Enzyme-linked Immunosorbent Assay For Detection Research Articles

Development of a blocking ELISA for detection of Japanese encephalitis virus antibodies in pig and horse sera

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA.

Straightforward high-temperature antibody immobilization for rapid and simple microfluidic paper-based ELISA for detection of β’-component (Onc k 5) protein, a major IgE-binding protein in salmon roe

Detection of allergenic proteins in food is required for maintaining the health of persons with food allergies. Enzyme-linked immunosorbent assay (ELISA) is a common technique for the detection of proteins. However, conventional ELISA is expensive, time-consuming, and needs technical expertise. Here, a microfluidic paper-based analytical device (µPAD) combined with a sandwich ELISA technique is developed for rapid, simple, and sensitive colorimetric detection of β’-component protein, a major allergen in salmon roe. The developed µPAD-ELISA employs a capture antibody and enzyme-labeled detection antibody. In addition to the µPAD, a technique for immobilization of the capture antibody has been developed, which completes the immobilization within 5 min by drying the antibody solution on a paper substrate in an oven at 50 °C without any chemical modification. The immobilized antibody exhibited higher binding capability to an antigen and stability against washing processes than those immobilized at room temperature. Since this technique allows simple and rapid preparation of the ELISA in the µPAD, the present µPAD-ELISA completed the analysis within only 15 min, including the immobilization step, compared to a conventional ELISA, which takes about 19 h. This µPAD-ELISA offered a good limit of detection of 1.59 ng mL−1 and reduced the amounts of reagents by a factor of about 17–67, which decreases the analysis cost. This µPAD-ELISA approach employing the present antibody immobilization is expected to be an effective strategy for the detection of allergenic proteins in food.

Toward the point-of-care testing of alkaline phosphatase in human serum

This work addresses challenges of simple but reliable determination of enzyme alkaline phosphatase (ALP) in μL-sized serum samples. The single-tube assay and plate-based immunoassay were developed by using a commercial glucose test strip (GTS) to transduce analytical signal from the ALP + glucose-6-phosphate (G6P) reaction. The glucose released from G6P contributed to the flow of anodic charge Q20s through GTS. The interferences from serum matrix including those from native glucose were eliminated by devising a signal ΔQ20s = Q20s (total) – Q20s (matrix). The ΔQ20s was proportional to ALP activity within a linear range of 7.4–720 IU L−1 for assay (R2, 0.995) and 10–300 IU L−1 for immunoassay (R2, 0.983), which far exceeded a normal clinical range for ALP in adult human serum (30–150 IU L−1). Both methods required no auxiliary enzymes or labels, and were accurate (93–99 % signal recovery) and precise (RSD ≤10%). The assay was a simpler option because it required only one 20-min incubation step to determine ALP. The immunoassay involved three steps but it was still less laborious (by ∼70%) than a traditional enzyme-linked immunosorbent assay (ELISA) of ALP due to the substitution of ELISA detection antibody with GTS. The GTS-G6P-based methods bring ALP testing closer to the point-of-care locations, which is important considering the growing role of ALP as a prophylactic, therapeutic, and anti-inflammatory agent, and a marker of human diseases.

Comparison of in-house enzyme-linked immunosorbent assay (ELISA) and six commercial ELISAs for Detection of antibodies to Bordetella pertussis

Enzyme-linked immunosorbent assays (ELISA) are currently the method of choice for the serodiagnosis of pertussis and play a key role in the diagnosis of pertussis in adolescents and adults, as well as in epidemiological studies. In the present study, the in-house developed indirect ELISA was comparatively evaluated with six commercial kits from various manufacturers. Antipertussis antibodies were measured in 40 serum samples from patients with clinical symptoms of respiratory tract infection, in two WHO standards, and in seven human ECDC control sera. IgA and IgG antibodies were detected at a diagnostically significant level by different ELISA kits of 5.0% to 27.0% and 12.0% to 70.0% of patients' sera, appropriately. The analysis of results carried out with six commercial kits showed only 17.5% consistent results in class IgG (either clearly positive or negative). The average percentage of errors in the level of antibodies determined in the control samples, reference serum samples, differed quite significantly and ranged from 9.5% to 35.4% depending on the kit. This poor correlation of the results obtained on various serological tests intended for the serodiagnosis of pertussis may cause very serious diagnostic problems, especially when examining a serum sample obtained once during the course of the disease.

