Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Abstract

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9-9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

Figure 1

Production of 11N11 scFv-SpyCatcher003 antibody in E. coli. (A) Schematic representation of the recombinant pHBSC3 expression vector. SC3, SpyCatcher003; variable light chain (V_L) and variable heavy chain (V_H) connected via a short linker form a single-chain variable fragment (scFv). (B) SDS-PAGE analysis of SEC-purified 11N11 scFv-SpyCatcher003 antibody. The input corresponds to the Ni-NTA elution sample. M, molecular weight markers.

Figure 2

Transient expression of IgG-SpyCatcher003 antibodies in mammalian cells. (A) Schematic representation of the recombinant pcDNA 3.4 expression vector. LC, light chain; HC, heavy chain; SC3, SpyCatcher003. (B) SDS-PAGE analysis of protein A purified IgG and IgG-SpyCatcher003 antibodies. M, molecular weight markers. A full image of the SDS-PAGE gel is provided in Supplementary Information.

Figure 3

Labelling and purification of SpyTag003-polyK peptide. (A) Schematic representation of the reaction between SpyTag003-polyK peptide and Eu(III)-N1-ITC chelate. (B) HPLC chromatogram of the Eu-labelled SpyTag003-polyK peptide.

Figure 4

Coupling of 11N11 scFv-SpyCatcher003 antibody with SpyTag003 and SpyTag003-polyK peptides. (A) SDS-PAGE analysis of 11N11 scFv-SpyCatcher003 antibody coupled with SpyTag003 and SpyTag003-polyK peptides. (B) SEC profiles of 11N11 scFv-SpyCatcher003 before (black) and after (grey) coupling with SpyTag003 peptide. (C) Differential scanning fluorimetry (DSF) curve of the 11N11 scFv-SpyCatcher003 before (black) and after (grey) coupling with SpyTag003 peptide.

Figure 5

Analysis of 11N11 IgG-SpyCatcher003 antibody complexation with the SpyTag003 and SpyTag003-polyK peptides. (A) SDS-PAGE analysis of 11N11 IgG-SpyCatcher003 antibody (lane 2) and as coupled with SpyTag003 peptide (lane 3) and SpyTag003-polyK peptide (lane 4). A full image of the SDS-PAGE gel is provided in Supplementary Information. (B) Isoelectric focusing (IEF) of 11N11 IgG-SpyCatcher003 antibody (lane 1) and as complex with SpyTag003 peptide (lane 2) and with SpyTag003-polyK peptide (lane 3). A full image of the IEF gel is provided in Supplementary Information. (C) DSF curve of the 11N11 IgG-SpyCatcher003 before (black) and after (grey) coupling with SpyTag003 peptide.

Figure 6

Performance evaluation for site-specifically labelled 11N11 scFv-SpyCatcher003/SpyTag003-polyK antibody in TRFIA. (A) Detection of the cTnI with site-specifically labelled 11N11 scFv-SpyCatcher003/SpyTag003-polyK antibody (black; 7.9 Eu-chelates/scFv) and non-specifically labelled scFv-SpyCatcher003/SpyTag003 antibodies (grey; 2.3 Eu-chelates/scFv and 7.2 Eu-chelates/scFv). (B) Detection of the cTnI with site-specifically labelled 11N11 IgG-SpyCatcher003/SpyTag003-polyK antibody (black; 17.8 Eu-chelates/IgG) and non-specifically labelled IgG-SpyCatcher003/SpyTag003 antibodies (grey; 2.8 Eu-chelates/IgG, 8.7 Eu-chelates/IgG and 25.7 Eu-chelates/IgG). Data represents mean ± SD (n = 3).