Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter

Scientific Reports
Nur Faezee Ismail, Theam Soon Lim

Abstract

Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diag...Continue Reading

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Citations

Nov 24, 2016·Methods : a Companion to Methods in Enzymology·Min ShenChandra K Dixit
Mar 11, 2020·Annual Review of Chemical and Biomolecular Engineering·Emily A BerckmanWilfred Chen
Oct 4, 2018·Analytical and Bioanalytical Chemistry·Linan ZhangLihua Li
Feb 26, 2019·Applied Microbiology and Biotechnology·Soo Khim Chan, Theam Soon Lim

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Methods Mentioned

BETA
biosensor
phage display
Protein Assay
enzyme-linked immunosorbent assay
ELISA
PCR
affinity purification

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