Premium
Immunoassay of serum α 1 ‐antitrypsin by affinity‐probe capillary isoelectric focusing using a fluorescence‐labeled recombinant antibody fragment
Author(s) -
Shimura Kiyohito,
Hoshino Makoto,
Kamiya Keiichiro,
Katoh Kohgoro,
Hisada Sunao,
Matsumoto Hiroyuki,
Kasai Kenichi
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200203)23:6<909::aid-elps909>3.0.co;2-f
Subject(s) - isoelectric focusing , chemistry , recombinant dna , chromatography , detection limit , immunoassay , agarose , capillary electrophoresis , fluorescence , antibody , microbiology and biotechnology , biochemistry , enzyme , biology , physics , quantum mechanics , immunology , gene
An immunoassay for human α 1 ‐antitrypsin based on affinity‐probe capillary isoelectric focusing (AP‐CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser‐induced fluorescence detection. A recombinant Fab' fragment of mouse immunoglobulin G1 (IgG1) against human α 1 ‐antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region. A single p I isomer of the labeled Fab' was purified by IEF in a slab of agarose gel and was then used as the affinity probe for α 1 ‐antitrypsin. The use of recombinant Fab’ considerably simplified the labeling process. Although there was some difficulty in the quantification of native α 1 ‐antitrypsin with the affinity probe, carbamylation of the antigen sample by heat treatment with urea made the complex peaks appear reproducibly and more distinct, thus facilitating the identification and quantification of the complex. The system provided an almost linear response to a pure sample of α 1 ‐antitrypsin over a concentration range of 5–1000 ng/mL and the detection limit extended down to around 2 ng/mL. α 1 ‐Antitrypsin in a serum sample was determined using this system to be 1.2 mg/mL which is comparable to the reported value for the protein.