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In this work, a miniaturized, automated, and cost-effective ELISA device is designed and implemented, without the utilization of conventional techniques such as pipetting or microfluidic valve technologies. The device has dimensions of 24 cm × 19 cm × 14 cm and weighs <3 kg. The total hardware cost of the device is estimated to be approximately $1200, which can be further reduced through optimization during scale-up production. Three-dimensional printed disposable parts, including the reagent reservoir disk and the microfluidic connector, have also been developed. IL-6 is used as a model system to demonstrate how the device provides an ELISA measurement. The cost per test is estimated to be <$10. The compactness, automated operation, along with the cost-effectiveness of this ELISA device, makes it suitable for point-of-care applications in resource-limited regions.

ABSTRACT Background Although high‐dose biotin interference in automated immunoassays is now considered, there are very few studies showing biotin interference in manually operated research kits, especially with enzyme‐linked immunosorbent assay (ELISA). The aims of our study were to determine the effects of biotin interference on various parameters, including leptin, leptin receptor (LEPR), ghrelin, acylated ghrelin, deacylated ghrelin, ghrelin receptor (GHSR), kisspeptin (KISS1), kisspeptin receptor (KISS1R), preptin, peroxisome proliferator activated receptor gamma (PPARγ), nod‐like receptor pyrin domain‐containing 3 (NLRP3) and interleukin‐18 (IL‐18), which contribute to energy homeostasis in healthy and obese children. Methods Serum pools were prepared from healthy and obese individuals, and biotin concentrations in samples containing different amounts of biotin were measured via sandwich and competitive ELISA methods. In addition, possible biotin interactions were investigated by determining the concentrations of all the study parameters in serum pools containing different amounts of biotin. Results We found that the biotin‐competitive, ghrelin‐competitive, KISS1‐competitive, GHSR, leptin and LEPR ELISA kits were less affected by biotin interference and the results of these assay kits were more reliable. Unexpectedly, high levels were also measured in the biotin sandwich ELISA kit, indicating that biotin interference can also occur in manually operated assay kits. Conclusions Biotin exhibited an interference effect even in well‐functioning, qualified kits, and this negative effect was less common in competitive kits. Biotin interference was closely associated with the quality of the research kit, the parameters studied, and the presence of high biotin concentrations in the blood. In this study, we aimed for the first time to demonstrate biotin interference in ELISA research kits used in the analysis of pediatric obesity parameters other than routine testing. Our study revealed that biotin interaction may occur in manual ELISA kits and may cause erroneous test results. It will raise awareness among health professionals, researchers, and medical companies on this issue. By encouraging manufacturers developing medical products to take measures to reduce the effect of biotin interaction, it will prevent erroneous results in scientific studies and contribute to more precise measurements.

Red deer (Cervus elaphus) are hosts of liver fluke (Fasciola hepatica); yet, prevalence is rarely quantified in wild populations. Testing fresh samples from remote regions by faecal examination (FE) can be logistically challenging; hence, we appraise frozen storage and the use of a coproantigen ELISA (cELISA) for F. hepatica surveillance. We also present cELISA surveillance data for red deer from the Highlands of Scotland. Diagnoses in faecal samples (207 frozen, 146 fresh) were compared using a cELISA and by FE. For each storage method (frozen or fresh), agreement between the two diagnostics was estimated at individual and population levels, where population prevalence was stratified into cohorts (e.g., by sampling location). To approximate sensitivity and specificity, 65 post-slaughter whole liver examinations were used as a reference. At the individual level, FE and cELISA diagnoses agreed moderately (κfrozen = 0.46; κfresh = 0.51), a likely reflection of their underlying principles. At the population level, FE and cELISA cohort prevalence correlated strongly (Pearson's R = 0.89, p < 0.0001), reflecting good agreement on relative differences between cohort prevalence. In frozen samples, prevalence by cELISA exceeded FE overall (42.8% vs. 25.8%) and in 9/12 cohorts, alluding to differences in sensitivity; though, in fresh samples, no significant difference was found. In 959 deer tested by cELISA across the Scottish Highlands, infection prevalence ranged from 9.6% to 53% by sampling location. We highlight two key advantages of cELISA over FE: i) the ability to store samples long term (frozen) without apparent loss in diagnostic power; and ii) reduced labour and the ability to process large batches. Further evaluation of cELISA sensitivity in red deer, where a range of fluke burdens can be obtained, is desirable. In the interim, the cELISA is a practicable diagnostic for F. hepatica surveillance in red deer, and its application here has revealed considerable geographic, temporal, sex and age related differences in F. hepatica prevalence in wild Scottish Highland red deer.

Infectious bursal disease is a highly contagious disease of young chickens caused by the infectious bursal disease virus. This disease poses an important threat to the commercial poultry industry globally. This study was designed to develop an In-House Indirect Enzyme-Linked Immune Sorbent Assay Kit for the serological detection of antibodies against infectious bursal disease viruses. An infectious bursal disease virus antigen dilution (1:2), sample serum (1:500), and mouse anti-chicken immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) (1:2,000) were used in this assay. The calculated cutoff value was 0.24. This in-house indirect ELISA method was compared with a commercial ELISA kit for the detection of antibodies against infectious bursal disease virus in chickens. The performance of the newly developed and commercial ELISA kit was evaluated as described by Samad et al. (1994). The sensitivity and specificity of the current ELISA method were 98% (95% CI: 92.96–99.76) and 97% (95% CI: 91.48–99.38), respectively. The average intra-assay % CV of the triplet of 2 samples was 7.6, and interassay comparisons indicated a CV of 5.45%. As indicated by the results, we described a valuable and cost-effective, sensitive and specific in-house indirect ELISA kit for the serological diagnosis of infectious bursal disease in Ethiopia.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

