Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment

Electrophoresis
Kiyohito ShimuraKen-ichi Kasai

Abstract

An immunoassay for human alpha(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 alpha(1)-antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region. A single pI isomer of the labeled Fab' was purified by IEF in a slab of agarose gel and was then used as the affinity probe for alpha(1)-antitrypsin. The use of recombinant Fab' considerably simplified the labeling process. Although there was some difficulty in the quantification of native alpha(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 alpha(1)-antitrypsin over a concentration range of 5-1000 ng/mL and the detection limit extended down to around 2 ng/mL. Alpha(1)-antitrypsin in a serum sample was determined using this system to be 1...Continue Reading

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Citations

May 4, 2005·Analytical Biochemistry·Shiming Lin, Su-Ming Hsu
Nov 25, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·W S B YeungJ P Ou
Jun 12, 2009·Electrophoresis·Niels H H Heegaard
Dec 25, 2008·Electrophoresis·Kiyohito Shimura
Dec 7, 2006·Electrophoresis·Lotta K Amundsen, Heli Sirén
Aug 24, 2006·Electrophoresis·Yu-Fen HuangHuan-Tsung Chang
Jul 12, 2007·Journal of Separation Science·Kiyohito ShimuraTakehiko Kitamori
Jan 25, 2020·Electrophoresis·Hana NevídalováZdeněk Glatz

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