Binding characteristics study for dengue virus non-structural protein 1 of antigen and its antibody by using circular dichroism technique

This paper presents the binding characteristics study of dengue non-structural protein 1 (NS1) antigen and its antibody using circular dichroism technique in far UV region. Circular dichroism (CD) is a spectroscopic technique which measures differences in the absorption of left-handed and right handed circularly polarized light. The CD spectrum can determine conformation of the NS1 antigen and its antibody, conformational changes of the antigen-antibody interaction and estimates the secondary structure of these proteins in far UV region. Firstly, CD spectrum of individual solutions of the antigen and the antibody were measured. Then, the solutions were mixed to produce a solution of complex dengue NS1 antigen and its antibody for measurement. The findings show that the antibody has the highest positive band of CD intensity follow by the complex antigen-antibody and antigen. The antibody is a chiral structure, has high helical conformation and more ordered epitope structure. Meanwhile, the NS1 antigen shows the negative and the lowest CD spectrum. The antigen is low chirality and has more random-like conformation. The complex (binding of the antigen and antibody) has the CD spectrum's shape similar to the antibody but in lower intensity. So, it has helical and beta conformations lower than the antibody. The binding characteristics of the complex solutions were also studied with increased in incubation time and with varied rotation applied. It is found that the immunoreactions between the antigen and its antibody are rapid processes which do not require too long incubation time. Besides, the applied rotation can increased the immunoreaction process

Performance of a Novel Low-Cost, Instrument-Free Plasma Separation Device for HIV Viral Load Quantification and Determination of Treatment Failure in People Living with HIV in Malaysia: a Diagnostic Accuracy Study

HIV viral load (VL) testing is the recommended method for monitoring the response of people living with HIV and receiving antiretroviral therapy (ART). The availability of standard plasma VL testing in low- and middle-income countries (LMICs), and access to this testing, are limited by the need to use fresh plasma. Good specimen collection methods for HIV VL testing that are applicable to resource-constrained settings are needed. We assessed the diagnostic performance of the filtered dried plasma spot (FDPS), created using the newly developed, instrument-free VLPlasma device, in identifying treatment failure at a VL threshold of 1,000 copies/ml in fresh plasma. Performance was compared with that of the conventional dried blood spot (DBS). Venous blood samples from 201 people living with HIV and attending an infectious disease clinic in Malaysia were collected, and HIV VL was quantified using fresh plasma (the reference standard), FDPS, and DBS specimens. VL testing was done using the Roche Cobas AmpliPrep/Cobas TaqMan v2.0 assay. At a threshold of 1,000 copies/ml, the diagnostic performance of the FDPS was superior (sensitivity, 100% [95% confidence interval {CI}, 89.1 to 100%]; specificity, 100% [95% CI, 97.8 to 100%]) to that of the DBS (sensitivity, 100% [95% CI, 89.4 to 100%]; specificity, 36.8% [95% CI, 29.4 to 44.7%]) (P 0.001). A stronger correlation was observed between the FDPS VL and the plasma VL (r 0.94; P 0.001) than between the DBS VL and the plasma VL (r 0.85; P 0.001). The mean difference in VL measures between the FDPS and plasma (plasma VL minus FDPS VL) was 0.127 log10 copies/ml (standard deviation [SD], 0.32), in contrast to – 0.95 log10 copies/ml (SD, 0.84) between the DBS and plasma. HIV VL measurement using the FDPS outperformed that with the DBS in identifying treatment failure at a threshold of 1,000 copies/ml and compared well with the quantification of VL in plasma. The FDPS can be an attractive alternative to fresh plasma for improving access to HIV VL monitoring among people living with HIV on ART in LMICs

Sequential push-pull pumping mechanism for washing and evacuation of an immunoassay reaction chamber on a microfluidic CD platform.

A centrifugal compact disc (CD) microfluidic platform with reservoirs, micro-channels, and valves can be employed for implementing a complete immunoassay. Detection or biosensor chambers are either coated for immuno-interaction or a biosensor chip is inserted in them. On microfluidic CDs featuring such multi-step chemical/biological processes, the biosensor chamber must be repeatedly filled with fluids such as enzymes solutions, buffers, and washing solutions. After each filling step, the biosensor chamber needs to be evacuated by a passive siphoning process to prepare it for the next step in the assay. However, rotational speed dependency and limited space on a CD are two big obstacles to performing such repetitive filling and siphoning steps. In this work, a unique thermo-pneumatic (TP) Push-Pull pumping method is employed to provide a superior alternative biosensor chamber filling and evacuation technique. The proposed technique is demonstrated on two CD designs. The first design features a simple two-step microfluidic process to demonstrate the evacuation technique, while the second design shows the filling and evacuation technique with an example sequence for an actual immunoassay. In addition, the performance of the filling and evacuation technique as a washing step is also evaluated quantitatively and compared to the conventional manual bench top washing method. The two designs and the performance evaluation demonstrate that the technique is simple to implement, reliable, easy to control, and allows for repeated push-pulls and thus filling and emptying of the biosensor chamber. Furthermore, by addressing the issue of rotational speed dependency and limited space concerns in implementing repetitive filling and evacuation steps, this newly introduced technique increases the flexibility of the microfluidic CD platform to perform multi-step biological and chemical processes

Performance evaluation of push-wash and pull-evacuation.

