Enzyme-linked Immunosorbent Assay Procedure Research Articles

A sensitive chemiluminescence based immunoassay for the detection of cortisol and cortisone as stress biomarkers

An electrochemically based antibody immobilization was used to perform environmentally and clinically relevant immunoassays for stress hormones biomarkers (cortisol and cortisone) using chemiluminescence (CL) detection. To achieve CL detection, the ferrocene tag on the antibodies was first oxidised, and this then acted as a catalyst for the luminol and hydrogen peroxide CL reaction. The conditions were optimised and measurements were made with an incubation time of 30 min. Using this approach limits of detection were obtained of 0.47 pg ml−1 and 0.34 pg ml−1 also R2 0.9912 and 0.9902 for cortisol and cortisone respectively with a linear concentration from 0 to 50 ng ml−1. The method was then applied to Zebrafish whole body and artificial saliva samples. For the Zebrafish sample recoveries of 91.0% and 90.0% were obtained with samples spiked with cortisol and cortisone, for artificial saliva the recoveries were 92.59% and 90.73% respectively. Interference studies showed only minor effects on the measurement of the analyte. A comparison between this procedure and the standard enzyme-linked immunosorbent assay (ELISA) procedure gave approximately the same R2 values.

Microfluidic Time-Delay Valve Mechanism on Paper-Based Devices for Automated Competitive ELISA.

Paper-based technologies have been drawing increasing attentions in the biosensor field due to their economical, ecofriendly, and easy-to-fabricate features. In this paper, we present a time-delay valve mechanism to automate a series of procedures for conducting competitive enzyme-linked immunosorbent assay (ELISA) on a paper-based device. The mechanism employs a controllable time-delay valve, which has surfactants to dissolve the hydrophobic barriers, in a fluid pathway. The valves can regulate the liquid and sequentially deliver the sample flow for automating ELISA procedures in microchannels. Competitive ELISA is achieved in a single step once the sample, or small molecule pesticide (e.g., Imidacloprid), is applied onto the paper-based device with a comparable sensitivity to plate-based competitive ELISA. The results further demonstrate the appositeness of using paper-based devices with the valve designs for on-the-go ELISA detection in agriculture and biomedical applications.

The Significance of Oxidized Low-Density Lipoprotein in Body Fluids as a Marker Related to Diseased Conditions.

Oxidatively modified low-density lipoprotein (oxLDL) is known to be involved in various diseases, including cardiovascular diseases. The presence of oxLDL in the human circulatory system and in atherosclerotic lesions has been demonstrated using monoclonal antibodies. Studies have shown the significance of circulating oxLDL in various systemic diseases, including acute myocardial infarction and diabetic mellitus. Several different enzyme-linked immunosorbent assay (ELISA) procedures to measure oxLDL were utilized. Evidence has been accumulating that reveals changes in oxLDL levels under certain pathological conditions. Since oxLDL concentration tends to correlate with low-density lipoprotein (LDL)-cholesterol, the ratio of ox-LDL and LDL rather than oxLDL concentration alone has also been focused. In addition to circulating plasma, LDL and oxLDL are found in gingival crevicular fluid (GCF), where the ratio of oxLDL to LDL in GCF is much higher than in plasma. LDL and oxLDL levels in GCF show an increase in diabetic patients and periodontal patients, suggesting that GCF might be useful in examining systemic conditions. GCF oxLDL increased when the teeth were affected by periodontitis. It is likely that oxLDL levels in plasma and GCF could reflect oxidative stress and transfer efficacy in the circulatory system.

Developmental Toxicity of TCE Exposure in Zebrafish: Analyzing Vitamin D Metabolism

Background and Hypothesis: Trichloroethylene (TCE) is an industrial solvent used since the 1940s. A known carcinogen, it is found in over half of Superfund sites where levels have reached 12,000 parts per billion (ppb; µg/L), though the US Environmental Protection Agency maximum contaminant level in drinking water is 5 ppb. In this study, the zebrafish model was used to investigate the developmental toxicity of TCE by assessing its effects on metabolism. Preliminary studies have shown a reduction in gene expression of the cytochrome P450 (CYP) enzymes responsible for breakdown of vitamin D. As a result, we expected to see lower levels of downstream metabolites, namely calcitriol, as detected by an Enzyme-Linked Immunosorbent Assay (ELISA). 
 Experimental Design: At 0-2 hours post fertilization (hpf), embryos were dosed with TCE at either 0 or 10 ppb. At 72 hpf, the larvae were removed from the TCE and prepared for the ELISA procedure. 
 Results: ELISA revealed similar levels of calcitriol between the control and 10 ppb groups. The difference between the two groups did not show statistical significance (p>0.05). 
 Conclusion: These results suggest that there may not be a direct relationship between CYP gene expression and certain downstream metabolic effects or that the organism was able to compensate for the biological changes. If the latter, additional time points for TCE exposure could be evaluated. As studies continue, we plan to evaluate CYP gene expression at higher concentrations to correlate with other biological effects and determine if there is consistency with results from lower concentrations.

