Recent developments in antibody-based assays for the detection of bacterial toxins

Abstract

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

Figure 1

(A) Schematic representation of bacterial toxins of low and high molecular weight (MW); and (B) examples for direct or indirect detection methods of bacterial toxins. LPS: lipopolysaccharides; HPLC: high-performance liquid chromatography; HPLC-MS: HPLC-mass spectrometry; MALDI-TOF: matrix-assisted laser desorption ionization time-of-flight; ELISA: enzyme-linked immunosorbent assay; and PCR: polymerase chain reaction.

Figure 2

Antibodies and assays: (A) types of antibodies used for immunoassay, immunoglobulin G (IgG) and related fragments (VL: variable light chain; VH: variable heavy chain; CL: constant light chain; CH: constant heavy chain; Fab: antigen binding fragment; and scFv: single-chain variable fragment); (B) heavy chain antibody (hcAb) and related fragments (VHH: variable domain of heavy chain antibodies; Nb: nanobody; and sdAb: single domain antibody); (C) the recognition of toxin targets by antibodies in competitive and noncompetitive assays; (D) labeling of primary antibodies for signal generation (Ab: antibody); (E) common readout techniques involved in immunoassays; and (F) examples for indirect assays. The amplification of toxin genes was done by PCR, the enrichment culture of the toxin producing organism and functional assays, such as cytotoxicity testing on mammalian cells.

Figure 3

(A) Schemes of gold and silver hetero dimer-based chiroplasmonic methods for (a) assaying proteins; (b) microcystin-LR. Reprinted with permission from [39]. Copyright 2013 American Chemical Society; (B) Scheme of streptavidin functionalized magnetic nanoparticles (MNPs) for the simultaneous detection of five bacterial toxins in a microarray. Reprinted with permission from [24]. Copyright 2013 American Chemical Society; (C) Scheme of indirect competitive immunoassay for the detection of microcystin-LR based on the conjugation of quantum dots (QDs) and aminoethyl microcystin-LR. Reprinted with permission from [44]. Copyright 2014 Elsevier; (D) Scheme of Ab-QD-based microarrays for pathogen detection. In the presence of captured pathogen by the Ab-QD probes, the fluorescence of QDs are in the ON state, whereas in the absence ofthe pathogen, QDs are quenched by fluorescence resonance energy transfer (FRET) interaction between QDs and graphene oxide (GO) (OFF state). Reprinted with permission from [45]. Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. CP: chiroplasmon; NPs: nanoparticles; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid; NHS: N-hydroxysuccinimide; and CD: circular dichroism.

Figure 4

(A) (a) Biochip layout; (b) scheme of electrode functionalization; and (c) Nyquist spectra of different cholera toxin (CT) concentrations. (B) (a) Scheme of the competitive immunoassay in the immune-reaction columns; and (b) an illustration of the chip operations for the immunoassay. Reproduced from [102,103] with permission from The Royal Society of Chemistry.

Figure 5

(A) (a) Synthesis of a novel aggregation-induced emission (AIE) probe (9,10-distyrylanthracene with two ammonium group, DSAI); and (b) a schematic description of a selective fluorescent aptasensor based on the DSAI/GO probe. Reprinted with permission from [115]. Copyright 2014 American Chemical Society; (B) Scheme of the key steps in the digital microfluidic (DMF) electroimmunoassay. Reproduced from [119] with permission from The Royal Society of Chemistry; (C) The immunochromatographic assay on a thread device assembly and assay protocol. Reprinted with permission from [120]. Copyright 2012 American Chemical Society; (D) The main types of assays for the detection of bacterial toxins in microtiter plates. Reproduced from [121] with permission from The Royal Society of Chemistry. ELISA for a single analyte; parallel detection of multiple analytes with multiple outputs; a single output for the detection of multiple analytes.