A plug-and-play, easy-to-manufacture fluidic accessory to significantly enhance the sensitivity of electrochemical immunoassays

Earlier access to patients’ biomarker status could transform disease management. However, gold-standard techniques such as enzyme-linked immunosorbent assays (ELISAs) are typically not deployed at the point-of-care due to their cumbersome instrumentation and complexity. Electrochemical immunosensors can be disruptive in this sector with their small size and lower cost but, without further modifications, the performance of these sensors in complex media (e.g., blood) has been limited. This paper presents a low-cost fluidic accessory fabricated using widely accessible materials and processes for boosting sensor sensitivity through confinement of the detection media next to the electrode surface. Liquid confinement first highlighted a spontaneous reaction between the pseudoreference electrode and ELISA detection substrate 3,3’,5,5’-tetramethylbenzidine (TMB) that decreases the amount of oxTMB available for detection. Different strategies are investigated to limit this and maximize reliability. Next, flow cell integration during the signal amplification step of sensor preparation was shown to substantially enhance the detection of cytokine interleukin-6 (IL-6) with the best sensitivity boost recorded for fresh human plasma (x7 increase compared to x5.8 in purified serum and x5.5 in PBS). The flow cell requires no specialized equipment and can be seamlessly integrated with commercial sensors, making an ideal companion for electrochemical signal enhancement.

Rapid Microfluidic Immuno-Biosensor Detection System for the Point-of-Care Determination of High-Sensitivity Urinary C-Reactive Protein.

A microfluidic immuno-biosensor detection system consisting of a microfluidic spectrum chip and a micro-spectrometer detection device is presented for the rapid point-of-care (POC) detection and quantification of high-sensitivity C-reactive protein (hs-CRP) in urine. The detection process utilizes a highly specific enzyme-linked immunosorbent assay (ELISA) method, in which capture antibodies and detection antibodies are pre-deposited on the substrate of the microchip and used to form an immune complex with the target antigen. Horseradish peroxidase (HRP) is added as a marker enzyme, followed by a colorimetric reaction using 3,3',5,5'-tetramethylbenzidine (TMB). The absorbance values (a.u.) of the colorimetric reaction compounds are measured using a micro-spectrometer device and used to measure the corresponding hs-CRP concentration according to the pre-established calibration curve. It is shown that the hs-CRP concentration can be determined within 50 min. In addition, the system achieves recovery rates of 93.8-106.2% in blind water samples and 94.5-104.6% in artificial urine. The results showed that the CRP detection results of 41 urine samples from patients with chronic kidney disease (CKD) were highly consistent with the conventional homogeneous particle-enhanced turbidimetric immunoassay (PETIA) method's detection results (R2 = 0.9910). The experimental results showed its applicability in the detection of CRP in both urine and serum. Overall, the results indicate that the current microfluidic ELISA detection system provides an accurate and reliable method for monitoring the hs-CRP concentration in point-of-care applications.

Protective Effect of Shenfu Injection against Sepsis-induced Acute Lung Injury by Suppressing Inflammation and Apoptosis Through the Regulation of the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway

Abstract Objective: Sepsis-induced acute lung injury (ALI) is a clinically critical condition with a high mortality rate. Shenfu injection (SFI) is a Chinese herbal medicine extracted from red ginseng and Aconite, Radix Aconiti, with various pharmacological activities. This study aimed to investigate the potential mechanism of action of SFI in preventing sepsis-induced ALI. Materials and Methods: We established a mouse model of sepsis-induced ALI by cecal ligation and puncture (CLP). The mice were randomly divided into three groups (n = 8): Sham, CLP, and SFI (10 mL/kg). Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for pathological analysis, enzyme-linked immunosorbent assay, immunohistochemistry (IHC), and protein detection. Results: Our results showed that SFI significantly ameliorated pathological damage caused by CLP-induced ALI. SFI treatment significantly decreased the lung wet-to-dry weight ratio. In addition, SFI treatment significantly reduced the protein levels and cell numbers in the BALF. SFI could significantly reduce the levels of tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1β in plasma and BALF. SFI significantly reduced the protein expression of Bax and cleaved caspase-3 and increased the protein levels of Bcl-2. Western blotting and IHC results showed that SFI reduced the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Conclusions: In a septic ALI mouse model, SFI inhibited apoptosis and inflammation through the JAK2/STAT3 pathway, providing a candidate drug for the treatment of septic ALI.

