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. 2022 Dec 11;13(12):2194.
doi: 10.3390/mi13122194.

Rapid Detection of Lipopolysaccharide and Whole Cells of Francisella tularensis Based on Agglutination of Antibody-Coated Gold Nanoparticles and Colorimetric Registration

Nadezhda A Byzova  1 , Anatoly V Zherdev  1 , Alexey A Gorbatov  2 , Anton G Shevyakov  2 , Sergey F Biketov  2 , Boris B Dzantiev  1
Affiliations

Rapid Detection of Lipopolysaccharide and Whole Cells of Francisella tularensis Based on Agglutination of Antibody-Coated Gold Nanoparticles and Colorimetric Registration

Nadezhda A Byzova et al. Micromachines (Basel). .

Abstract

The paper presents development and characterization of a new bioanalytical test system for rapid detection of lipopolysaccharide (LPS) and whole cells of Francisella tularensis, a causative agent of tularemia, in water samples. Gold nanoparticles (AuNPs) coated by the obtained anti-LPS monoclonal antibodies were used for the assay. Their contact with antigen in tested samples leads to aggregation with a shift of absorption spectra from red to blue. Photometric measurements at 530 nm indicated the analyte presence. Three preparations of AuNPs with different diameters were compared, and the AuNPs having average diameter of 34 nm were found to be optimal. The assay is implemented in 20 min and is characterized by detection limits equal to 40 ng/mL for LPS and 3 × 104 CFU/mL for whole cells of F. tularensis. Thus, the proposed simple one-step assay integrates sensitivity comparable with other immunoassay of microorganisms and rapidity. Selectivity of the assay for different strains of F. tularensis was tested and the possibility to choose its variants with the use of different antibodies to distinguish virulent and non-virulent strains or to detect both kinds of F. tularensis was found. The test system has been successfully implemented to reveal the analyte in natural and tap water samples without the loss of sensitivity.

Keywords: agglutination; colloidal gold; express diagnostics; immunoanalysis; lipopolysaccharide; tularemia.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Electron microphotographs (A) of AuNPs1 (1), AuNPs2 (2), and AuNPs3 (3) and histograms of diameter distribution (B).
Figure 1
Figure 1
Electron microphotographs (A) of AuNPs1 (1), AuNPs2 (2), and AuNPs3 (3) and histograms of diameter distribution (B).
Figure 2
Figure 2
Dependences of OD450 on the concentration of MAbs (A) of clones Fb11 (I) and T143 (II) and the OD of AuNPs1–Fb11 ((B), III), AuNPs1–T143 ((B), IV), AuNPs3–Fb11 ((C), V), and AuNPs3–T143 ((C), VI) conjugates. F. tularensis LPS was immobilized in the microplate wells at the concentration of 3 µg/mL. All measurements were performed in triplicate.
Figure 3
Figure 3
Agglutination of AuNPs1–Fb11 (A), AuNPs1–T143 (B), AuNPs2–Fb11 (C), AuNPs2–T143 (D), AuNPs3–Fb11 (E), and AuNPs3–T143 (F) conjugates in the presence of various concentrations of F. tularensis LPS. Curves 1–4 correspond to 1, 10, 20, and 30 min of the reaction.
Figure 3
Figure 3
Agglutination of AuNPs1–Fb11 (A), AuNPs1–T143 (B), AuNPs2–Fb11 (C), AuNPs2–T143 (D), AuNPs3–Fb11 (E), and AuNPs3–T143 (F) conjugates in the presence of various concentrations of F. tularensis LPS. Curves 1–4 correspond to 1, 10, 20, and 30 min of the reaction.
Figure 4
Figure 4
Agglutination of AuNPs2–Fb11 conjugate in the presence of various concentrations of thermally inactivated F. tularensis subsp. holarctica 15 NIIEG (A) and 503 (B), F. tularensis subsp. mediasiatica 120 (C), F. tularensis subsp. tularensis Schu (D) and A-Cole B-399 (E), and F. tularensis subsp. novicida Utah112 (F). Curves 1 and 2 correspond to 1 and 20 min of the reaction.
Figure 4
Figure 4
Agglutination of AuNPs2–Fb11 conjugate in the presence of various concentrations of thermally inactivated F. tularensis subsp. holarctica 15 NIIEG (A) and 503 (B), F. tularensis subsp. mediasiatica 120 (C), F. tularensis subsp. tularensis Schu (D) and A-Cole B-399 (E), and F. tularensis subsp. novicida Utah112 (F). Curves 1 and 2 correspond to 1 and 20 min of the reaction.
Figure 5
Figure 5
Agglutination of AuNPs2–T143 conjugates in the presence of various concentrations of thermally inactivated F. tularensis subsp. holarctica 15 NIIEG (A) and 503 (B), F. tularensis subsp. mediasiatica 120 (C), F. tularensis subsp. tularensis Schu (D) and A-Cole B-399 (E), and F. tularensis subsp. novicida Utah112 (F). Curves 1 and 2 correspond to 1 and 20 min of reaction.
Figure 5
Figure 5
Agglutination of AuNPs2–T143 conjugates in the presence of various concentrations of thermally inactivated F. tularensis subsp. holarctica 15 NIIEG (A) and 503 (B), F. tularensis subsp. mediasiatica 120 (C), F. tularensis subsp. tularensis Schu (D) and A-Cole B-399 (E), and F. tularensis subsp. novicida Utah112 (F). Curves 1 and 2 correspond to 1 and 20 min of reaction.
Figure 6
Figure 6
Calibration curves for AuNPs2–T143 agglutination at 530 nm in the presence of different concentrations of F. tularensis LPS (A) and F. tularensis subsp. holarctica 15 NIIEG (B). Curves 1 and 2 correspond to 1 and 20 min of the reaction. Measurements were made in triplicate.
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