title: Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter.
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 diagnostic assays for various diseases on the PGM platform.
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Nur Faezee
authors.initials: NF
authors.lastname: Ismail
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Theam Soon
authors.initials: TS
authors.lastname: Lim
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005947:Glucose; D049452:Green Fluorescent Proteins; D006801:Humans; D007118:Immunoassay; D018796:Immunoconjugates; D019095:Point-of-Care Systems; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; D018796:Immunoconjugates; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D005947:Glucose