Premium
High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains
Author(s) -
Zhou Fangyu,
Kroetsch Andrew,
Nguyen Vyncent P.,
Huang Xiao,
Ogoke Ogechi,
Parashurama Natesh,
Park Sheldon
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201800647
Subject(s) - linker , antibody , chemistry , peptide , bivalent (engine) , combinatorial chemistry , intein , small molecule , epitope , biophysics , biochemistry , microbiology and biotechnology , biology , genetics , computer science , rna , organic chemistry , rna splicing , gene , metal , operating system
Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody‐binding protein as an adapter can simplify antibody functionalization by forming a specific antibody‐bound complex and introducing site‐specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody‐binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein (“lasso”), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 n m and a dissociation rate that is 55‐ to 84‐fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme‐linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12‐fold. The small size of the lasso and a long half‐life of dissociation make the peptide a useful tool in antibody detection and immobilization.