Introduction
Vaccine‐induced immune thrombocytopenia and thrombosis (VITT) following ChAdOx1 nCOV‐19 vaccine has been described, associated with unusual site thrombosis, thrombocytopenia, raised ...D‐dimer, and high‐titer immunoglobulin‐G (IgG) class anti‐platelet factor 4 (PF4) antibodies. Enzyme‐linked immunosorbent assays (ELISA) have been shown to detect anti‐PF4 in patients with VITT, but chemiluminescence assays do not reliably detect them. ELISA assays are not widely available in diagnostic laboratories, and, globally, very few laboratories perform platelet activation assays.
Methods
Assays that are commercially available in the United Kingdom were evaluated for their ability to identify anti‐PF4 antibodies in samples from patients with suspected VITT. Four IgG‐specific ELISAs, two polyspecific ELISAs, and four rapid assays were performed on samples from 43 patients with suspected VITT from across the United Kingdom. Cases were identified after referral to the UK Expert Haematology Panel multidisciplinary team and categorized into unlikely, possible, or probable VITT.
Results and Discussion
We demonstrated that the HemosIL AcuStar HIT‐IgG, HemosIL HIT‐Ab, Diamed PaGIA gel, and STic Expert assays have poor sensitivity for VITT in comparison to ELISA. Where these assays are used for heparin‐induced thrombocytopenia (HIT) diagnosis, laboratories should ensure that requests for suspected VITT are clearly identified so that an ELISA is performed. No superiority of IgG‐ELISAs over polyspecific ELISAs in sensitivity to VITT could be demonstrated. No single ELISA method detected all possible/probable VITT cases; if a single ELISA test is negative, a second ELISA or a platelet activation assay should be considered where there is strong clinical suspicion.
Cyclopiazonic acid (CPA) is an indole‐tetramine mycotoxin commonly produced by Penicillium and Aspergillus and is widely found in agricultural products, fermented food, and feed. Food contaminated ...with CPA poses a substantial health risk to consumers. Therefore, eco‐friendly immunoassays, including an indirect competitive enzyme‐linked immunosorbent assay (ic‐ELISA) and a lateral flow immunochromatographic strip (LFICS), were developed to monitor CPA in maize and rice samples. For this purpose, a monoclonal antibody (3H12) posed highly resistant to pH (5.6 to 9.6) and ethanol (≤50%) was generated by mouse immunization. Negative maize and rice samples or samples spiked with CPA were extracted with ethanol/0.01 M sodium borate buffer (4/1, v/v, pH 8.4). For ic–ELISA analysis, the limits of detection (LODs) were 0.48 and 0.28 ng/g for maize and rice samples, respectively. The recovery for spiked maize was 76.9% to 83.5% with the highest variable coefficient (CVmax) being 9.32%. For spiked rice, the recovery was 85.3% to 105.1% with a CVmax of 8.56%. For LFICS analysis, the visible LODs were 2.5 and 1 ng/g and cutoff values were 5 and 2.5 ng/g for maize and rice samples, respectively. The LFICS method gave results within 5 to 10 min, providing an auxiliary analytical tool for the rapid, sensitive, and portable screening of the massive samples onsite.
•A sensitive and broad-spectrum mAb against 4 avermectins was obtained.•Molecular modelling was used to hapten design and antibody analyze.•A mAb-based ic-ELISA was developed for avermectins in ...various samples.
Avermectins (AVMs) are a group of anti-parasitic agents that have been widely used in food-producing animals. To monitor the residue of the AVMs, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure. Conjugates of 4″-HS-IVM/AVM on three different proteins were used to raise a broad-spectrum monoclonal antibody (mAb), 6D4, that had IC50 values for avermectin, ivermectin, eprinomectin and emamectin of 7.2, 10.4, 19.8 and 20.8 µg L−1, respectively. The limit of detection and limit of quantitation of this method for AVMs in various matrix samples ranged from 0.5 to 5.4 µg L−1 and 1.0 to 10.3 µg L−1, respectively. The recoveries of the samples spiked with AVMs were in the range of 78.1–110.5% with coefficients of variation below 14%. The ic-ELISA was applied to monitor ivermectin in the milk samples, and the results showed a good correlation with that of HPLC-MS/MS.
Introduction
Glabridin is a unique isoflavonoid found only in Glycyrrhiza glabra L. The pharmacological effects of glabridin are well established, especially for beauty‐ and wellness‐related uses, ...such as antioxidant, anti‐inflammatory, ultraviolet (UV) protection, and skin‐lightening effects. Therefore, glabridin is often found in commercial products such as creams, lotions, and dietary supplements.
