Serum Enzyme-linked Immunosorbent Assay Research Articles

The role of Ca2+ signalling and InsP3R in the pathogenesis of intrahepatic cholestasis of pregnancy

Background In order to contribute new insights for future prevention and treatment of intrahepatic cholestasis of pregnancy (ICP), and to promote positive pregnancy outcomes, we evaluated serum Ca2+ levels and inositol 1,4,5-trisphosphate receptor (InsP3R) expression in the liver tissue of a rat ICP model. Methods After establishing the model by injection of oestradiol benzoate and progesterone into pregnant rats, animals were divided into normal control (n = 5) and ICP model groups (n = 5). The expression of InsP3R protein in the liver, and serum levels of Ca2+, glycocholic acid and bile acid were detected. Results InsP3R mRNA and protein were significantly lower in the ICP model group compared to the normal group, as determined by qPCR and immunohistochemistry, respectively. Serum enzyme-linked immunosorbent assay results revealed significantly higher levels of glycocholic acid and bile acid in the ICP model group compared to the normal group, while Ca2+ levels were significantly lower. The levers of Ca2+ were significantly and negatively correlated with the levels of glycocholic acid. The observed decrease in Ca2+ was associated with an increase in total bile acids, but there was no significant correlation. Conclusions Our results revealed that the expression of InsP3R and serum Ca2+ levels was significantly decreased in the liver tissue of ICP model rats. Additionally, Ca2+ levels were found to be negatively correlated with the level of glycocholic acid.

Quercetin alleviates chronic unpredictable mild stress-induced depression-like behavior by inhibiting NMDAR1 with α2δ-1 in rats.

Depression is a serious mental disorder and the most prevalent cause of disability and suicide worldwide. Chronic unpredictable mild stress (CUMS) can lead to a significant acceleration of depression development. Quercetin (Que) is a flavonoid compound with a wide range of pharmacological effects. Recent studies have shown that quercetin can improve CUMS-induced depression-like behavior, but the mechanism of its improvement is still unclear. α2δ-1 is a regulatory subunit of voltage-gated calcium channel, which can interact with N-methyl-D-aspartate receptor (NMDAR) to form a complex. In this study, we found that Que could inhibit the increase of α2δ-1 and NMDAR expression in rat hypothalamus induced by CUMS. In pain, chronic hypertension and other studies have shown that α2δ-1 interacts with the NMDAR to form a complex, which subsequently affects the expression level of NMDAR. Consequently, the present study aimed to investigate the antidepressant effect of Que invivo and invitro and to explore its mechanism of action in terms of the interaction between α2δ-1 and NMDAR. Rats were randomly exposed to two stressors every day for 4 weeks to establish a CUMS rat model, then sucrose preference test (SPT), forced swimming test (FST), tail suspension test (TST), and open field test (OFT) were performed to detect the behavior of CUMS rats, so as to evaluate whether the CUMS rat model was successfully established and the improvement effect of Que on CUMS-induced depression-like behavior in rats. Experimental techniques such as serum enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blot, and co-immunoprecipitation, as well as invitro experiments, were used to investigate the mechanisms by which Que exerts its antidepressant effects. Behavioral and ELISA test results showed that Que could produce a reduction in the excitability of the hypothalamic-pituitary-adrenal (HPA) axis in CUMS rats and lead to significant improvements in their depressive behavior. Western blot, immunofluorescence, and co-immunoprecipitation experiments showed that Que produced a decrease in NMDAR1 and α2δ-1 expression levels and interfered with α2δ-1 and NMDAR1 binding. In addition, the neural regulation mechanism of Que on antidepressant effect in PC12 cells knocked out α2δ-1 gene was further verified. Cellular experiments demonstrated that Que led to a reversal of up-regulation of NMDAR1 and α2δ-1 expression levels in corticosterone-injured PC12 cells, while Que had no effects on NMDAR1 expression in PC12 cells with the α2δ-1 gene knockout. Que has a good antidepressant effect and can significantly improve the depression-like behavior caused by CUMS. It exerts antidepressant effects by inhibiting the expression level of α2δ-1, interfering with the interaction between α2δ-1 and NMDAR, and then reducing the excitability of the HPA axis.

