Optimal Assay Conditions Research Articles

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18

High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.

Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus.

BackgroundResearch with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices.MethodsTotal nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV.ResultsOptimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors.ConclusionsPTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0509-3) contains supplementary material, which is available to authorized users.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Batai Virus in Cattle and Mosquitoes.

Batai virus (BATV) is an arthropod-borne single-stranded RNA virus belonging to the genus Orthobunyavirus of the family Bunyaviridae that is primarily transmitted by mosquitoes. Methods for detecting BATV are currently limited to serological surveillance, virus isolation, and conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. In this study, we sought to develop a BATV detection assay that needs no specialized equipment and is highly specific, sensitive, and simple. We first developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of BATV that uses two pairs of primers to amplify a conserved region of the BATV M gene. The optimal reaction conditions for this RT-LAMP BATV detection assay were 40 min at 65°C. The amplification products could be visualized directly for color changes. This RT-LAMP method has a detection limit of 2.86 copies/μL and a sensitivity that was approximately 10- and 100-fold greater than real-time and conventional RT-PCR, respectively. RT-LAMP for BATV detection showed no cross-reactivity with other viruses and its sensitivity was validated with cattle blood and mosquito specimens. Our results suggest that this RT-LAMP method was simpler and faster than conventional RT-PCR or real-time RT-PCR. Moreover, RT-LAMP represents a potential tool to test for BATV in clinical and mosquito samples, especially in rural areas of China. This method also shows promise as a diagnostic tool due to its rapid and sensitive detection without the need for sophisticated equipment or complicated protocols.

Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals.

The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight the difficulty in adapting in vitro methods to high-throughput format for screening the skin sensitization potential of large numbers of chemicals whilst ensuring that the data produced are both accurate and reproducible.

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

N6-methyladenosine (m6A) is the most common reversible internal modification in mammalian RNA. Changes in m6A levels have been implicated in a variety of cellular processes, including nuclear RNA export, control of protein translation, and protein splicing, and they have been linked to obesity, cancer, and other human diseases. METTL3 and METTL14 are N6-adenosine methyltransferases that work more efficiently in a stable METTL3-METTL14 heterodimer complex (METTL3-14). ALKBH5 is an m6A-RNA demethylase that belongs to the AlkB family of dioxygenases. We report the development of radioactivity-based assays for kinetic characterization of m6A-RNA modifications by METTL3-14 complex and ALKBH5 and provide optimal assay conditions suitable for screening for ligands in a 384-well format with Z′ factors of 0.78 and 0.77, respectively.

Artificial Synthesis of Conserved Segment S Gene Fragment of Rift Valley Fever Virus and Preliminary Study of Its Reverse Transcription Loop-Mediated Isothermal Amplification Detection Method

Purpose: To develop a rapid detection method for Rift Valley fever virus (RVFV) diagnosis.Methods: According to the reference sequences of RVFV published in GenBank, nine overlapping polymerase chain reaction (PCR) primers and four specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) primers were designed using DNAStar and LAMP primer design software, respectively. Based on the synthesis of a conserved part of the RVFV S segment gene sequence using overlapping PCR, RT-LAMP assay was first established and evaluated after a series of tests, including, optimization of reaction conditions, and sensitivity and specificity tests.Result: A target RVFV S segment gene fragment of 288 bp was synthesised. The optimal reaction conditions for RT-LAMP assay were 63 °C for 45 min: the assay has a specific ladder-like pattern of amplification bands from about 120 bp. The lowest target gene copy number of RT-LAMP for RVFV detection was 70 copies. The assay showed good specificity as only the synthesised target RVFV gene was amplified with no amplification for the detection of Peste des petits ruminants virus, Epidemic encephalitis B virus, E. coli, Pasteurella multocida, or Salmonella.Conclusion: This study provides a rapid, sensitive, specific RT-LAMP method for RVFV detection.Keywords: Rift valley fever virus, Overlapping polymerase chain reaction, Reverse transcription loopmediated isothermal amplification, Rapid diagnosis test

Comparative Study of Novel Ratio Spectra and Isoabsorptive Point Based Spectrophotometric Methods: Application on a Binary Mixture of Ascorbic Acid and Rutin.