Objective
This study aimed to develop an enzyme‐linked immunosorbent assay (ELISA) using a glabridin‐specific antibody.
Method
Immunogen conjugation of glabridin‐bovine serum albumin was performed via the Mannich reaction, and the resulting conjugates were injected into BALB/c mice. Subsequently, hybridomas were produced. An ELISA method for glabridin determination was developed and validated.
Result
A highly specific antibody against glabridin was produced using clone 2G4. The assay range for the determination of glabridin was 0.28–7.02 μg/ml, with a detection limit of 0.16 μg/ml. The validation parameters in terms of accuracy and precision met the acceptable criteria. Standard curves of glabridin in various matrices were compared to evaluate the matrix effect on human serum using ELISA. Standard curves of the human serum and water matrix were obtained in the same manner, and the measurement range was 0.41–10.57 μg/ml.
Conclusion
The developed ELISA method was used to quantify glabridin in plant materials and products with high sensitivity and specificity, and has potential applications in quantifying compounds in plant‐derived products and human serum samples.
In this study, specific antibodies against glabridin, which is an authentic marker in Glycyrrhiza glabra, were used to develop an ELISA method. A highly specific antibody against glabridin was produced using clone 2G4. The ELISA method was performed for glabridin determination in natural plants and human serum. The validation parameters, in terms of accuracy and precision, met the acceptable criteria. The developed ELISA for glabridin has potential applications in quantifying compounds in plant‐derived products and human serum samples.
As one of the leading causes of food poisoning, staphylococcal enterotoxins (SEs) secreted by Staphylococcus aureus pose a serious threat to human health. The immunoassay has become the dominant tool ...used for the rapid detection of harmful bacteria and toxins as a result of its excellent specificity. However, with regard to SEs, staphylococcal protein A (SpA) is likely to bind with the fragment crystallizable (Fc) terminal of the traditional antibody and result in a false positive, limiting the practical application of this method. Therefore, to eliminate the bottleneck problem, the sandwich immunoassay was development by replacing the traditional antibody with a nanobody (Nb) that lacked a Fc terminal. Using 0.5 × 107 colony-forming units, the Nb library was constructed using Bactrian camels immunized with staphylococcal enterotoxin B (SEB) to obtain a paired Nb against SEB with good affinity. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using one Nb as the capture antibody and a phage-displayed Nb with signal-amplifying properties as the detection antibody. In optimal conditions, the current immunoassay displayed a broad quantitative range from 1 to 512 ng/mL and a 0.3 ng/mL limit of detection. The recovery of spiked milk, milk powder, cheese, and beef ranged from 87.66 to 114.2%. The Nbs-ELISA was not influenced by SpA during the detection of SEB in S. aureus food poisoning. Therefore, the Nb developed here presented the perfect candidates for immunoassay application during SE determination as a result of the complete absence of SpA interference.
We set out to investigate the interference factors that led to false-positive novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM detection results using gold immunochromatography ...assay (GICA) and enzyme-linked immunosorbent assay (ELISA) and the corresponding solutions. GICA and ELISA were used to detect SARS-CoV-2 IgM in 86 serum samples, including 5 influenza A virus (Flu A) IgM-positive sera, 5 influenza B virus (Flu B) IgM-positive sera, 5
IgM-positive sera, 5
IgM-positive sera, 6 sera of HIV infection patients, 36 rheumatoid factor IgM (RF-IgM)-positive sera, 5 sera from hypertensive patients, 5 sera from diabetes mellitus patients, and 14 sera from novel coronavirus infection disease 19 (COVID-19) patients. The interference factors causing false-positive reactivity with the two methods were analyzed, and the urea dissociation test was employed to dissociate the SARS-CoV-2 IgM-positive serum using the best dissociation concentration. The two methods detected positive SARS-CoV-2 IgM in 22 mid-to-high-level-RF-IgM-positive sera and 14 sera from COVID-19 patients; the other 50 sera were negative. At a urea dissociation concentration of 6 mol/liter, SARS-CoV-2 IgM results were positive in 1 mid-to-high-level-RF-IgM-positive serum and in 14 COVID-19 patient sera detected using GICA. At a urea dissociation concentration of 4 mol/liter and with affinity index (AI) levels lower than 0.371 set to negative, SARS-CoV-2 IgM results were positive in 3 mid-to-high-level-RF-IgM-positive sera and in 14 COVID-19 patient sera detected using ELISA. The presence of RF-IgM at mid-to-high levels could lead to false-positive reactivity of SARS-CoV-2 IgM detected using GICA and ELISA, and urea dissociation tests would be helpful in reducing SARS-CoV-2 IgM false-positive results.