Immunochromatography Lateral Flow Strip Enhancement Based on Passive Gold Nanoparticles Conjugation to Detect Schistosma haematobium Antigens in Human Serum

PurposeThis study aimed to develop and evaluate a lateral flow card for the detection of active Schistosoma haematobium infection.MethodsIn order to prepare the immunochromatography lateral flow strip (ICLFS), antibodies purified from schistosomiasis were conjugated passively with gold nanoparticles using a potassium carbonate buffer.ResultsThe novel ICLFS was able to correctly identify 64 out of 67 samples of schistosomiasis, 6 out of 90 samples of other parasites, and 0 out of 27 control samples. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were 95.5%, 93.3%, 90%, and 91.4% respectively. Comparatively, the sensitivity, specificity, NPV, and PPV of sandwich enzyme-linked immunosorbent assays (ELISA) conjugated with gold nanoparticles (AuNPs) were 91.1%, 88.8%, 85.9%, and 84.4% respectively. The increased sensitivity and specificity of ICLFS produced superior results to those of sandwich ELISA.ConclusionIn conclusion, ICLFS is more beneficial and precise than sandwich ELISA for detection of S. haematobium infection at early stage.

4-Oxo-2-Nonenal- and Agitation-Induced Aggregates of α-Synuclein and Phosphorylated α-Synuclein with Distinct Biophysical Properties and Biomedical Applications.

α-Synuclein (α-syn) can form oligomers, protofibrils, and fibrils, which are associated with the pathogenesis of Parkinson's disease and other synucleinopathies. Both the lipid peroxidation product 4-oxo-2-nonenal (ONE) and agitation can induce aggregation of α-syn and phosphorylated α-syn. Thus, clarification of the characteristics of different α-syn species could help to select suitable aggregates for diagnosis and elucidate the pathogenesis of diseases. Here, we characterized ONE-induced wild-type (WT) α-syn aggregates (OW), ONE-induced phosphorylated α-syn (p-α-syn) aggregates (OP), agitation-induced α-syn preformed fibrils (PFF), and agitation-induced p-α-syn preformed fibrils (pPFF). Thioflavin T (ThT) dying demonstrated that OW and OP had fewer fibrils than the PFF and pPFF. Transmission electron microscopy revealed that the lengths of PFF and pPFF were similar, but the diameters differed. OW and OP had more compact structures than PFF and pPFF. Aggregation of p-α-syn was significantly faster than WT α-syn. Furthermore, OW and OP were more sodium dodecyl sulfate-stable and proteinase K-resistant, suggesting greater stability and compactness, while aggregates of PFF and pPFF were more sensitive to proteinase K treatment. Both ONE- and agitation-induced aggregates were cytotoxic when added exogenously to SH-SY5Y cells with increasing incubation times, but the agitation-induced aggregates caused cell toxicity in a shorter time and more p-α-syn inclusions. Similarly, p-proteins were more cytotoxic than non-p-proteins. Finally, all four aggregates were used as standard antigens to establish sandwich enzyme-linked immunosorbent assay (ELISA). The results showed that the recognition efficiency of OW and OP was more sensitive than that of PFF and pPFF. The OW- and OP-specific ELISA for detection of p-α-syn and α-syn in plasma samples of Thy1-α-syn transgenic mice showed that the content of aggregates could reflect the extent of disease. ONE and agitation induced the formation of α-syn aggregates with distinct biophysical properties and biomedical applications.

Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena

BackgroundAntigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA.MethodsWe report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland–Altman (BA) analysis and Lin’s concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA.ResultsThe TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin’s CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin’s CCC: 0.24) but increased on days 28 and 90 PI (Lins’ CCC: 0.88 and 0.98, P < 0.001).Conclusions/significanceThe TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.Graphical

Efficacy of Sailuotong on neurovascular unit in amyloid precursor protein/presenilin-1 transgenic mice with Alzheimer's disease.

