A sensitive and specific enzyme-linked immunoadsorbent assay (ELISA) was developed to detect ricin in biological fluids. The assay is based on the sandwich format using monoclonal antibodies (MAbs) of two distinct specificities. An affinity-purified anti-ricin B chain MAb (1G7) is utilized to adsorb ricin from solution and the second anti-ricin A chain MAb (5E11) conjugated with peroxidase is then used to form a sandwich, and peroxidase allows color development and measurement of optical density at 450 nm. Standard curves were linear over the range of 2.5-100 ng/mL ricin. The limit of detection was below 5 ng/mL in assay buffer as well as in a 1:10 dilution of urine or 1:50 dilution of human serum spiked with ricin.
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.
A rapid lateral flow assay was developed to detect botulinum neurotoxin type A (BoNT/A). The assay was based on the sandwich format using monoclonal antibodies (MAbs) of two distinct specificities. One anti-BoNT/A heavy chain MAb (150-3) was immobilized to a defined detection zone on a porous nitrocellulose membrane, while the other anti-BoNT/A heavy chain MAb (44.1) was conjugated to colloidal gold particles, which served as a detection reagent. The BoNT/A-containing sample was added to the strip and allowed to react with MAb (44.1)-coated particles. The mixture was then passed along the porous membrane by capillary action past the MAb (150-3) in the detection zone, which binds the particles that had BoNT/A bound to their surface, giving a red color within this detection zone with intensity proportional to BoNT/A concentration. In the absence of BoNT/A, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/mL of BoNT/A were detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 1 ng/mL. The developed BoNT/A assay also showed no cross-reaction to type B neurotoxin (BoNT/B) and type E neurotoxin (BoNT/E).
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