Porcine circovirus type 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome which leads to significant economic losses in the global swine industry. In China, there is a widespread dissemination of PCV2 infection in the pig population. Serologi­cal diagnosis of the disease is considered as an effective control measure. Here, we developed a capsid protein (Cap)-based enzyme-linked immunosorbent assay (Cap-ELISA) for the detection of PCV2 antibodies in swine serum using a nuclear localization signal-truncated capsid protein produced in Escherichia coli. The Cap protein was expressed as water-soluble and purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography. After the optimization of the working conditions of the Cap-ELISA using chessboard titrations, a total of 649 serum samples were tested using the Cap-ELISA and a commercial ELISA kit. The diagnostic sensitivity (DSN), diagnostic specificity (DSP) and accuracy of the Cap-ELISA were determined to be 96.7%, 94.1% and 99.5%, respectively. Cross-reactivity analysis indicated that the Cap-ELISA was PCV2-specific and possessed no cross-reactions with antibodies against other common swine pathogens including porcine circovirus type 1 (PCV1), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine parvovirus (PPV), foot and mouth disease virus (FMDV), porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV). Repeatability of the experiment showed that Cap-ELISA was highly repeatable with the intra- and inter-plate coefficients of variation less than 10%. Hence, the Cap-ELISA has the potential for the swine industry to monitor PCV2 epidemiology and to evaluate PCV2 vaccine efficacy.

Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKine TM , Promega-Emax ® , R&D-System-Quantikine ® ) and one multiplexing assay (Millipore-Milliplex ® ). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications.

The enzyme-linked immunosorbent assay (ELISA) detects antigen-antibody interactions by using enzyme-labelled conjugates and enzyme substrates that generate colour changes. This review aims to provide an overview of ELISA, its various types, and its applications in detecting metabolites in biological fluids. The article discusses the history of the assay, its underlying principles and procedures, common ELISA protocols, and the most accurate and reliable techniques for measuring peptide molecules in biological fluids. Additionally, we emphasize best laboratory practices to achieve consistent, high-quality results and outline the essential materials for setting up an ELISA laboratory, drawing from our over 30 years of experience in the field.

ABSTRACT Background Bordetella bronchiseptica is an essential bacterial pathogen characterized by chronic respiratory disease in dogs known as Kennel cough. The presence of causative antibodies in animals can also be detected by lipopolysaccharide antigen‐based enzyme linked immunosorbent assay (ELISA). In recent years, it has been determined that there is a significant relationship between acute phase proteins and diseases, and disease follow‐up can be done within the framework of this relationship. Methods In this study, blood sera from 150 dogs in an animal shelter in Van province were evaluated for B. bronchiseptica by the homemade ELISA method, and their correlations with serum amyloid A (SAA) were investigated. Blood serum samples were analysed for antibodies against B. bronchiseptica using a homemade ELISA method. Positive animals were also molecularly confirmed using nasal swabs by PCR. A commercial ELISA kit determined SAA levels in blood sera. Results Eighteen (12%) of the analysed blood serum samples were found positive by the homemade ELISA method. SAA concentrations in the positive blood sera were elevated from 12.7 to ≤38.98 mg/L. SAA concentrations in blood sera serologically positive for B. bronchiseptica were statistically significant. Conclusions In this study, in which the relationship between SAA concentration and B. bronchiseptica was investigated for the first time in Turkey, it was concluded that SAA concentration analysis may help diagnose and monitor the disease. In addition, the presence and prevalence of this critical and zoonotic agent causing chronic respiratory tract disease in dogs in Van province was revealed for the first time in this study. A study in Van, Turkey examined Bordetella bronchiseptica in shelter dogs using ELISA and PCR. Overall, 12% of blood samples tested positive. Serum amyloid A (SAA) levels were significantly elevated in positive cases, suggesting SAA could be a useful diagnostic marker for this chronic respiratory disease‐causing bacterial pathogen.

ABSTRACT Porcine deltacoronavirus (PDCoV) is an infectious disease that causes diarrhoea in pigs of different ages; however, piglets are more susceptible. PDCoV was first reported in 2012 in China and Hong Kong. Later, it was first reported in the USA in 2014 and in Mexico in 2019. Several studies have shown that M protein is highly conserved and, therefore, suitable for diagnostic systems. In this study, for the first time, an indirect enzyme‐linked immunosorbent assay (iELISA) based on a recombinant M protein (rM‐PDCoV) was developed to evaluate the seroprevalence of PDCoV in four states in Mexico. High sensitivity (83%) and specificity (100%) were observed for the iELISA. The kappa index calculated a nearly perfect agreement (0.8831) compared to the Western blot (gold standard test), suggesting acceptable statistical value support. In this study, 50.38% of the serum samples from backyard pigs were PDCoV‐positive. The serological comparison showed that PDCoV/PEDV coinfections occurred in 31.98% of the analysed sera. These results can enrich our understanding of how this virus spreads and enable the evaluation of PDCoV infections. Moreover, it highlights the importance of continually investigating the seroprevalence of PDCoV in Mexico because there is also no information about the current prevalence of the disease. Developed an iELISA with the PDCoV recombinant protein M to sero‐evaluate 516 samples from 4 different states of Mexico and serologically compare PDCoV/PEDV.