To discuss the influence of Sailuotong (, SLT) on the Neurovascular Unit (NVUs) of amyloid precursor protein (APP)/presenilin-1(PS1) mice and evaluate the role of gas supplementation in activating blood circulation during the progression of Alzheimer's disease (AD). The mice were allocated into the following nine groups: (a) the C57 Black (C57BL) sham-operated group (control group), (b) ischaemic treatment in C57BL mice (the C57 ischaemic group), (c) the APP/PS1 sham surgery group (APP/PS1 model group), (d) ischaemic treatment in APP/PS1 mice (APP/PS1 ischaemic group), (e) C57BL mice treated with aspirin following ischaemic treatment (C57BL ischaemic + aspirin group), (f) C57BL mice treated with SLT following ischaemic treatment (C57BL ischaemic + SLT group), (g) APP/PS1 mice treated with SLT (APP/PS1 + SLT group), (h) APP/PS1 mice treated with donepezil hydrochloride following ischaemic treatment (APP/PS1 ischaemic + donepezil hydrochloride group) and (i) APP/PS1 mice treated with SLT following ischaemic treatment (APP/PS1 ischaemic + SLT group). The ischaemic model was established by operating on the bilateral common carotid arteries and creating a microembolism. The Morris water maze and step-down tests were used to detect the spatial behaviour and memory ability of mice. The hippocampus of each mouse was observed by haematoxylin and eosin (HE) and Congo red staining. The ultrastructure of NVUs in each group was observed by electron microscopy, and various biochemical indicators were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression level was detected by Western blot. The mRNA expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The results of the Morris water maze and step-down tests showed that ischemia reduced learning and memory in the mice, which were restored by SLT. The results of HE staining showed that SLT restored the pathological changes of the NVUs. The Congo red staining results revealed that SLT also improved the scattered orange-red sediments in the upper cortex and hippocampus of the APP/PS1 and APP/PS1 ischaemic mice. Furthermore, SLT significantly reduced the content of Aβ, improved the vascular endothelium and repaired the mitochondrial structures. The ELISA detection, western blot detection and qRT-PCR showed that SLT significantly increased the vascular endothelial growth factor (VEGF), angiopoietin and basic fibroblast growth factor, as well as the levels of gene and protein expression of low-density lipoprotein receptor-related protein-1 (LRP-1) and VEGF in brain tissue. By increasing the expression of VEGF, SLT can promote vascular proliferation, up-regulate the expression of LRP-1, promote the clearance of Aβ and improve the cognitive impairment of APP/PS1 mice. These results confirm that SLT can improve AD by promoting vascular proliferation and Aβ clearance to protect the function of NVUs.

Evaluation of a Commercial Serum Competitive Enzyme-Linked Immunosorbent Assay for Detection of Neospora caninum-Specific Antibodies in Raw Milk of Ruminants

Bovine neosporosis is an infection caused by the protozoan parasite Neospora caninum and has substantial veterinary hazards. Neosporosis cannot be controlled by vaccination or chemotherapy. Thus, accurate diagnosis followed by isolation and culling of infected animals is regarded as the most efficient method of control. In vivo diagnosis often relies on serologic testing of the animals, and milk represents a non-invasive and easy-to-collect sample matrix. However, indirect enzyme-linked immunosorbent assay (ELISA) specifically designed for antibody detection in milk are sometimes not easily available and it is tempting to use ELISA kits that are originally designed for use in serum in milk samples instead. Herein, we evaluated a widely used commercial ELISA (ID Screen® Neospora caninum competition Multispecies ELISA (ID. Vet, Grabels, France)), developed for detection of N. caninum antibodies in serum samples, for its performance on milk samples. Milk samples from dairy ruminants (cows, buffaloes, sheep, and goats; n = 149) were tested in parallel with the serum ELISA and a commercial milk ELISA as a standard test (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France). The detected prevalence values were 28.2% (42/149), 17.4% (26/149), and 17.4% (26/149) using milk ELISA, serum ELISA, and both ELISAs, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for the serum ELISA used with milk samples were 61.9%, 100%, 100%, and 87%, respectively. The agreement and kappa value between the two ELISAs were 89.3% and 0.70, respectively, suggesting substantial agreement. High values of Pearson correlation coefficient (0.904, p ≥ 0.0001) and area under the receiver operating characteristic (ROC) curve (0.789, p ≥ 0.0001) demonstrated the high diagnostic performance of the serum ELISA in milk samples. Also, a Bland–Altman Plot and histogram describing the frequency of distribution of ELISA optical densities confirmed the high agreement of both serum and milk ELISAs. The current results revealed the high specificity but moderate sensitivity of the serum ELISA used for milk samples compared with the milk ELISA. However, the excellent positive predictive value of the serum ELISA makes it an alternative option in case of the unavailability of milk ELISAs. With this study, we provided additional evidence that a widely used serum ELISA test kit may also be used for the detection of N. caninum antibodies in milk samples.