Relationship of fruit and vegetable intake to dietary antioxidant capacity and markers of oxidative stress: A sex-related study.

Oxidative stress contributes to the development of chronic diseases. Fruits and vegetables contain several phytonutrients (carotenoids, polyphenols) that exert antioxidant effects. The aim of this study was to investigate sex differences in fruit and vegetable intake, and the relationship to plasma levels of carotenoids as well as to total antioxidant capacity (pTAC). We studied also sex differences in the relationship between fruit and vegetables intake and plasma levels of lipid hydroperoxides, as well as of oxidized low-density lipoprotein (ox-LDL). This study included 83 healthy adults (35 men and 48 women, mean age 40 ± 10 y). Dietary intake of carotenoids and total antioxidant capacity (dTAC) were evaluated on the basis of a 15-d food frequency questionnaire. Plasma levels of β-carotene, lutein, and pTAC were studied. Moreover, levels of plasma lipid hydroperoxides and ox-LDL were evaluated using the ferrous oxidation-xylenol orange 2 (FOX2) assay and a monoclonal antibody-based enzyme-linked immunosorbent assay procedure, respectively. Dietary habits were sex-related with a higher intake of fruits and vegetables (P < 0.05) and β-carotene (P < 0.001) in women than in men. Mean values of plasma lutein and β-carotene were higher in women than in men. Mean values of ox-LDL and lipid hydroperoxides were higher in men than in women (P < 0.05). Significant negative correlations were established between the individual values of ox-LDL and the levels of lutein versus β-carotene and versus pTAC values in plasma in both groups. Individuals belonging to the tertile with the highest daily intake of fruits and vegetables or the highest daily dTAC showed the lowest levels of plasma ox-LDL. In each category, sex-related differences were observed with men showing higher levels of ox-LDL than women. Moreover, lower levels of plasma β-carotene were observed in men in each tertile of daily intake of fruits and vegetables compared with females. Based on the data obtained, we confirm that high fruit and vegetable consumption exerts a positive effect on antioxidant defenses and decreases oxidative damage of plasma lipoproteins for both sexes. The results suggest that the protective effect can be found to a higher extent in women than in men. Sex-based differences are apparent in many chronic diseases. Thus, a higher consumption of antioxidant-rich fruits and vegetables should be recommended in efforts to prevent diseases in which sex-related differences in oxidative stress play a considerable role.

Elevated Salivary Il-1β, Pge2 And Mmp-3 Levels And Their Significant Reduction Post Therapy In Patients With Chronic Periodontitis Compared To Healthy Individuals

Background: The role of IL-1β, PGE2 and MMP-3 in the pathogenesis of periodontal disease is well researched. This study aimed to asses and compared the salivary IL-1β, PGE2 and MMP-3 levels in patients with untreated chronic severe periodontitis and those treated with periodontal phase I therapy and periodontally healthy individuals as controls, in relationship to the presence of salivary anti-β1 IgA. Methods: A total of 30 subjects participate in the study: 15 subjects had chronic severe periodontitis and 15 were healthy individuals used as a control. After saliva collection and its purification, we quantify by enzyme-linked immunosorbent assay (ELISA) procedure using as coating antigen a synthetic β1 peptide with an amino acid sequence identical to the second extracellular loop of the human β1 adrenoceptor (β1 AR), the presence of anti β1 AR antibody (IgA) in the saliva of patients and healthy individuals. Also, IL-1β, PGE2, nitrites and metalloproteinase 3 (MMP-3) were assessed using ELISA) assay. Results: Our data indicated that IL-1β, PGE2, nitrites and MMP-3 levels are elevated in the saliva of patients with untreated chronic severe periodontitis and were significantly higher than in healthy subjects. Also, the amounts of anti- β1 IgA in the saliva was significantly higher compared with that of healthy individuals. After periodontal phase I therapy these levels of inflammatory biomarkers are significantly reduced but the titres of the antibody did not change, suggesting a close association between salivary IL-1β, PGE2, nitrites and MMP-3 and periodontitis without any changes in the levels of anti β1 IgA. Conclusions: These results suggest that the abnormal amount of these cytokines and enzymes in saliva has potential monitoring applications as a risk marker of the disease progression but the raised levels of anti β1 IgA present in the saliva of chronic severe periodontitis patient, are not directly associated with the course of the disease. Additional studies are needed to validate this assumption.

