MALDI-TOF MS–based immunoassay using switching peptide for sensitive detection of intact foodborne bacteria

Abstract A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)–based immunoassay was developed for quantitative detection of five foodborne bacterial species: Escherichia coli O157:H7, Salmonella typhimurium , Listeria monocytogenes , Staphylococcus aureus , and Bacillus cereus . The assay was conducted in a homogeneous solution without washing steps and enabled quantification through detection of switching peptides (antibody-binding peptides released upon antigen binding) released upon antigen–antibody binding, which served as mass tags for MALDI-TOF MS readout. Target-specific antibodies were isolated from commercially available unimmunized pig serum using a stepwise filtration approach involving target and filter bacteria, yielding 130.4 ± 20.0 µg and 95.2 ± 26.6 µg for the first and second isolated antibodies from 1 mL of pig serum ( n = 5), respectively. Quantitative analysis of the switching peptides using an internal standard (P14R) exhibited good linearity over the concentration range of 0.01–10.00 pmol/µL, with relative standard deviations of 1.4%–11.3% and coefficients of determination exceeding 0.983. Compared with fluorescence- and high-performance liquid chromatography–based methods, MALDI-TOF MS achieved a lower limit of detection, demonstrating improved sensitivity for detecting low-abundance bacterial targets. The developed immunoassay enabled quantitative detection of the five bacterial species at lower bacterial cell numbers per test than fluorescence-based assays.