The utilization of ELISA in early detection of cancer

The early detection of cancer is crucial in improving treatment success rates and survival rates. The primary focus of this research is the application of enzyme-linked immunosorbent assay (ELISA) in early cancer detection, as well as the measurement methods of cancer biomarkers. It can provide an overview of the currently used cancer biomarkers and their application in ELISA. Furthermore, this research will utilize the ELISA technique to determine whether a specific substance can serve as a cancer biomarker, with a particular emphasis on its application in early cancer detection. Through a comprehensive analysis of relevant research, this research elucidates the significance of ELISA in the measurement of cancer biomarkers and its potential as a robust cancer screening tool. Finally, this research will highlight the future directions of research, focusing on the implementation of ELISA technology in detecting and identifying a wider range of cancer biomarkers for achieving more accurate early cancer diagnosis.

AQP4-DARPin1: A Chimeric Antigen Based on Scaffold Protein DARPin for Efficient Detection of AQP4-IgG in NMOSD.

AQP4-IgG is an autoantibody associated with neuromyelitis optica spectroscopic disorder (NMOSD), a central nervous system inflammatory disease that requires early diagnosis and treatment. We designed two fusion proteins, AQP4-DARPin1 and AQP4-DARPin2, comprising the complete antigenic epitopes of aquaporin-4 (AQP4) and the constant region of the scaffold protein DARPin. These fusion proteins were expressed and purified from Escherichia coli and coated on microplates to develop an efficient method for detecting AQP4-IgG. Molecular dynamics simulation revealed that the fusion of AQP4 extracellular epitopes with DARPin did not alter the main structure of DARPin. The purified AQP4-DARPins bound recombinant antibody rAb-53 (AQP4-IgG) with affinities of 135 and 285 nM, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation demonstrated that AQP4-DARPin1 specifically recognized AQP4-IgG in the NMOSD patient serum. AQP4-DARPin1 as a coated antigen showed higher ELISA signal and end point dilution ratio than full-length AQP4. Our AQP4-DARPin1-coated AQP4-IgG ELISA had 100% specificity and 90% sensitivity. These results indicate that AQP4-DARPin1, compared to existing detection strategies that use full-length or extracellular loop peptides of AQP4, provides a new and more effective approach to the ELISA detection of NMOSD.

Anti-HBsAg IgY polyclonal antibodies potential as capture antibody for HBsAg Detection Kit development

Hepatitis B, affecting about 296 million globally, is a significant concern, with Indonesia ranking second in Southeast Asia for case numbers. The disease's latent initial phase, devoid of early symptoms upon hepatitis B virus (HBV) infection, highlights the demand for precise diagnostics. This research aimed to develop anti-HBsAg polyclonal antibodies (pAb) for application as capture agents within a sandwich enzyme-linked immunosorbent assay (ELISA). Chicken egg-derived IgY antibodies have advantages over mammalian ones due to simpler extraction and higher yield. In a study involving 21-week-old chickens, four intramuscular injections of 500 µg HBsAg antigen in Freund's adjuvant were administered at two-week intervals. Eggs as IgY source were collected daily and then IgY was isolated from eggyolk using polyethylene glycol (PEG) precipitation. The Bradford method was used to measure the total protein concentration, while the existence of IgY and pAb specific IgY against Anti-HBsAg was verified through SDS-PAGE and sandwich ELISA using HRP as a reporter, respectively. The resulting SDS-PAGE showed two distinct IgY bands: a 68 kDa heavy chain and a 23 kDa light chain. Using these anti-HBsAg IgY antibodies as capturing agents, the slightly elevation of IgY pAb against HBsAg level has been identified within the second week following the initial immunization. Subsequently, from the third to the eighth week, antibody levels escalated significantly, ranging from 2 to 13-fold higher than those observed in the second week. These findings suggest the potential use of IgY pAb as effective capture antibodies in sandwich ELISA for HBsAg antigen detection.

