Colloidal silver-based lateral flow immunoassay for detection of profenofos pesticide residue in vegetables.

A colloidal silver nanoparticle (AgNP)-based lateral flow immunoassay (LFIA) was evaluated in terms of the rapid detection of profenofos (PEO) pesticide residue in vegetables. Colloidal AgNPs, of a diameter of approximately 20 nm, were surface-modified with trisodium citrate dehydrate (TSC) in order to improve their stability and dispersion. An anti-profenofos polyclonal antibody (pAb) was successfully immobilized on the surface of the AgNPs by ionic interaction and characterized using UV-vis, SEM, TEM, FTIR and XPS analyses. Surface modification of Ag-pAb conjugates of varying pH, pAb content and cross-reactivity was employed to design and prepare labels for use in an LFIA to examine whether these factors affect the performance of the assay. The visible detection limit and optical detection limit of the PEO test strip were 0.20 and 0.01 ppm, respectively, in PEO standard solution. This assay showed no cross-reaction with omethoate, methamidophos or pyraclofos. Finally, the PEO test strip was effectively applied for the detection of PEO in liquid vegetables A and B, with optical detection limits of 0.09 and 0.075 ppm, respectively.

Silver nanoparticle-base lateral flow immunoassay for rapid detection of Staphylococcal enterotoxin B in milk and honey.

A silver nanoparticle (AgNP)-based sandwich-type lateral flow immunoassay (LFIA) was evaluated for rapid detection of Staphylococcal enterotoxin B (SEB) in milk and honey. The role of trisodium citrate dihydrate (TSC) in the formation of Ag/TSC nanoparticles was established using UV-Vis spectroscopy. The association of silver with TSC in Ag/TSC nanoparticles was studied by the decrease in the intensity of anodic peak potential at 0.47 V and shift to 0.30 V in cyclic voltammetry (CV). The morphological, compositional and interaction studies of the AgNPs conjugated with the anti-SEB polyclonal antibody (Ag-sAb) was established using transmission electron microscopy (TEM) and X-ray photo electron spectroscopy (XPS) measurements. The visible detection limit and optical detection limit of the SEB test strip were 0.5 and 0.125 ppm, respectively, in SEB standard solution. This assay showed no cross-reaction with Staphylococcal enterotoxin A, Staphylococcal enterotoxin C or Salmonella typhi. Finally, the SEB test strip was effectively applied for the detection of SEB in spiked liquid milk and viscous honey, with optical detection limits of 0.25 and 0.5 ppm, respectively.

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

BACKGROUND: Yersinia 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. METHODS: Recombinant 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. RESULTS: By 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. CONCLUSION: Based 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.

Rapid detection of Yersinia pestis recombinant fraction 1 capsular antigen.

Yersinia pestis, an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10(5) CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10(5) CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge.

Comparison of LFA with PCR and RPLA in detecting SEB from isolated clinical strains of Staphylococcus aureus and its application in food samples.

Three sensitive and specific assays, the lateral flow assay (LFA), polymerase chain reaction assay (PCR) and reversed passive latex agglutination assay (RPLA), were selected for detection of staphylococcal enterotoxin B (SEB) from 77 clinical Staphylococcus aureus strains isolated from humans. Analytical results revealed that the LFA has almost the same detection sensitivity as that of PCR and RPLA. The concordances between the 3 assays were as follows: LFA-PCR, 92.2%; LFA-RPLA, 94.8%; and PCR-RPLA, 97.4%. For further evaluation, the LFA was used for the detection of SEB in different food matrices. The assay was able to successfully identify SEB in a wide variety of food samples at levels as low as 10 ng/mL in less than 10 min. This study proved that the LFA is an excellent tool for detection of SEB both in isolated clinical S. aureus strains and in food specimens and may prove particularly important as an early warning tool to prevent food poisoning in consumers.

Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2µg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2µg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC.

Monoclonal antibody-based lateral flow assay for detection of botulinum neurotoxin type A.

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).

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B.

A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).

Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.

Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

Colloidal gold-based immunochromatographic assay for detection of ricin.

A rapid immunochromatographic assay was developed to detect ricin. The assay was based on the sandwich format using monoclonal antibodies (Mabs) of two distinct specificities. One anti-ricin B chain Mab (1G7) was immobilized to a defined detection zone on a porous nitrocellulose membrane, while the other anti-ricin A chain Mab (5E11) was conjugated to colloidal gold particles which served as a detection reagent. The ricin-containing sample was added to the membrane and allowed to react with Mab (5E11)-coated particles. The mixture was then passed along the porous membrane by capillary action past the Mab (1G7) in the detection zone, which will bind the particles that had ricin bound to their surface, giving a red color within this detection zone with an intensity proportional to ricin concentration. In the absence of ricin, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of ricin was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 100 pg/ml.