There has been increased interest in the measurement of melatonin in plasma and saliva recently either as a marker of circadian phase or to understand the physiological role of melatonin. For both ...situations, there is a need for a specific assay for melatonin that is sensitive enough to detect low concentrations (<2 pg/mL). Since the mid‐1970s, there have been many assays developed to measure melatonin in blood and saliva. Radioimmunoassays and ELISA have predominated because of their relative simplicity and high throughput. In this review, I show that the early radioimmunoassays while providing valuable information about nocturnal melatonin levels in humans, generally produced inaccurate basal (daytime) levels. Mass spectrometry assays, however, have provided us with the target values that immunoassays need to achieve, that is, daytime plasma melatonin levels <1 pg/mL. There are now many contemporary commercial assays available utilising both RIA and ELISA technologies, but not all achieve the standards set by the mass spectrometry assays. The performance of these assays is reviewed. I conclude with recommendations on issues researchers need to consider when conducting melatonin studies, including the importance of time of day of collection, validation of assays, the potential causes of poor assay specificity at low levels, the advantages/disadvantages of using saliva vs plasma and extraction assays vs direct assays, kit manufacturers responsibilities and the reporting requirements when publishing melatonin studies.
•The invention of ELISA and its types are presented in a sequence.•Laboratory experiences with peptide analyses are provided.•The need to use protease inhibitors to protect peptides in peptide ...analyses is emphasized.•It is underlined that ELISA kit manufacturers have to standardize kits.•The significance of ELISA in peptide analyses is noted.
Playing a critical role in the metabolic homeostasis of living systems, the circulating concentrations of peptides/proteins are influenced by a variety of patho-physiological events. These peptide/protein concentrations in biological fluids are measured using various methods, the most common of which is enzymatic immunoassay EIA/ELISA and which guide the clinicians in diagnosing and monitoring diseases that inflict biological systems. All the techniques where enzymes are employed to show antigen–antibody reactions are generally referred to as enzymatic immunoassay EIA/ELISA method. Since the basic principles of EIA and ELISA are the same. The main objective of this review is to present an overview of the historical journey that had led to the invention of EIA/ELISA, an indispensible method for medical and research laboratories, types of ELISA developed after its invention direct (the first ELISA method invented), indirect, sandwich and competitive methods, problems encountered during peptide/protein analyses (pre-analytical, analytical and post-analytical), rules to be followed to prevent these problems, and our laboratory experience of more than 15 years.
Long-term gastric acid suppression has been associated with complications in mucosa integrity. But the roles for gastric acid in extragastral damage manifestation have not been reported. We examined ...the effects of acid suppression on oesophageal and oral mucosa (OM). Previously we showed that an extract of Cucurbita maxim sweet seed (eCSE) plays an important role in non-erosive lesions (NEOL) in proximal part of GI tract. The present study was designed to study effects of eCSE on oesophageal mucosa (OEM) and OM under modification of enterosalivary nitrites recirculation (mESNR). The study was done on rats subdivided to the first group (control) injected with vehicle at a dose 1 ml, i.p.; the second group of animals was subdivided into three subgroups and rats were sacrificed after administration of R (Ranitidine, "Darnycya Ltd", Ukraine) at a dose of 100 mg/kg i.p., or over 2 and 3 days drug applying. The third group was similarly subdivided and A (Atropine, "Darnycya Ltd", Ukraine), at a dose of 3 mg/kg, i.p., the same time and doses. The fourth and fifth groups were administered R with A, and R with A and L-NAME (in dose 10 mg/kg, i.p.) In another series of experiments eCSE (Institute of Molecular Biology and Genetics of NASU) was introduced per os in 0.5 ml/200 g/day. IL-1beta, GRO/CINC-1 were detected by ELISA, NO serum content--by biochemical study. Rats with mESNR regulation by A blocking had an increase of IL-1beta by 32.8% over 2 days, 3 days a similar pretreatment level of GRO/CINC-1 increased by 13.9% vs control. Dual blocking of cholinergic and histaminergic mechanisms by combination of R with A--significant increase in the level of GRO/CINC-1 (by 137.1%). In the group with mESNR with combined blocking (R, A and L-NAME) accelerated production of IL-1beta (by 14.3%) and GRO/CINC-1 (by 191.4%) appeared after 3 days vs control. The NO serum content showed an increasing tendency with a maximum rise after 3 days of blocking of histaminergic and both cholinergic and histaminergic and a combination of triple blocking. The pre-treatment of eCMS showed an anti-inflammatory effect via a significantly decreased level of IL-1beta in rats with blocked cholinergic mechanism after one-day treatment. The results of the present study revealed that gastric acid suppression caused NEOL in rat OM, OEM. Plant-originated eCMS represented a mucosal protective effect on integrity of extragastral lesions manifestation, can be used as novel preventive therapeutic agent.
