Noncompetitive immunoassay of small analytes at the femtomolar level by affinity probe capillary electrophoresis: direct analysis of digoxin  using a uniform-labeled scFv immunoreagent.
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.
Hafner
F T
FT
Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, USA.
Kautz
R A
RA
Iverson
B L
BL
Tim
R C
RC
Karger
B L
BL
D000345:Affinity Labels; D002316:Cardiotonic Agents / Q000032:analysis* / Q000097:blood / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D004077:Digoxin / Q000032:analysis* / Q000097:blood / Q000652:urine; D019075:Electrophoresis, Capillary; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D002316:Cardiotonic Agents; D007140:Immunoglobulin Fab Fragments; D004077:Digoxin