Evaluating cross-reactivity of new psychoactive substances (NPS) in human whole blood by enzyme-linked immunosorbent assay (ELISA).

Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies to detect NPS in biological matrices. Enzyme-linked immunosorbent assays (ELISA) are among the most popular screening methods. To evaluate the effectiveness of ELISA for NPS detection, five subclasses of NPS (novel synthetic opioids, fentanyl analogs, stimulants, benzodiazepines and hallucinogens) were evaluated in whole blood for their cross-reactivity on commercially available ELISA kits. A variety of novel synthetic opioids were tested at concentrations of 1-80 ng/mL and 50-2000 ng/mL and demonstrated no cross-reactivity to a morphine ELISA plate at either concentration range. Fentanyl analogs were tested at concentrations ranging from 0.01 to 1 ng/mL and had cross-reactivities ranging from 8% to 178% on the fentanyl ELISA kit used. Both para-chloro fentanyl (178%) and acryl fentanyl (164%) showed cross-reactivities well above that of fentanyl. Novel stimulants were tested at concentrations of 0.5-40 ng/mL and 20-2,000 ng/mL. 4-Fluoroamphetamine was the only novel stimulant with cross-reactivity (3,354%) to the amphetamine ELISA plate. Novel benzodiazepines were tested at concentrations of 1-40 ng/mL on a benzodiazepine plate. Cross-reactivities ranged from 36.1% to 263%, with desalkylflurazepam having the highest cross-reactivity. Finally, novel hallucinogens were tested at concentrations of 0.5-10 ng/mL on a phencyclidine (PCP) ELISA plate, which produced no cross-reactivity and then with 10-1,000 ng/mL, which gave results from 56.6% to 151%. Both hydroxy-PCP (151%) and chloro-PCP (137%) showed cross-reactivities above that of PCP. This research has demonstrated the utility of using ELISA-based screening for novel benzodiazepines, hallucinogens and for fentanyl analogs; however, there is limited application and risk of false-negative results for the other drug classes due to low or non-existent cross-reactivities.

Cell-based vs enzyme-linked immunosorbent assay for detection of anti-Tribbles homolog 2 autoantibodies in Chinese patients with narcolepsy.

Narcolepsy type 1 is attributed to a deficiency in cerebrospinal fluid orexin and is considered linked to autoimmunity. The levels of anti-Tribbles homolog 2 (TRIB2) autoantibodies are elevated in the sera of some patients with narcolepsy with cataplexy. Additionally, injecting mice with serum immunoglobulin from patients with narcolepsy with positive anti-TRIB2 antibodies can induce hypothalamic neuron loss and alterations in sleep patterns. Consequently, we hypothesized the existence of a potential association between anti-TRIB2 antibodies and narcolepsy. To test this possibility, we used cell-based assays (CBAs) and enzyme-linked immunosorbent assays (ELISAs) to detect the presence of anti-TRIB2 antibodies in Chinese patients with narcolepsy. We included 68 patients with narcolepsy type 1, 39 patients with other central disorders of hypersomnolence, and 43 healthy controls. A CBA and a conventional ELISA were used to detect anti-TRIB2 antibody levels in patients' sera. CBA was used to detect serum anti-TRIB2 antibodies in Chinese patients with narcolepsy, and the results were negative. However, when the ELISA was used, only 2 patients with narcolepsy type 1 had TRIB2 antibody titers higher than the mean titer plus 2 standard deviations of the healthy controls. In our study, ELISA identified TRIB2 autoantibodies in sera of patients with narcolepsy where CBA failed to demonstrate them. Contrary to our hypothesis, this intriguing finding deserves further research to elucidate the potential association between TRIB2 and narcolepsy type 1. Exploring the implications of TRIB2 autoantibodies in narcolepsy and disparate outcomes between ELISA and CBA could provide crucial insights. Zhong X, Yuan Y, Zhan Q, etal. Cell-based vs enzyme-linked immunosorbent assay for detection of anti-Tribbles homolog 2 autoantibodies in Chinese patients with narcolepsy. J Clin Sleep Med. 2024;20(6):941-946.

