Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment.
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.
Shimura
Kiyohito
K
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan. shimurak@pharm.teikyo-u.ac.jp
Hoshino
Makoto
M
Kamiya
Kei-ichiro
K
Katoh
Kohgoro
K
Hisada
Sunao
S
Matsumoto
Hiroyuki
H
Kasai
Ken-ichi
K
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
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