This paper presents novel methods for spectrophotometric determination of ascorbic acid (AA) in presence of rutin (RU) (coformulated drug) in their combined pharmaceutical formulation. The seven methods are ratio difference (RD), isoabsorptive_RD (Iso_RD), amplitude summation (A_Sum), isoabsorptive point, first derivative of the ratio spectra (1DD), mean centering (MCN), and ratio subtraction (RS). On the other hand, RU was determined directly by measuring the absorbance at 358 nm in addition to the two novel Iso_RD and A_Sum methods. The work introduced in this paper aims to compare these different methods, showing the advantages for each and making a comparison of analysis results. The calibration curve is linear over the concentration range of 4–50 μg/mL for AA and RU. The results show the high performance of proposed methods for the analysis of the binary mixture. The optimum assay conditions were established and the proposed methods were successfully applied for the assay of the two drugs in laboratory prepared mixtures and combined pharmaceutical tablets with excellent recoveries. No interference was observed from common pharmaceutical additives.

Identification of an Mg<sup>2+</sup>-Independent Soluble Phosphatidate Phosphatase in Cottonseed (<i>Gossypium hirsutum L.</i>)

Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg2+-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the Vmax and Km values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.

A comparative study of tissue factor and kaolin on blood coagulation assays using rotational thromboelastometry and thromboelastography.

Rotational thromboelastometry (ROTEM) and thromboelastography (TEG) have been increasingly used to diagnose acute coagulopathy and guide blood transfusion. The tests are routinely performed using different triggering activators such as tissue factor and kaolin, which activate different pathways yielding different results. To optimize the global blood coagulation assays using ROTEM and TEG, we conducted a comparative study on the activation methods employing tissue factor and kaolin at different concentrations as well as standard reagents as recommended by the manufacturer of each device. Key parameter values were obtained at various assay conditions to evaluate and compare coagulation and fibrinolysis profiles of citrated whole blood collected from healthy volunteers. It was found that tissue factor reduced ROTEM clotting time and TEG R, and increased ROTEM clot formation time and TEG K in a concentration-dependent manner. In addition, tissue factor affected ROTEM alpha angle, and maximum clot firmness, especially in the absence of kaolin activation, whereas both ROTEM and TEG clot lysis (LI30, CL30, and LY30) remained unaffected. Moreover, kaolin reduced ROTEM clotting time and TEG R and K, but to a lesser extent than tissue factor, in-tem and ex-tem. Correlations in all corresponding parameters between ROTEM and TEG were observed, when the same activators were used in the assays compared with lesser correlations between standard kaolin TEG and ROTEM (INTEM/EXTEM). The two types of viscoelastic point-of-care devices provide different results, depending on the triggering reagent used to perform the assay. Optimal assay condition was obtained to reduce assay time and improve assay accuracy.

Rapid enzyme-linked immunosorbent assay and immunochromatographic strip for detecting ribavirin in chicken muscles

ABSTRACTThe use of ribavirin (RBV) as an antiviral drug for livestock has been prohibited in China, the USA, and many other countries. In this study, we developed a rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a gold nanoparticle immunochromatographic (ICA) strip test for detecting RBV in chicken muscles. Under the optimum assay conditions, where the assay employed phosphate-buffered saline at pH 7.4, no acetonitrile, and an ionic strength of 0.8%, the quantitative working range was 1.43–26.47 ng/ml with an IC50 of 6.15 ng/ml. The recovery rate for RBV in real samples ranged from 82.1% to 112.3%. The immunochromatographic test strip method had a visual cutoff value of 50 μg/kg. Given their high recovery rates and good sensitivity, the proposed ic-ELISA and ICA methods could be useful for the RBV analysis in chicken tissue samples.

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.

Ultrasound-based protein determination in maize seeds

The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by 1H and 13C NMR spectroscopy, SDS–PAGE electrophoresis, and UV–visible spectrophotometry (UV–vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline–alcoholic solution. Proteins in both types of extracts were well characterized by UV–vis spectroscopy. However, the 545nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.

