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doi: 10.1371/journal.pone.0054947. Epub 2013 Jan 29.

Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci

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Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci

Stéphanie Simon et al. PLoS One.2013.

Abstract

Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Test of the four most sensitive mAbs pairs against BL21(pla).
Sensitivity in two-site immunoassays was determined using 10 fold serial dilutions of BL21(pla) or BL21 as negative control. The * indicates the tracer antibody.
Figure 2
Figure 2. Detection ofY. pestis strains of various biovars by the Pla45/Pla35
*immunoassay. Six different strains ofY. pestis were grown at 28°C and 2×107 cfu/ml of each strain were used as antigen in the sandwich immunoassay. Absorbances at 414 nm were normalized using the absorbance of CO92 as reference (100%). Biovars were A: Antiqua, M: Medievalis and O, Orientalis. AY. pestis strain cured of pPla (6/69ΔpPla) was used as a negative control.
Figure 3
Figure 3. Epitope mapping of Pla45 and Pla35 mAbs.
(A) Pepscan epitope mapping of Pla45 and Pla35 mAbs was performed with synthetic peptides covering the 5 extracellular loops (L1 to L5) and the amino acids bordering these loops in PLA. The epitopes recognized by Pla45 are shown in a pale pink box, and those recognized by Pla35 in a darker pink box. (B) Amino acid sequence alignment of loop 5 from PLA, Epo, PgtE, OmpT and OmpP. Epitopes recognized by Pla45 and Pla35 mAbs on PLA, and the corresponding epitopes on the other molecules are in pink boxes.
Figure 4
Figure 4. Specificity of the Pla45/Pla35* mAb pair for PLA.
(A) Sandwich immunoassay againstSalmonella enterica serovar Typhimurium,Erwinia pyrifoliae,Escherichia coli and BL21(pla). (B) Sandwich immunoassay againstY. pestis CO92 and six strains ofY. pseudotuberculosis of various serotypes (for serotypes I to VI). (C) Reactivity of the Pla45/Pla35* pair againstY. pestis strain CO92 harboring pPla, and strain 6/69ΔpPla cured of the plasmid. (D) Western-blotting with Pla35 against recombinant PLA (lane 1, 1 µg), whole cell extracts ofY. pestis CO92 at a concentration of 4×106 cfu/well (lane 2), or 4×105 cfu/well (lane 3), IP516 at a concentration of 4×106 cfu/well (lane 4), or 4×105 cfu/well (lane 5), and 6/69ΔpPla (lane 6, 4×107 cfu/well),Y. pseudotuberculosis IP32953 (lane 7, 4×107 cfu/well), andE coli BL21 (lane 8, 4×107 cfu/well). Numbers on the left indicate the molecular weight markers (in kDa). Greek letters on the right indicate the various forms of PLA.
Figure 5
Figure 5. Impact of the growth temperature on PLA detection by sandwich ELISA.
(A)Y. pestis CO92 (blue lines) andY. pseudotuberculosis IP32953 (orange lines) were grown at three temperatures: 37°C (▪), 28°C (•) and 20°C (▴). Ten-fold serial dilutions of the cultures were used as antigens for the Pla45/Pla35* sandwich ELISA. (B) Comparison ofY. pestis detection using an anti-PLA or an anti-F1 tracer antibody. 109 cfu/ml ofY. pestis cultivated at three different growth temperatures (20°C, 28°C and 37°C), were incubated with Pla45 as capture antibody, and with either Pla35* (dark blue), or anti-F1* (light blue) conjugates.
Figure 6
Figure 6. Sensitivity of the optimized Pla45/Pla35* immunoassay.
Y. pestis CO92 was grown at 37°C (dark blue), 28°C (medium blue), or 20°C (light blue), and subjected to the optimized sandwich ELISA. The graphs represent the lowest AU values. The limit of detection (LoD) is represented by a horizontal black line. Vertical lines represent the standard deviation of duplicates.
Figure 7
Figure 7. Lateral flow immunoassays.
(A) Specificity of the PLA-dipstick forY. pestis harboring pPla. Both CO92 and 6/69ΔpPla were grown at 28°C and serially diluted. The bacterial suspensions were incubated for 10 min with colloidal gold-labeled Pla35 mAb, and the PLA-dipsticks were then dipped for 30 min into 100 µl of the bacterial suspensions for upward migration of the liquid. Numbers below the dipsticks indicate the number of cfu/well. (B). Influence of the temperature of culture on CO92 detection. Bacteria were grown at 20°C, 28°C or 37°C, and the tests were performed as described in (A).
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