Immuno Nanosensor for the Ultrasensitive Naked Eye Detection of Tuberculosis.

In the present study, a beneficial approach for the ultrasensitive and affordable naked eye detection and diagnosis of tuberculosis (TB) by utilizing plasmonic enzyme-linked immunosorbent assay (ELISA) via antibody-antigen interaction was studied. Here, the biocatalytic cycle of the intracellular enzymes links to the formation and successive growth of the gold nanoparticles (GNPs) for ultrasensitive detection. The formation of different colored solutions by the plasmonic nanoparticles in the presence of enzyme labels links directly to the existence or non-existence of the TB analytes in the sample solutions. For disease detection, the adapted protocol is based mainly on the conventional ELISA procedure that involves catalase-labeled antibodies, i.e., the enzymes consume hydrogen peroxide and further produce GNPs with the addition of gold (III) chloride. The amount of hydrogen peroxide remaining in the solution determines whether the GNPs solution is to be formed in the color blue or the color red, as it serves as a confirmation for the naked eye detection of TB analytes. However, the conventional ELISA method only shows tonal colors that need a high concentration of analyte to achieve high confidence levels for naked eye detection. Also, in this research, we proposed the incorporation of protein biomarker, Mycobacterium tuberculosis ESAT-6-like protein esxB (CFP-10), as a means of TB detection using plasmonic ELISA. With the use of this technique, the CFP-10 detection limit can be lowered to 0.01 µg/mL by the naked eye. Further, our developed technique was successfully tested and confirmed with sputum samples from patients diagnosed with positive TB, thereby providing enough evidence for the utilization of our technique in the early diagnosis of TB disease.

Prognostic value of interferon-gamma, interleukin-6, and tumor necrosis factor-alpha in the radiation response of patients diagnosed with locally advanced non-small-cell lung cancer and glioblastoma multiforme

Background/aim: This study aimed to investigate the effect of chemoradiotherapy (CRT) on interferon-gamma (IFN-γ), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), which are critical markers of the clinical radiation response of patients with locally advanced non-small-cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). Materials and methods: Thirty patients who were treated with CRT and 20 healthy controls were prospectively evaluated. Circulating levels of cytokines were measured by enzyme-linked immunosorbent assay procedure. Post-CRT and pre-CRT levels were compared. Results: Post-CRT, TNF-α and IFN-γ levels were significantly lower than pre-CRT levels in the NSCLC and GBM groups, respectively. The statistical analysis did not show any significant difference between the post- and pre-CRT IL-6 levels. However, the pre-CRT IL-6 levels in the GBM group and post-CRT IL-6 levels in the NSCLC group were significantly higher than those of the control group. Conclusion: CRT affected TNF-α levels in NSCLC and IFN-γ levels in GBM, with the levels of both decreasing significantly. The IL-6 levels of the post-CRT NSCLC group were higher than those of the post-CRT GBM group. Irradiation-induced IL-6 may be responsible for tumor regrowth. Therefore, treatment with IL-6 inhibitors could be a potential therapeutic strategy for sensitizing NSCLC to irradiation in the clinic.

Detection of Fumonisins in Fresh and Dehydrated Commercial Garlic.

An epidemic fungal disease caused by Fusarium proliferatum, responsible for fumonisin production (FB1, FB2, and FB3), has been reported in the main garlic-producing countries in recent years. Fumonisins are a group of structurally related toxic metabolites produced by this pathogen. The aim of this work was to establish an enzyme-linked immunosorbent assay (ELISA) procedure, mostly applied to cereals, that is suitable for fumonisin detection in garlic and compare these results to those obtained by high-performance liquid chromatography (HPLC) and screening of fresh and dehydrated garlic for toxicological risk. The results show good correlation between the two analytical methods. In fresh symptomatic garlic, fumonisin levels were higher in the basal plates than those in the portions with necrotic spots. Among the 56 commercially dehydrated garlic samples screened, three were positive by ELISA test and only one was above the limit of quantitation. The same samples analyzed by HPLC showed the presence of FB1 in trace amounts that was below the limit of quantitation; FB2 and FB3 were absent. The results are reassuring, because no substantial contamination by fumonisins was found in commercial garlic.