Ultrasensitive Detection and Subtyping of Porcine Endogenous Retrovirus Provirus Based on Magnetic Nanoparticles and Chemiluminescence.

Porcine endogenous retrovirus (PERV) is commonly integrated in pig genomes, and could cause a cross-species infection by xenotransplantation. In this study, we developed a rapid and ultrasensitive approach for detection and subtyping of PERV provirus based on magnetic nanoparticles (MNPs) and chemiluminescence (CL). The carboxylated MNPs (CMNPs) were covalently coupled with aminated probes for capturing biotinylated target fragments of PERV, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated fragments. The MNPs composites were incubated with streptavidin-alkaline phosphatase (SA-ALP) and CL signal intensities were determined by subsequently adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD). The optimal assay conditions of this approach were 1 mM for SA modification, 10 µM for probe modification, 55 (PERV), 54 (PERV-A), 50 (PERV-B), and 56 °C (PERV-C) for hybridization temperatures respectively, and 30 min for hybridization time. This approach was specific and highly sensitive, and the limit of detection (LOD) was 100 amol, which has the potential for screening out safe pig donors for xenotransplantation as well as to examine clinical samples from human patients treated with porcine xenotranplantation.

Dual-labeled time-resolved fluoroimmunoassay for simultaneous detection of clothianidin and diniconazole in agricultural samples

Europium (Eu3+) and samarium (Sm3+) were used as fluorescent labels to develop a highly sensitive dual-labeled time-resolved fluoroimmunoassay (TRFIA) for detect clothianidin and diniconazole in food samples. Under the optimized assay conditions, 50% inhibition concentration (IC50) and the limit of detection (LOD, IC10) of clothianidin were 5.08 and 0.021μg/L, and 13.14 and 0.029μg/L for diniconazole. The cross-reactivities (CRs) were negligible except dinotefuran (9.4%) and uniconazole (4.28%). The recoveries of clothianidin and diniconazole ranged from 79.3% to 108.7% in food samples. The results of TRFIA for the authentic samples were validated by gas chromatography (GC) analyses, and a satisfactory correlations were obtained. These results indicated that the method was an alternative tool for simultaneous detection of clothianidin and diniconazole in food samples.

Determination of Furaltadone Metabolite in Fish by Chemiluminescence Enzyme Immunoassay

Abstract An indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) detection for the furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ) was established. The effects of the coating antigen concentration, antibody dilution, immunoreaction time, reaction buffer and its ion concentration on the performance of the assay were studied and optimized. The results showed that the optimized assay conditions were as follows: the coating antigen concentration was 10 ng mL−1, the polyclonal antibody was diluted 60000 times, immunoreaction time was 50 min and the buffer solution was 0.01 M phosphate buffer solution (PBS, pH 7.4). Under the optimized conditions, the linear detection range of the developed icCLEIA was 0.026–3.52 μg L−1, the half inhibitory concentration (IC50) was 0.29 μg L−1 and the limit of detection (IC10) was 0.012 μg L−1. The average recoveries of AMOZ of spiked fish ranged from 101.0% to 115.5%. In conclusion, the icCLEIA is applicable for detection of trace AMOZ in fish samples.

Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

Simultaneous detection of imidacloprid and parathion by the dual-labeled time-resolved fluoroimmunoassay.

A highly sensitive direct dual-labeled time-resolved fluoroimmunoassay (TRFIA) to detect parathion and imidacloprid simultaneously in food and environmental matrices was developed. Europium (Eu(3+)) and samarium (Sm(3+)) were used as fluorescent labels by coupling separately with L1-Ab and A1P1-Ab. Under optimal assay conditions, the half-maximal inhibition concentration (IC50) and limit of detection (LOD, IC10) were 10.87 and 0.025 μg/L for parathion and 7.08 and 0.028 μg/L for imidacloprid, respectively. The cross-reactivities (CR) were negligible except for methyl-parathion (42.4 %) and imidaclothiz (103.4 %). The average recoveries of imidacloprid ranged from 78.9 to 104.2 % in water, soil, rice, tomato, and Chinese cabbage with a relative standard deviation (RSD) of 2.4 to 11.6 %, and those of parathion were from 81.5 to 110.9 % with the RSD of 3.2 to 10.5 %. The results of TRFIA for the authentic samples were validated by comparison with gas chromatography (GC) analyses, and satisfactory correlations (parathion: R (2) = 0.9918; imidacloprid: R (2) = 0.9908) were obtained. The results indicate that the dual-labeled TRFIA is convenient and reliable to detect parathion and imidacloprid simultaneously in food and environmental matrices.

