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

• Published in: Analytical chemistry (Washington) Vol. 72; no. 23; pp. 5779 - 5786
• Main Authors: Hafner, Frank T, Kautz, Roger A, Iverson, Brent L, Tim, Roger C, Karger, Barry L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-12-2000
• Subjects: Affinity Labels; Analysis; Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cardiotonic Agents - analysis; Cardiotonic Agents - blood; Cardiotonic Agents - urine; Chemistry; Chromatography, High Pressure Liquid; Digoxin - analysis; Digoxin - blood; Digoxin - urine; Electrophoresis, Capillary; Fundamental and applied biological sciences. Psychology; Humans; Immunoassay; Immunoglobulin Fab Fragments; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Analysis method; Digoxin; Capillary electrophoresis; Quantitative analysis; Single chain antibody; Immunological method
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac000853+

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-μm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 °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.