Improvement of ELISA procedures through simultaneous addition of antigen and detection antibody and elimination of washing steps

ABSTRACTThe enzyme-linked immunosorbent assay (ELISA) is extensively used for measurement of proteins utilized for various research and diagnostic purposes in the biological disciplines. However, it is a labor-intensive and lengthy procedure due to a number of incubation and washing steps required for performing the assay. The ELISA procedure in the current study has been simplified through the simultaneous addition of antigen and detection antibody and elimination of washing steps. This resulted in a decreased time required to perform the procedure and without affecting assay capability. This approach offers the possibility of increasing ELISA productivity in low throughput laboratories without the need for alternative analytical platforms which would require significant assay redevelopment and capital expense.

Application of the Enzyme-linked Immunosorbent Assay Procedure to the Detection of Grapevine Fanleaf Virus

Enzyme-linked immunosorbent assay was used to detect grapevine fanleaf virus (GFV) directly in grapevine leaf tissue. The addition of nicotine or nicotine and sodium diethyldithiocarbamate to the extracting buffer solution greatly enhanced the sensitivity of the procedure and enabled detection of GFV concentrations as low as 12 ng/m t The conjugated GFV gamma-globulin detected the GFV strains, grapevine vein banding and grapevine yellow mosaic but failed to detect the GFV serotype arabis mosaic in grapevine leaf tissue. The technique makes possible the collection and processing of numerous samples throughout the growing season and is, therefore, particularly suitable for studies on the incidence and spread of GFV in the field. It also facilitates the resolution of disease syndromes with which GFV is associated.

Evaluation of risk factors in MTCT among HIV-seropositive pregnant women in selected centers in Akure, South Western Nigeria

Background: The burden of mother-to-child transmission (MTCT) of HIV remains a major challenge in Nigeria, the country ranks highest globally. About 10% of global HIV-infected individuals live in Nigeria, of which 58% are women. The study analyzed risk and cofactors of MTCT in healthcare centers of Akure, Ondo State, Nigeria, between November 2014 and April 2016. Methods: A total of 240 pregnant women aged 19–43 years were recruited for the study, 114 HIV-seropositive mean age 31.81 years and 126 HIV-seronegative mean age 29.05 years as controls. High vaginal sawbs, breast milk, oropharynx, and neonates' nares were collected using sterile cotton-tipped applicator and introducing each into thioglycollate fluid medium for growth. Bacterial isolates were characterized by the standard microbiological methods and API kits. HIV serostatus of each participant was determined by HIV-1/2 strip and confirmed by Abbott enzyme-linked immunosorbent assay procedure. Sexually transmitted infections were detected by the enzyme immunoassays using commercial kits. Results: A total of 2,148 bacterial isolates were recovered from both cohorts (911 from HIV-seropositive, 864 HIV-seronegative, and 373 from neonates' nares). Predominant pathogens recovered were Staphylococcus aureus, Pseudomonas aeruginosa, and diverse corynebacteria as commensals. Coinfection of HIV with other sexually transmitted diseases (STDs) was prominent. About 92.1% of patients received combination ART. Neonates mortality rate was 10.5 and 7.8% mothers were potential transmitters. Spontaneous vaginal delivery accounted 91.7% deliveries. Conclusion: Incidence of STDs among HIV-seropositive was 77.5 and 22.5% for HIV-seronegative. Incidence of spontaneous vaginal delivery was 91.7% in HIV-seropositive women and 10.5% mortality rate recorded for neonates and 7.8% transmission for mothers posed high risk of MTCT, which is epidemiological significant.

The applicability of ELISA detection of gastric mucosa-expressing proteins for the identification of vomit.

The identification of vomit stains may be helpful for crime scene reconstruction. However, there is no specific and convenient method for identifying vomit stain. Therefore, to establish the procedure for forensic identification of vomit stains, we focused on four gastric mucosa-expressing proteins, pepsinogen I (PGA), pepsinogen II (PGC), gastrin (GAST), and mucin 5AC (MUC5AC). We developed enzyme-linked immunosorbent assay (ELISA) procedures for the detection of these four candidate proteins. The specificity and sensitivity of ELISA detection of these proteins were analyzed, and applicability for the identification of vomit in forensic casework samples was also investigated. We found the sensitivities of ELISA for detection of PGA, PGC, GAST, and MUC5AC from the standard protein (peptide) and from diluted gastric mucosa extract were 10.0-100.0ng/ml and 1:200-1:1600, respectively. PGA and PGC were successfully detected in stomach contents and gastric mucosa samples; however, these also cross-reacted with some urine and semen samples, respectively, because of low level expression in these fluids. MUC5AC was positive for most gastric mucosa samples; however, it was difficult to detect in stomach contents. ELISA detection of GAST was not suitable for the identification of vomit. All aged samples stored up to 90days gave positive results for ELISA procedures for PGA, PGC, and MUC5AC. Therefore, ELISA detection of these proteins might be applicable to aged samples. PGA was also detected in all actual vomit samples tested. These results suggest that ELISA for the detection of gastric mucosa-expressing proteins, especially PGA, could be an effective tool for the forensic identification of vomit.