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.

Evaluation of influence of Butea monosperma floral extract on inflammatory biomarkers

Abstract Butea monosperma is a deciduous tree, widely distributed throughout India, Burma, and Ceylon. The present study was intended to investigate the anti-inflammatory effects of Butea monosperma on the induced inflammatory model by evaluating pro-inflammatory biomarkers and their computational analysis. The anti-inflammatory activity may be attributed to the phyto-constituents for inhibitory effects on the two pro-inflammatory mediators (IL-8 and TNF-α). For this purpose, rats (n = 48) were equally divided in each group, i.e., 8 each in the negative and positive control and 32 in the experimental group with 8 rats for each dose, i.e., 50, 100, 200, and 400 mg/kg. TNF-α and IL-8 were tested by serum enzyme-linked immunosorbent assay (ELISA). The ELISA results showed 400 mg/kg dose as the potent anti-inflammatory. The binding sites of target proteins (TNF-α and IL-8) were docked with the active compounds (butrin and butein) of Butea monosperma. The butrin (target: TNF-α) and butein (target: IL-8) showed −8.4 and −6.0 kcal/mol binding energies, respectively, compared to the (diclofenac) standard drug with −6.8 kcal/mol binding energy. Hence, we concluded that Butea monosperma can be subjected to as a useful anti-inflammatory drug.

Screening of heat stress-related biomarkers in chicken serum through label-free quantitative proteomics

Heat stress (HS) can result in sudden death and is one of the most stressful and costly events in chicken. Currently, biomarkers used clinically to detect heat stress state in chickens are not optimal, especially for living ones. Analysis of changes in serum proteins of heat-stressed chickens can help to identify some novel convenient biomarkers for this. Twenty-four chickens were exposed to HS at 42°C ± 1°C with a relative humidity of 65% for continuous 5 h in a single day, and 10 birds were used as controls (Con). During HS, 15 dead chickens were categorized as heat stress death group (HSD), and 9 surviving ones served as heat stress survivor group (HSS). Label-free quantitative proteomics (LFQP) was used to analyze differentially expressed proteins (DEPs) in serum of tested animals. Candidate proteins associated with HS were validated by enzyme-linked immunosorbent assay (ELISA). Diagnostic value of candidate biomarkers was assessed using receiver operating characteristic (ROC) curve analysis. Source of the selected proteins was analyzed in liver tissues with immunohistochemistry and in cell culture supernatant of primary chicken hepatocytes (PCH) using ELISA. In this study, compared to Con, LFQP identified 123 and 53 significantly different serum proteins in HSD and HSS, respectively. Bioinformatics analysis showed that XDH, POSTN, and HSP90 were potential HS biomarkers in tested chickens, which was similar with results from serum ELISAs and immunohistochemistry in liver tissues. The ROC values of 0.793, 0.752, and 0.779 for XDH, POSTN, and HSP90, respectively, permitted the distinction of heat-stressed chickens from the control. Levels of 3 proteins above in the cell culture supernatant of PCH showed an increasing trend as HS time increased. Therefore, considering that mean concentration of POSTN in serum was higher than that of HSP90, XDH, and POSTN may be optimal biomarkers in serum for detecting HS level in chickens, and mainly secreted from hepatocytes. The former indicates that heat-stressed chickens are in a damaged state, and the latter implies that chickens can repair heat stress damage.

Study the technical validity and feasibility of serum HER-2/Neu and its correlation with tissue HER-2 status in female breast cancer patients only

