title: Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment.
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 1.2 mg/mL which is comparable to the reported value for the protein.
authors.affiliation: Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan. shimurak@pharm.teikyo-u.ac.jp
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Shimura
authors.forename: Makoto
authors.initials: M
authors.lastname: Hoshino
authors.forename: Kei-ichiro
authors.initials: K
authors.lastname: Kamiya
authors.forename: Kohgoro
authors.initials: K
authors.lastname: Katoh
authors.forename: Sunao
authors.initials: S
authors.lastname: Hisada
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Matsumoto
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Kasai
mesh_terms: D000178:Acrylamides; D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012235:Rhodamines; D000515:alpha 1-Antitrypsin / Q000032:analysis* / Q000276:immunology
chemical_list: D000178:Acrylamides; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D000515:alpha 1-Antitrypsin; C429790:poly(N,N-dimethylacrylamide); C034996:tetramethylrhodamine iodoacetamide