Concept: HIV test
This work demonstrates that a full laboratory-quality immunoassay can be run on a smartphone accessory. This low-cost dongle replicates all mechanical, optical, and electronic functions of a laboratory-based enzyme-linked immunosorbent assay (ELISA) without requiring any stored energy; all necessary power is drawn from a smartphone. Rwandan health care workers used the dongle to test whole blood obtained via fingerprick from 96 patients enrolling into care at prevention of mother-to-child transmission clinics or voluntary counseling and testing centers. The dongle performed a triplexed immunoassay not currently available in a single test format: HIV antibody, treponemal-specific antibody for syphilis, and nontreponemal antibody for active syphilis infection. In a blinded experiment, health care workers obtained diagnostic results in 15 min from our triplex test that rivaled the gold standard of laboratory-based HIV ELISA and rapid plasma reagin (a screening test for syphilis), with sensitivity of 92 to 100% and specificity of 79 to 100%, consistent with needs of current clinical algorithms. Patient preference for the dongle was 97% compared to laboratory-based tests, with most pointing to the convenience of obtaining quick results with a single fingerprick. This work suggests that coupling microfluidics with recent advances in consumer electronics can make certain laboratory-based diagnostics accessible to almost any population with access to smartphones.
In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10(-18) g ml(-1). p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.
We reviewed recent literature on the cascade of HIV care from HIV testing to suppression of viral load, which has emerged as a critical focus as HIV treatment programs have scaled up.
- Biochemical and biophysical research communications
- Published over 3 years ago
Polystyrene is a common substrate material for protein adsorption in biosensors and bioassays. Here, we present a new method for multilayered, site-directed immobilization of antibody on polystyrene surface through the linkage of a genetically engineered ligand and the assembly of staphylococcal protein A (SPA) with immunoglobulin G (IgG). In this method, antibodies were stacked on polystyrene surface layer by layer in a potential three-dimensional way and exposed the analyte-binding sites well. Enzyme-linked immunosorbent assay (ELISA) revealed that the new method showed a 32-fold higher detection sensitivity compared with the conventional one. Pull-down assay and Western blot analysis further confirmed that it is different from the ones of monolayer adsorption according to the comparison of adsorption capacity. The differentiated introduction of functional ligands, which is the key of this method, might offer a unique idea as a way to interfere with the dynamic behavior of a protein complex during the process of adsorption.
The recombinant viral protein based indirect enzyme-linked immunosorbent assay (ELISA) is cheap, safe, specific and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The Background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since, specificity is high for western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and Specificity of indirect ELISA was 100% and 98.7% respectively in comparison with western blotting. Recombinant N protein based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.
Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.
The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings.
BACKGROUND: People concentrated in congregated systems, such as prisons, are important but often neglected reservoirs for TB transmission, and threaten those in the outside community. Therefore, this study was conducted to determine the prevalence of tuberculosis in a prison system of North Gondar Zone. METHODS: An active case-finding survey in North Gondar Prison was carried out from March to May 2011. All prison inmates who had history of cough for at least a week were included in the study. Three morning sputum samples were collected from suspected inmates and examined through fluorescence microscopy. Fine needle aspiration cytology was done for those having significant lymphadenopathy. Pre and post HIV test counseling was provided after written consent. Binary logistic and multivariable analysis was performed using SPSS version 16. RESULTS: A total of 250 prisoners were included in the survey. Among these, 26 (10.4%) prisoners were found to have TB giving a point prevalence of 1482.3 per 100,000 populations of smear positive TB among the TB suspects. All the inmates who participated in the study volunteered for HIV testing and a total of 19(7.6%) inmates were found to be reactive for the HIV antibody test amongst of which 9(47.4%) had TB co-infection. The prevalence of HIV infection in the TB infected inmates was found to be 34.6% (9/26). From the 26 TB cases identified 12 (46.2%) were having under nutrition (BMI < 18.5kg/m2). CONCLUSIONS: There is high prevalence of TB in North Gondar Prison with possible active transmission of TB within the prison. There was a high prevalence of HIV among the TB suspects. Strong cooperation between prison authorities and the national tuberculosis control programmes is urgently required to develop locally appropriate interventions to reduce transmission. The determinants for poor nutrition in the prison need also further investigation.
Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer’s disease (AD). To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice) and the age- and sex-matched non-transgenic (non-Tg) littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA) to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot), was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin) Ig gamma-2B chain C region (IGH-3), Ig lambda-2 chain C region (IGLC2), Ig kappa chain C region (IGKC), and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.
Microglia-mediated neuroinflammation induced by α-synuclein in the substantianigra likely either initiates or aggravates nigral neuro degeneration in Parkinson’s disease (PD). We aimed to explore the effects of α-mangostin (α-M), a polyphenolicxanthone derivative from mangosteen on α-synuclein-stimulated DA neurodegeneration. Primary microglia, mesencephalic neuron, mesencephalic neuron-glianeuronal cultures, and transwell co-cultures were prepared separately. Liquid scintillation counting was used to determine the radioactivity in DA uptake. Enzyme-linked immunosorbent assay (ELISA) was performed in the IL-1β, IL-6, and TNF-α assay. The expression of proteins was analyzed by Western blot. α-M inhibited the increased levels of pro-inflammatory cytokines, NO, and ROS in α-synuclein-stimulated primary microglia. Mechanistic study revealed that α-M functioned by inhibition of nuclear factor kappa B (NF-κB) and NADPH oxidase. Further, α-M protected α-synuclein-induced microglial and direct neurotoxicity. Although detailed mechanisms remain to be defined, our observations suggest a potential compound, which inhibits microglial activation induced by α-synuclein by targeting NADPH oxidase, might be a therapeutic possibility in preventing PD progression.