<p>Performance evaluation of implementing evacuation, rinse and wash using push-wash and pull-evacuation in an immunoassay.</p

Demonstration of sequential biosensor chamber pull-evacuation:

<p><b>(i)</b> Blue and Red liquids are loaded into source chamber A1 and A2. <b>(ii—iv)</b> Blue liquid bursts from source chamber A1 into biosensor chamber B, then pull-evacuated into waste chamber W. <b>(v—viii)</b> Sequentially Red liquid bursts from source chamber A2 into biosensor chamber B, then pull-evacuated into waste chamber W</p

Microfluidic CD layers and demonstration CD designs.

<p><b>(a)</b> Layered fabrication of multi-level 3D microfluidic CDs. <b>(b)</b> A design to demonstrate sequential biosensor chamber pull-evacuation. Liquid bursts from source chamber A1 into biosensor chamber B, then pull-evacuated into waste chamber W, followed by liquid bursting from source chamber A2 into biosensor chamber B, then pull-evacuated into waste chamber W. <b>(c)</b> A design to demonstrate biosensor chamber push-wash and pull-evacuation for an immunoassay. Target antigen in biosensor chamber B is washed off into waste chamber W, followed by the bursting of the blocking solution from source chamber A1 into biosensor chamber B, then rinsed and washed off into waste chamber W, and finally the bursting of flourescent labelled antibody solution from source chamber A2 to biosensor chamber B, then rinsed and double volume washed into waste chamber W</p

Demonstration of biosensor chamber push-wash and pull-evacuation for an immunoassay.

<p><b>(i)</b> Yellow (representing test sample containing target antigen), Red (representing blocking solution), Blue (representing fluorescent labelled secondary antibodies) liquids, and de-ionized water (representing washing solution) are respectively loaded into biosensor chamber B, source chambers A1 and A2, washing solution chamber C. <b>(ii—vi)</b> Yellow liquid is pull-evacuated into waste chamber W, wash solution chamber is sealed and biosensor chamber is washed twice. <b>(vii—xi)</b> Red liquid is burst into biosensor chamber B, followed by a rinse and a wash of the biosensor chamber B. <b>(xii—xvii)</b> Blue liquid is burst into biosensor chamber B, followed by a rinse and a double volume wash of the biosensor chamber B.</p

Sarcocystis nesbitti causes acute, relapsing febrile myositis with a high attack rate: description of a large outbreak of muscular sarcocystosis in Pangkor Island, Malaysia, 2012.

BACKGROUND: From the 17th to 19th January 2012, a group of 92 college students and teachers attended a retreat in a hotel located on Pangkor Island, off the west coast of Peninsular Malaysia. Following the onset of symptoms in many participants who presented to our institute, an investigation was undertaken which ultimately identified Sarcocystis nesbitti as the cause of this outbreak. METHODOLOGY/PRINCIPAL FINDINGS: All retreat participants were identified, and clinical and epidemiological information was obtained via clinical review and self-reported answers to a structured questionnaire. Laboratory, imaging and muscle biopsy results were evaluated and possible sources of exposure, in particular water supply, were investigated. At an average of 9-11 days upon return from the retreat, 89 (97%) of the participants became ill. A vast majority of 94% had fever with 57% of these persons experiencing relapsing fever. Myalgia was present in 91% of patients. Facial swelling from myositis of jaw muscles occurred in 9 (10%) patients. The median duration of symptoms was 17 days (IQR 7 to 30 days; range 3 to 112). Out of 4 muscle biopsies, sarcocysts were identified in 3. S. nesbitti was identified by PCR in 3 of the 4 biopsies including one biopsy without observed sarcocyst. Non-Malaysians had a median duration of symptoms longer than that of Malaysians (27.5 days vs. 14 days, p = 0.001) and were more likely to experience moderate or severe myalgia compared to mild myalgia (83.3% vs. 40.0%, p = 0.002). CONCLUSIONS/SIGNIFICANCE: The similarity of the symptoms and clustered time of onset suggests that all affected persons had muscular sarcocystosis. This is the largest human outbreak of sarcocystosis ever reported, with the specific Sarcocystis species identified. The largely non-specific clinical features of this illness suggest that S. nesbitti may be an under diagnosed infection in the tropics