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•A nanozyme and aptamer-based immunosorbent assay (NAISA) was developed for the sensitive detection of AFB1 (5 pg mL−1).•m-APS NPs replaced horseradish enzyme with better catalytic ...activity and stability towards H2O2.•Aptamer was utilized to specifically recognize aflatoxin B1 with easier preparation.•The method showed a 600 and 12-fold improvement in sensitivity than that of t-ELISA (3 ng mL−1) and e-ELISA (0.06 ng mL−1).
Traditional enzyme-linked immunosorbent assay (ELISA) suffers from the limitations of relatively low sensitivity and stability, and enzyme-labelled antibodies are hard to be prepared and purified. Based on a nanozyme, an aptamer and Fe3O4 magnetic nanoparticles (MNP), a nanozyme and aptamer-based immunosorbent assay (NAISA) was developed for aflatoxin B1 (AFB1) detection with simpler operation and separation. In this work, mesoporous SiO2/Au-Pt (m-SAP) were prepared to act as signal labels, which showed high catalase-like activity and was denoted as nanozyme. Aptamer was adopted to specifically recognize with AFB1, and MNP facilitated to realize magnetic separation. To verify the performance of NAISA, traditional ELISA (t-ELISA) and enhanced ELISA (e-ELISA) using MNP and m-SAP nanozyme were applied in AFB1 detection. The NAISA method showed the lowest limit of detection (LOD) with 5 pg mL−1 (n = 3, ±4.2 %), 600 and 12-fold lower than that of t-ELISA (3 ng mL−1) and e-ELISA (0.06 ng mL−1), respectively. In the interference tests, AFB1 can be identified among six different interfering substances. The NAISA method, thus, can be of great importance as it allows selective and sensitive AFB1 detection, while providing the simplicity of use and need for screening hazardous materials.
Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing ...agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dengue virus is an important arbovirus infection which transmitted by the
Aedes
female mosquitoes. The attempt to control and early detection of this infection is a global public health issue at ...present. Because of the clinical importance of its detection, the main focus of this review is on all of the methods that can offer the new diagnosis strategies. The advantages and disadvantages of reported methods have been discussed comprehensively from different aspects like biomarkers type, sensitivity, accuracy, rate of detection, possibility of commercialization, availability, limit of detection, linear range, simplicity, mechanism of detection, and ability of usage for clinical applications. The optical, electrochemical, microfluidic, enzyme linked immunosorbent assay (ELISA), and smartphone-based biosensors are the main approaches which developed for detection of different biomarkers and serotypes of Dengue virus. Future efforts in miniaturization of these methods open the horizons for development of commercial biosensors for early-diagnosis of Dengue virus infection.
Graphical abstract
Transmission of Dengue virus by the biting of an
Aedes aegypti
mosquito, the symptoms of Dengue hemorrhagic fever and the structure of Dengue virus and application of biosensors for its detection.
A uniquemulticomponent vaccine against serogroup B meningococci incorporates the novel genome-derived proteins fHbp, NHBA, and NadA that may vary in sequence and level of expression. Measuring the ...effectiveness of such vaccines, using the accepted correlate of protection against invasive meningococcal disease, could require performing the serum bactericidal assay (SBA) against many diverse strains for each geographic region. This approach is impractical, especially for infants, where serum volumes are very limited. To address this, we developed the meningococcal antigen typing system (MATS) by combining a unique vaccine antigen-specific ELISA, which detects qualitative and quantitative differences in antigens, with PorA genotyping information. The ELISA correlates with killing of strains by SBA and measures both immunologic cross-reactivity and quantity of the antigens NHBA, NadA, and fHbp. We found that strains exceeding a threshold value in the ELISA for any of the three vaccine antigens had ≥80% probability of being killed by immune serum in the SBA. Strains positive for two or more antigens had a 96% probability of being killed. Inclusion of multiple different antigens in the vaccine improves breadth of coverage and prevents loss of coverage if oneantigen mutates or is lost. The finding that a simple and high-throughput assay correlates with bactericidal activity is a milestone in meningococcal vaccine development. This assay allows typing of large panels of strains and prediction of coverage of protein-based meningococcal vaccines. Similar assays may be used for protein-based vaccines against other bacteria.
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