector for our CE instrument.…”

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser‐induced fluorescence detection

“…Separation of free ligand from ligand-analyte complex by either capillary electrophoresis (CE) [1][2][3][4][5] or capillary isoelectric focusing (CIEF) [6,7] enables quantification of analyte. The highly selective complex formation combined with sensitive laser-induced fluorescence (LIF) detection enables trace analysis in complex mixtures.…”

Recent Progress in Noncompetitive Hapten Immunoassays: A Review

“…The performances of this method are good, with a limit of detection of 5 pM for human growth hormone (Shimura and Karger, 1994) and 10pM for digoxin (Hafner et al 2000), respectively. However, the correct separation appears difficult for some neutral analytes and the use of a charged analogue of the analyte is necessary to differentiate the complex from the unbound antibody.…”

“…This method is named as affinity probe capillary electrophoresis (APCE) and first described by Shimura and Karger (1994) using capillary isoelectric focusing for human growth hormone assay and further applied to small analytes by Hafner et al (2000). In this case, the immune complex is separated from excess labeled antibody since the complexation of the antigen with the antibody induces small changes of electrophoretic behavior of the labeled antibody.…”