Homogeneous sandwich immunoassay based on the enzymatic complementation induced by single-chain Fv fragments.
We describe a novel homogeneous sandwich immunoassay based on beta-galactosidase (beta-gal) complementation (the crab-claw sandwich enzymatic complementation immunoassay, CS-ECIA). We chose a high-molecular-weight antigen human serum albumin (HSA) as a model and constructed two chimeric proteins, in which a pair of single-chain Fvs (scFvs) recognizing distant epitopes of HSA was fused to either an N-terminal deletion mutant of beta-gal (deltaalpha) or a C-terminal deletion mutant of beta-gal (deltaomega). Upon simple mixing of the reagents with the sample, the two chimeric proteins became associated through binding separate epitopes on HSA that allowed reassociation of the two mutant enzymes. The resulting enzymatic complementation was measured as an increase in beta-gal activity using a luminescent substrate. With this CS-ECIA, a HSA concentration of 10-1000 pg/mL could be determined. In addition, the assay was easy to operate and required less time, handling, and sample volume than conventional sandwich enzyme-linked immunoassays. The assay will have general utility by substituting scFvs with other pairs of scFvs recognizing any polyvalent antigens.
Komiya
Naoto
N
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Ueda
Hiroshi
H
Ohiro
Yoshiyuki
Y
Nagamune
Teruyuki
T
D000941:Antigens / Q000032:analysis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007124:Immunoenzyme Techniques / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D008163:Luminescent Measurements; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012709:Serum Albumin / Q000032:analysis / Q000276:immunology; D001616:beta-Galactosidase / Q000235:genetics
D000941:Antigens; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C055966:immunoglobulin Fv; D001616:beta-Galactosidase