Development and validation of an ELISA kit for the detection of ricin toxins from biological specimens and environmental samples.

Ricin is a toxin that can be easily extracted from seeds of Ricinus communis plants. Ricin is considered to be a major bio-threat as it can be freely and easily acquired in large quantities. A deliberate release of such toxin in civilian populations would very likely overwhelm existing public health systems, resulting in public fear and social unrest. There is currently no commercially available or FDA-approved prophylaxis such as vaccines, or therapeutic antitoxins or antidotes, available for ricin intoxication. Patient treatment is typically supportive care based on symptoms, often designed to reinforce the body's natural response. This paper describes the development and validation of a robust ELISA test kit, which can be used to screen for ricin in biological specimens such as whole blood and faeces. Faecal specimens are shown in this study to have better diagnostic sensitivity and a wider diagnostic window compared to whole blood. From these results, it is concluded that faeces is the most suitable clinical specimen for diagnosis of ricin poisoning via the oral route. The ELISA test kit can also detect ricin in environmental samples. An advantage of this ELISA kit over other commercial off-the-shelf (COTS) detection kits currently on the market that are developed to screen environmental samples only is its ability to diagnose ricin poisoning from clinical specimens as well as detect ricin from environmental samples.

The cytotoxicity of mercury chloride to the keratinocytes is associated with metallothionein expression.

There are trace amounts of heavy metals in cosmetics. Heavy metals such as mercury (Hg), which is added to skin-whitening cosmetics, may cause acute or chronic damage to human cells. The aim of this study was to investigate the cytotoxicity of mercury chloride (HgCl2) to human keratinocytes. The keratinocytes were treated with various concentrations of HgCl2 and the cell survival fractions were found to be 38.08, 17.59, 12.76, 3.29 and 0.77% when the cells were treated with 0.25, 0.5, 0.75, 1 and 1.5 µM of HgCl2, respectively. Moreover, we observed that the greatest damage was to the cell membrane. The metallothionein (MT) protein expression was also investigated. MT expression levels increased with increasing concentrations of HgCl2. The results indicated that MT protects the keratinocytes against HgCl2-induced toxicity.

Double-antigen enzyme-linked immunosorbent assay for detection of hepatitis E virus-specific antibodies in human or swine sera.

A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n = 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications.

Evaluation of a new rapid immunochromatographic assay for serodiagnosis of acute hepatitis E infection.

A rapid and reliable diagnostic assay for acute hepatitis E virus (HEV) infection is needed. We evaluated a rapid, immunochromatographic assay for IgM antibodies to HEV (ASSURE HEV IgM Rapid Test) using acute-phase HEV samples (n = 200) from Indonesia and Nepal and convalescent-phase HEV samples (n = 70) from Nepal. Blood donors in Thailand (n = 100), individuals with hepatitis A (n = 80), hepatitis B (n = 45), and hepatitis C (n = 50) in Thailand and Nepal, acute-phase sera of individuals with Epstein-Barr virus infection (n = 20), and rheumatoid factor-positive blood (n = 26) served as negative controls. The assay had a sensitivity of 93% (95% confidence interval [CI] = 88.5-96.1%) and a specificity of 99.7% (95% CI = 98.3-100%). The positive and negative predictive values were 99.5% (95% CI = 97.1-100%) and 95.8% (95% CI = 93.1-97.7%), respectively. These results suggest that this assay is a sensitive and specific tool for the rapid diagnosis of acute HEV infection.

Comparison of a new immunochromatographic test to enzyme-linked immunosorbent assay for rapid detection of immunoglobulin m antibodies to hepatitis e virus in human sera.

An immunochromatographic test for rapid detection of IgM antibodies in patients with acute hepatitis E infection was developed utilizing the well-characterized recombinant protein EP2.1 and monoclonal antibody 4B2. The new rapid test based on a novel reverse-flow technology was able to generate a positive result within 2 to 3 min. Our study showed that this test was able to detect anti-HEV IgM antibodies in 96.7% of the patient samples tested (n = 151) while maintaining an excellent specificity of 98.6% with samples from various patient or healthy control groups (total n = 208). Furthermore, this rapid test gave a good specificity of 90.9% when tested with rheumatoid factor (RF)-positive sera (RF value of < or =850 IU/ml; n = 11) although a higher concentration of RF in samples might cause cross-reactivity. The new test has a good agreement of 97.2% with a kappa value of 0.943 when compared with a reference enzyme-linked immunosorbent assay. The positive predictive value and the negative predictive value for the rapid test thus reached 98.0 and 97.6%, respectively. This is the first rapid, point-of-care test for hepatitis E and will be especially useful for the diagnosis of acute hepatitis E virus infection in field and emergency settings and in resource-poor countries.