Development and use of specific ELISA methods for quantifying the biological activity of bevacizumab, cetuximab and trastuzumab in stability studies.

Bevacizumab (BVZ), cetuximab (CTX) and trastuzumab (TTZ) are monoclonal antibodies (mAbs) used worldwide for the treatment of several widespread kinds of cancer. They are marketed as medicines under their respective tradenames: Avastin(®), Erbitux(®) and Herceptin(®). The aim of this research was to develop in-house specific enzyme-linked immunosorbent assays (ELISA) to assess the long-term stability of these three mabs. These assays assess the biological functionality of the mAbs by quantifying their biological activity. For this purpose, we developed an indirect ELISA procedure whereby the specific antigens against which the mAbs are directed are used as specific "capturing" antibodies on the ELISA plates. We therefore used vascular endothelial growth factor (VEGF) in the ELISA for BVZ; human epidermal growth factor receptor (hEGFR) in the ELISA for CTX and human receptor HER2 (hHER2) in the ELISA for TTZ. After the mAbs had attached to their antigen, we used an anti-human IgG (whole molecule) peroxidase-conjugate and o-phenylenediaminedihydrochloride substrate. The reaction was stopped using sulphuric acid and absorbance was recorded at a wavelength of 450nm. The three ELISA methods were validated in terms of calibration models, range of the assay, limits of detection and quantitation, intra and interday precision and accuracy, and specificity by cross reactions. Forced degradation studies were also conducted on the medicines, providing useful information. Finally, the proposed ELISA were successfully used in a long-term stability study to quantify the remaining biological activity in medicines that had been opened and then stored under two different storage conditions, i.e. refrigerated at 4°C and frozen at -20°C. Results indicated that BVZ (Avastin(®)) is the most stable of the three in terms of its biological functionality.

Determination of Bisphenol A by a Gold Nanoflower Enhanced Enzyme-Linked Immunosorbent Assay

ABSTRACTThe use of nanomaterials to improve the sensitivity of enzyme-linked immunosorbent assays (ELISA) for small molecules allows wider application of this method. In this work, gold nanoflowers were synthesized by a seed-mediated procedure and used to prepare a horseradish peroxidase-immunoglobulin G-gold nanoflower complex. The complex was used as the signal amplification probe in a novel ELISA for bisphenol A. The gold nanoflower-enhanced ELISA demonstrated excellent detection sensitivity of 0.05 ng/mL, which was 20 times more sensitive than a standard ELISA procedure. A linear dynamic range from 0.1 to 5.0 ng/mL was obtained by this approach. The gold nanoflower-enhanced ELISA was used to determine bisphenol A in lake water, with recoveries from 92.0 to 102.0% in samples fortified at concentrations of 0.5, 1.0, and 3.0 ng/mL. The gold nanoflower-enhanced ELISA was shown to offer good sensitivity, simplicity, and low cost.

Coinfections of Human Immunodeficiency Virus and Sexually Transmitted Infections among HIV Seropositive Pregnant Women at Healthcare Centres in Akure, Southwestern Nigeria