Diagnosis of bovine tuberculosis in cattle is challenging due to complex immune host response to infection that limit the performance of available diagnostic tests. In this study, performance of two ...commercial serological assays developed to detect bovine tuberculosis were evaluated: Enferplex Bovine TB antibody kit including 11 antigens (EnferGroup, Ireland) and IDEXX M. bovis Ab kit (IDEXX, USA). The specificity value obtained with the ELISA IDEXX M. bovis Ab test was 97.1%, whereas it was 97.1% and 95.1% for the high specificity and sensitivity settings, respectively, with the Enferplex Bovine TB antibody kit. The sensitivity of the multiplexed Enferplex Bovine TB antibody test for SICCT-positive animals was higher (N = 172; 51.7% and 58.7% with high specificity and sensitivity settings, respectively) compared to the ELISA IDEXX M. bovis Ab test (sensitivity of 36.6%). “Antigen profiles” generated by the multiplexed Enferplex method showed that five out of 11 antigens present in the test were mostly identified as positive sera in cattle originating from bTB-outbreaks. In comparison, unique profiles appeared to be correlated with false positive results. However additional studies are needed to confirm the observed antigen profiles, and their potential use as an additional diagnostic tool. Serial interpretation of the two serological tests produced higher diagnostic specificity (>99%), reducing false positive results, which is essential for a screening test when the prevalence of bovine tuberculosis is low.
•First evaluation of performance of commercial multiplexed kit with 11 antigens.•Commercial multiplexed kit was more sensitive than ELISA IDEXX M. bovis Ab kit.•Serial interpretation of both serological tests offers high diagnostic specificity.
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.
In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design ...pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL−1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1–100ngmL−1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.
•A novel wax-printed paper-based lateral flow device was proposed as an automated and one-step ELISA.•A wax-printed pad consisted of non-delayed channel, wax-delayed channel, test and control zones.•The device provides fast point-of-care testing of AFP in human serum (10min).
Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate ...batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.
In August 2020, anew West Nile virus (WNV) outbreak affected 71 people with meningoencephalitis in Andalusia (Spain). Samples from these individuals were received in our laboratory, a regional Virus ...Referral Centre. The aim of this study was to compare the agreement, sensitivity and specificity of findings between the WNV VIRCLIA IgG and IgM assay (Vircell, Spain) and the WNV ELISA IgM and IgG assay (Euroimmun, Germany) and to compare the performance of WNV VIRCLIA IgM and Euroimmun ELISA for cerebrospinal fluid (CSF) diagnosis. The study included 24 CSF samples (paired with serum samples) and 247 serum samples from 217 patients with suspected WNV infection (1 or 2 per patient). The agreement between ELISA and CLIA tests for IgM and Ig G detection in serum was 93% (kappa index = 0.85) and 96% (kappa index = 0.89) respectively. Sensitivity values of ELISA and CLIA tests for IgM in serum samples were 96.7% and 98.9%, respectively, and specificity values were 96.4% and 95.4% respectively. Sensitivity values of ELISA and CLIA test for IgG in serum samples were 91.1% and 97%, respectively, and specificity values were 100% and 98.8% respectively. Results obtained with ELISA and CLIA tests in CSF samples showed 75% agreement between them (kappa index = 0.51). According to these findings, the WNV VIRCLIA IgM and IgG monotest offers an accurate qualitative detection of WNV in serum and CSF specimens.
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