Optimization, Validation and Application of Spectrophotometric Assay for 3-Hydroxy-3-methylglutarylcoenzyme A Reductase Activity

Purpose : To improve the sensitivity and specificity of spectrophotometric 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity assay. Methods : Spectrophotometric HMG-CoA reductase detection in male Wistar rat liver microsomes was optimized by applying different conditions, such as reaction buffer pH, NADPH and protein concentration, and preincubation and reaction times. The optimal set of conditions was validated using HMG-CoA reductase inhibitors, namely, pravastatin, fluvastatin, and rosuvastatin. IC 50 was calculated and compared with that of a radiochemical assay. Ginkgo biloba extract’s (GBE50) inhibitory effect on HMG-CoA reductase activity was evaluated using the optimized spectrophotometric protocol. Results : The optimum assay conditions were as follows: reaction buffer pH 7.0, 100 μM NADPH, 50 μM HMG-CoA, and 200 μg/mL microsomal protein. The preincubation and reaction times were 20 and 60 min, respectively, at 37 oC. The IC50 of pravastatin, fluvastatin, and rosuvastatin under the optimum condition was 0.026, 0.015, and 0.007 μM while for radioisotope assay, it was 0.034, 0.049 and 0.0119 μM, respectively. GBE50 significantly inhibited HMG-CoA reductase activity in a concentrationdependent manner (p < 0.05). Conclusion : These results suggest that HMG-CoA reductase activity can be detected using the improved spectrophotometric assay. This assay can facilitate the discovery and development of new HMG-CoA reductase inhibitors in vitro. Keywords : Spectrophotometry, 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity, Cholesterol metabolism, Ginkgo biloba, Pravastatin, Fluvastatin, Rosuvastatin

Reliable and sensitive adenovirus-based reporter system for high-throughput screening of dioxins

Dioxin-responsive element (DRE)-driven luciferase reporter assay is a rapid and cost-effective screening method for detection and semi-quantitation of dioxins and dioxin-like chemicals in samples from a variety of matrices. However, most of such stable cell lines gradually lose their dioxin responsiveness over time, possibly due to cellular degradation and/or silence of the transfected reporter gene. The study aimed to establish a recombinant adenovirus-based DRE-driven luciferase reporter system for easy maintenance and improvement of dioxin sensitivity/responsiveness. Both the Ad-4xDR adenovirus-based and the Huh7-4xDRE-Luc cell-based reporter systems carry a luciferase reporter gene driven by the same 4xDRE-TATA cassette, i.e., 4 copies of DRE and a TATA box. Comparison of the two reporter systems indicated that the adenovirus-based system was much more sensitive and responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than the cell-based system. Among the four recombinant adenoviruses, Ad-4xDR, Ad-6xDR, Ad-9xDR, and Ad-15xDR carrying 4, 6, 9, and 15 copies of DRE, respectively, the Ad-6xDR-infected H4IIE cells showed the highest TCDD responsiveness. Following optimization of assay conditions, including temperature, dimethyl sulfoxide (DMSO) concentration, and multiplicity of infection (MOI) of adenovirus, the Ad-6xDR-based system exhibited markedly superior performance in TCDD sensitivity (limit of detection (LOD) = 0.02 pM) and responsiveness (the half maximal effective concentration (EC50)=10.41 pM and a maximum of 409.34 folds of luciferase induction at 300 pM TCDD). Analysis of soil samples showed a high correlation between the Ad-6xDR-based reporter assay and high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). The study reveals that, as compared with our previous cell-based system, the Ad-6xDR-based reporter system is more reliable and sensitive for high-throughput screening of dioxins.