High-Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody-Binding Domains

Biotechnology Journal
Fangyu ZhouSheldon Park

Abstract

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 ...Continue Reading

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Citations

Jun 3, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Kristina WesterlundAmelie Eriksson Karlström
Aug 10, 2021·Bioconjugate Chemistry·Emma von WittingSara Kanje

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