Have we attended herd immunity against COVID? A cross-sectional analytical study of seroprevalence among blood donors

Abstract BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID-19) is one of the worst health-care problem humankind has faced in the last few centuries. Understanding the population’s antibody responses is crucial for effective decision-making while implementing public health interventions in view of the COVID-19 pandemic. The study was done to know the immune status of healthy donors to COVID-19 infections and to know the duration of persistence of immunoglobulin G (IgG) antibodies with respect to infections and vaccination. AIMS: This study aimed to determine the prevalence of SARS-CoV-2 IgG antibodies in healthy blood donors in our tertiary care center. MATERIALS AND METHODS: This was a cross-sectional analytical study conducted for a period of 1 year in our blood center. Our study had a sample size of 809 healthy blood donors. Samples of the first 81 donors of each month were chosen. Donor details such as previous COVID positivity, symptoms, hospitalization, comorbidities, contact history, and vaccination status were collected using a structured questionnaire. A 5-ml sample was collected and was tested using Qualisa COVID-19 IgG enzyme-linked immunosorbent assay detection kits. The antigens used for coating the kits were recombinant antigens representing SARS-CoV-2 S1 protein. Data were analyzed using SPSS version 21. The prevalence of positive IgG seroprevalence of COVID-19 antibodies was reported using proportion along with 95% confidence interval. Association between all the categorical variables and the presence of seropositive status for IgG antibodies was assessed using the Chi-square test. P &lt;0.05 was considered statistically significant. RESULTS: The seroprevalence of COVID-19 antibodies in our study participants was 82.3%. There is a significant association between COVID-19 infectivity, vaccination, with seroprevalence. There was a seropositivity of 51% among those who were neither infected nor vaccinated, which may be due to the development of herd immunity in our population. CONCLUSIONS: Population who were neither vaccinated nor had a history of infection also had a significant prevalence of antibodies suggesting the development of herd immunity in our community. The present study will add to the knowledge and better understanding of COVID-19 antibodies in the existing literature. Further studies will be required to verify our findings and to know the persistent nature of COVID antibodies.

Circ-phkb promotes cell apoptosis and inflammation in LPS-induced alveolar macrophages via the TLR4/MyD88/NF-kB/CCL2 axis

BackgroundCircular RNAs (CircRNAs) have been associated with acute lung injury (ALI), but their molecular mechanisms remain unclear.MethodsThis study developed a rat model of lipopolysaccharide (LPS)-induced ALI and evaluated the modeling effect by hematoxylin and eosin staining, Masson’s trichrome staining, lung wet-to-dry weight ratio, terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA) detection of inflammatory factors (interleukin-1β, tumor necrosis factor alpha, and interleukin-6). Using lung tissues from a rat model of LPS-induced ALI, we then conducted circRNA sequencing, mRNA sequencing, and bioinformatics analysis to obtain differential circRNA and mRNA expression profiles as well as potential ceRNA networks. Furthermore, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays to screen for circ-Phkb in ALI rat lung tissues, alveolar macrophages, and LPS-induced NR8383 cells. We conducted induction with or without LPS with circ-Phkb siRNA and overexpression lentivirus in NR8383. Cell Counting Kit-8, C5-Ethynyl-2′-deoxyuridine (Edu), TUNEL, and cytometry were used to identify proliferation and apoptosis, respectively. We detected inflammatory factors using ELISA. Finally, we used Western blot to detect the apoptosis-related proteins and TLR4/MyD88/NF-kB/CCL2 pathway activation.ResultsOur results revealed that both circRNA and mRNA profiles are different from those of the Sham group. We observed a significant circ-Phkb upregulation in NR8383 cells and LPS-exposed rats. Apoptosis and inflammation were greatly reduced when circ-Phkb expression was reduced in NR8383 cells, cell proliferation was increased, and circ-Phkb overexpression was decreased.ConclusionsIn terms of mechanism, circ-Phkb suppression inhibits CCL2 expression via the TLR4/MyD88/NF-kB pathway in LPS-induced alveolar macrophages.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.