Background: Sexually transmitted infections (STIs) represent significant risk factors for HIV transmission and adverse fetal complications in pregnancy. We tested 240 pregnant women for their HIV serostatus and for the effects of HIV-STI coinfections on CD4+ cell counts and on HIV plasma viral load. The study was conducted at four antenatal clinics in Akure, Ondo State of Nigeria between November 2014 and December 2015. Methods: The HIV-1/2 strip was used for preliminary determination of HIV serostatus and this was followed by a confirmatory Abbott enzyme-linked immunosorbent assay (ELISA) procedure. CD4+ T-cell counts were done by cytometric analysis while viral load was determined by COBAS® AmpliPrep TaqMan HIV-1 procedure. Liver enzymes were determined by an automated chemistry analyzer. Detection of hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus-6 (HPV-6) and herpes simplex virus type-2 (HSV-2) were determined by ELISA. The syphilis VDRL test was done by the rapid plasma reagin procedure, while Trichomonas vaginalis infection was demonstrated by the wet mount procedure. Infections with Chlamydia trachomatis and Neisseria gonorrhoeae were determined respectively by ELISA and by culture on proteose peptone agar supplemented with 5% serum. Results: The results show prevalence of STIs in 33.3% of the 240 study participants; 77.5% for HIV seropositive and 22.5% for seronegative subjects. Trichomonas vaginalis constituted the highest isolate Coinfections in 50% of HIV seropositive subjects and 22.2% for seronegative controls. HIV-HBV coinfection occurred among 7 individuals while HIVHBV-TV coinfection occurred in 4 individuals. The results also showed elevated titers of alanine aminotransferase (ALT), creatinine and total bilirubin liver enzymes in both HIV-HBV coinfection and HBV monoinfection. HIV viral loads were twice as high in HIV-HBV coinfection relative to HBV mono- infection. Conclusion: The prevalence of STIs was significantly high at 54.4% among HIV seropositive pregnant women as compared to 14.3% for HIV seronegative pregnant women. T. vaginalis was the highest isolate at 50% in HIV seropositive subjects and may therefore be a driving factor for HIV transmission and susceptibility as very few T. vaginalis infections were recorded in control subjects.

Facilitating the Validation of Novel Protein Biomarkers for Dementia: An Optimal Workflow for the Development of Sandwich Immunoassays.

Different neurodegenerative disorders, such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD), lead to dementia syndromes. Dementia will pose a huge impact on society and thus it is essential to develop novel tools that are able to detect the earliest, most sensitive, discriminative, and dynamic biomarkers for each of the disorders. To date, the most common assays used in large-scale protein biomarker analysis are enzyme-linked immunosorbent assays (ELISA), such as the sandwich immunoassays, which are sensitive, practical, and easily implemented. However, due to the novelty of many candidate biomarkers identified during proteomics screening, such assays or the antibodies that specifically recognize the desired marker are often not available. The development and optimization of a new ELISA should be carried out with considerable caution since a poor planning can be costly, ineffective, time consuming, and it may lead to a misinterpretation of the findings. Previous guidelines described either the overall biomarker development in more general terms (i.e., the process from biomarker discovery to validation) or the specific steps of performing an ELISA procedure. However, a workflow describing and guiding the main issues in the development of a novel ELISA is missing. Here, we describe a specific and detailed workflow to develop and validate new ELISA for a successful and reliable validation of novel dementia biomarkers. The proposed workflow highlights the main issues in the development of an ELISA and covers several critical aspects, including production, screening, and selection of specific antibodies until optimal fine-tuning of the assay. Although these recommendations are designed to analyze novel biomarkers for dementia in cerebrospinal fluid, they are generally applicable for the development of immunoassays for biomarkers in other human body fluids or tissues. This workflow is designed to maximize the quality of the developed ELISA using a time- and cost-efficient strategy. This will facilitate the validation of the dementia biomarker candidates ultimately allowing accurate diagnostic conclusions.

Using the DAS-ELISA Test to Establish an Effective Distance Between Bait Stations for Control of Linepithema humile (Hymenoptera: Formicidae) in Natural Areas.

Linepithema humile (Mayr), the Argentine ant, is an invasive pest that has spread throughout the United States and is a problem in natural and managed habitats in South Carolina. Foraging patterns and the effectiveness of liquid baits for control of this pest have been studied in urban areas. However, similar studies have not been conducted in natural areas such as parks, picnic grounds, or campsites. L. humile populations can be large and widespread, making them a major nuisance pest for visitors to these natural areas. The primary objective of this study was to determine an effective distance between bait stations for control of L. humile in a natural area. A double antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) procedure was used to detect individual ants that consumed rabbit immunoglobin (IgG) protein for marking and tracking. In both lab and field conditions, there was a significant difference in the detection of IgG in ants fed protein marker mixed with sugar water compared with ants only fed sugar water. Additional field studies revealed that an individual ant could retain detectable levels of protein marker for 3 d and that an ant feeding on IgG containing bait could be detected over 15 m from the original bait source. Overall, we found that using liquid ant baits, with a placement of 20 m between stations, was effective in reducing L. humile numbers between April to October, 2012 in a natural park area of Lake Greenwood State Park, SC.

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc.

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.