Sleep-related vagotonic effect of zolpidem in rats.

RATIONALE: Zolpidem is a relatively new nonbenzodiazepine sedative-hypnotic. The effects of zolpidem on autonomic functions remain unclear. OBJECTIVES: The aim of this study was to evaluate the effects of zolpidem on sleep and related cardiac autonomic modulations as compared with triazolam in Wistar-Kyoto rats. METHODS: Continuous power spectral analyses of electroencephalogram (EEG), electromyogram, and heart rate variability were performed on freely moving rats during daytime sleep. The consciousness states were classified into active waking (AW), quiet sleep (QS), and paradoxical sleep (PS). Drugs were administered via gavage and data within 2 h were analyzed. RESULTS: All zolpidem (ZP3, 3 mg/kg; ZP30, 30 mg/kg) and triazolam (TZ0.075, 0.075 mg/kg; TZ0.75, 0.75 mg/kg) groups had longer accumulated QS time and averaged QS duration as compared with the vehicle control. The accumulated QS time and averaged QS duration of ZP3 were similar to those of TZ0.075. Significant suppressions of PS time were noted in all drug groups except ZP3. During QS, ZP3 and ZP30 exhibited significant increases of magnitude and percentage of EEG delta power, whereas TZ0.075 and TZ0.75 did not. Heart period and high-frequency power of heart rate variability increased significantly in ZP3 during all sleep-wake states. Both parameters, however, did not increase but even decreased in ZP30, TZ0.075, and TZ0.75. CONCLUSIONS: Zolpidem not only caused a longer and deeper sleep but also led to an elevated cardiac vagal activity at a specific dose in the rat.

Use of viral lysate antigen combined with recombinant protein in Western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome.

A Western immunoblot assay for confirmatory serodiagnosis of severe acute respiratory syndrome (SARS) was developed utilizing viral lysate antigens combined with a recombinant nucleocapsid protein, GST-N (glutathione S-transferase-nucleocapsid) of the SARS coronavirus (SARS-CoV). The viral lysate antigens were separated by electrophoresis and transblotted onto nitrocellulose membranes. The resultant membrane was subsequently added with the GST-N recombinant protein at a specific location. The positions of bands corresponding to some of the structural proteins immobilized on the membrane were then located and verified with mouse or rabbit antisera specific to the respective proteins. The Western immunoblot assay was able to detect antibodies to SARS-CoV in all 40 serum specimens from SARS patients and differentiate the SARS-positive samples from those of the healthy donor or non-SARS patient controls (150 samples) when set criteria were followed. In addition, when the immunoblot was used to test samples considered falsely positive by an in-house-developed SARS-specific enzyme-linked immunosorbent assay, band patterns different from those with samples from SARS patients were obtained.

Evaluation and validation of an enzyme-linked immunosorbent assay and an immunochromatographic test for serological diagnosis of severe acute respiratory syndrome.

A newly developed severe acute respiratory syndrome (SARS)-specific enzyme-linked immunosorbent assay (ELISA) was further validated to confirm cutoff values and evaluate its diagnostic performance with clinical samples. In parallel, an immunochromatographic test was also evaluated. A total of 227 clinical serum specimens collected from SARS patients were used in the study, together with 385 samples from healthy donors. By use of an immunofluorescent (IF) test as the "gold standard", both the ELISA and the immunochromatographic test were able to detect immunoglobulin G antibodies to SARS not only from late-convalescent-stage samples (>21 days from the onset of clinical symptoms), as previously established, but also from early-acute-phase samples (1 to 10 days from onset). The ELISA, using an optical density (OD) of 0.25 as its cutoff value, produced the best sensitivity while maintaining high specificity. It detected SARS-specific antibodies in 58, 70, 75, and 95%, respectively, of the four groups of samples collected from patients 1 to 10 days, 11 to 20 days, 21 to 30 days, and more than 30 days after the onset of clinical symptoms. Similarly, the immunochromatographic test detected SARS-specific antibodies in 55, 68, 81, and 79% of the four groups, respectively. The overall specificities for the ELISA and the rapid test were 99.5 and 97.7%, respectively. Although the positive correlation observed between the ELISA OD values and the IF titers was moderate (r = 0.6915; P < 0.001), the detection rates of both the ELISA and the rapid test were found well in agreement with the IF titers.

Recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients.

An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively.