Rapid and sensitive detection of Yersinia pestis by lateral-flow assay in simulated clinical samples

Abstract

BackgroundYersinia pestis is a contributing agent to the epidemic disease, plague, which killed an estimated 200 million people during historical times. In this study, a rapid, cheap, sensitive, and specific technique, the lateral flow assay (F1 strips), has been successfully developed to detect this pathogen, by using paired monoclonal antibodies (MAbs) against Y. pestis capsule like fraction 1 (F1) protein. Compared with the polyclonal antibody (PAb) based F1 strips, the Mab-based F1 strips have a remarkable increased detection limitation (10 to 100 folds). Furthermore, besides the limitation and specificity evaluation, the application of this F1 strip on simulated clinical samples indicate the LFA can be a good candidate to detect plague.MethodsRecombinant F1 antigen was expressed and purified from a series of works. The various anti-F1 monoclonal antibodies generated from hybridoma cells were screened with the ELISA technique. To evaluate the feasibility of this Y. pestis F1 test strip, the F1 protein/Y. pestis was spiked into simulated clinical samples such as human serum, mouse bronchoalveolar lavage fluids, and mouse blood to mimic natural infection status. Additionally, this technique was applied to detect the Y. pestis in the environment-captured rats, to evaluate the practical usefulness of the strips.ResultsBy using this MAb-based-LFA technique, 4 ng/ml of recombinant F1-protein and 103 CFU/ml of Y. pestis could be detected in less than 10 mins, which is at least 10-folds than that of the PAb format. On the other hand, although various Yersinia strains were applied to the strips, only Y. pestis strain showed a positive result; all other Yersinia species did not produce a positive signal, indicating the high efficiency and specificity of the MAb-based F1-strips.ConclusionBased on our findings, we suggest that the MAb-format-LFA will be valuable as a diagnostic tool for the detection of Y. pestis. This report shows that the F1 strip is sufficient to support not only the detection of plague in simulated clinical samples, but also it may be a good candidate to meet the epidemiological surveillance during an outbreak of the biological warfare.

Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

“…The capsule-like fraction 1 (F1) antigen produced by Y. pestis is a specific marker for pathogen identification. It is thus not surprising that the F1 antigen is the basis of a variety of detection methods [ 31 ]. F1 antigen biosynthesis is unique to Y. pestis and upregulated at 37 °C (mammalian phase of the infection cycle) [ 5 , 6 ].…”

“…More ideally, rapid and specific confirmatory detection techniques are needed for unambiguous pathogen identification. For Y. pestis , lateral flow immunochromatographic assays employing F1 capsule antibodies have been in successful use for many years and new derivatives are still being developed [ 10 , 31 , 39 ]. For instance, such invaluable rapid tests have been instrumental for on-site plague diagnostics in Madagascar since 2002 [ 40 ].…”

Development of a double‐antibody sandwich ELISA for sensitive detection of Yersinia pestis

“…The F1 protein, along with the V protein, is being developed as a major candidate for a plague vaccine . As the F1 protein is found only in Y. pestis , it has been used as a specific marker for identification of the bacteria …”

“…All animal experiments were performed in compliance with the recommendations (KCDC‐008‐14‐2A) of the Institutional Animal Care and Use Committee of the Korea Centers for Disease Control and Prevention. Monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs) were generated against recombinant F1 as described previously . Biotinylation of the F1 PAbs was performed using an EZ‐LINK NHS‐PEG4‐Biotinylation kit (Pierce, Rockford, IL, USA) according to the manufacturer's protocols.…”

“…Previous studies have demonstrated that lateral flow assay–based rapid diagnostic test (RDT) kits and ELISA methods could detect F1 antigen in the range of 0.5–50 ng and the bacteria in the range of 1 × 10 3 to 1 × 10 5 CFU/ml . By contrast, Tomaso et al detected 10 ng/ml of the F1 protein in serum or urine samples and 6 × 10 3 CFU/ml of Y. pestis in PBS buffer when using a commercially available ELISA kit.…”