Background: Breast cancer is one of the leading causes of death in women worldwide. The future burden of breast cancer is predicted to increase over 3 million new cases and 1 million deaths in 2040. In India, it is being increasingly diagnosed at a younger age as compared to the west. Human epidermal growth factor receptor 2 (HER-2) transmembrane protein, with a molecular weight 185 kDa, plays a key role in cell growth and its overexpression favors cell proliferation by inhibiting apoptosis. HER-2 extracellular domain, released into blood before appearing into tissue, can be easily detected by enzyme-linked immunosorbent assay (ELISA) using serum. HER-2 has many roles in assisting diagnosis, targeted therapy, and evaluating prognosis. Aims and Objectives: The study aimed to monitor the serum HER-2 level and its correlation with tissue HER-2 status in breast cancer patients. Materials and Methods: We performed a prospective study on technical validity and feasibility of serum HER-2 level by ELISA in breast cancer patients and its correlation with tissue HER-2 status by immunohistochemistry (IHC) method. Results: We found the serum ELISA level was raised and within the reference range in all breast cancer patients. A higher level of serum HER-2 level was seen in high tumor stage, high Nottingham grade, and with axillary lymph node metastasis. Serum HER-2 level was significantly higher in IHC score 3+ as compared to score 2+and 1+. Conclusion: This study concluded that serum HER-2 ELISA is a valid and feasible test. Association of serum HER-2 level with tumor stage; Nottingham grade; and axillary nodal metastasis was significant. The correlation between serum HER-2 and tissue HER-2 was found significant. Thus, serum HER-2 may be used as an early diagnostic and prognostic biomarker to guide in clinical practice.

Cerebral sparganosis in a child with corpus callosum invasion: a case report

BackgroundInvasion of the corpus callosum by sparganosis is rare in children. After invading the corpus callosum, sparganosis has various migration modes, which can break through the ependyma and enter the ventricles, thus causing secondary migratory brain injury.Case presentationA girl aged 4 years and 7 months presented with left lower limb paralysis for more than 50 days. Blood examination showed that the proportion and absolute number of eosinophils in the peripheral blood were increased. Furthermore, enzyme-linked immunosorbent assay of serum and cerebrospinal fluid samples revealed positivity for IgG and IgM antibodies for sparganosis. Initial magnetic resonance imaging (MRI) revealed ring-like enhancements in the right frontoparietal cortex, subcortical white matter, and splenium of the corpus callosum. Within 2 months, a fourth follow-up MRI showed that the lesion had spread to the left parietal cortex, subcortical white matter, and deep white matter in the right occipital lobe and right ventricular choroid plexus, with left parietal leptomeningeal enhancement.ConclusionMigratory movement is one of the characteristics of cerebral sparganosis. When sparganosis invades the corpus callosum, clinicians should be aware that it may then break through the ependyma and enter the lateral ventricles, leading to secondary migratory brain injury. Short-term follow-up MRI is necessary to evaluate the migration mode of sparganosis and dynamically guide treatment strategies.

Preventive effects of quercetin against foot-and-mouth disease virus in vitro and in vivo by inducing type I interferon.

Foot-and-mouth disease (FMD) is an acute contagious infectious disease that affects cloven-hoofed animals. Although current emergency FMD vaccines only take effect 7 days after vaccination, antiviral agents, such as quercetin, which is a common flavonoid, could reduce the spread of FMD virus (FMDV) during outbreaks. We investigated the in vitro and in vivo antiviral effects of quercetin against FMDV. Analysis of viral copy numbers showed that quercetin had a dose-dependent inhibitory effect on FMDV at concentrations between 19.5 and 1,250 μM in porcine cells. In addition, we observed a quercetin-induced interferon (IFN)-α protein and interferon-stimulated gene (ISG) upregulation in swine cells. Enzyme-linked immunosorbent assay of sera revealed that quercetin induces the production of IFN-α, IFN-β, IFN-γ, interleukin (IL)-12, and IL-15 in mice. Inoculation of mice with quercetin or a combination of quercetin with an inactivated FMD vaccine enhanced both the survival rate and neutralizing antibody titer. Therefore, we suggest the use of quercetin as a novel and effective antiviral agent for controlling FMDV infection; however, further investigation of its application in livestock is required.

Safety and Immunogenicity of the ID93 +GLA-SE Tuberculosis Vaccine in BCG-Vaccinated Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

This randomized, double-blind, placebo-controlled, phase 2a trial was conducted to evaluate the safety and immunogenicity of the ID93+ glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE) vaccine in human immunodeficiency virus (HIV)-negative, previously Bacillus Calmette-Guérin (BCG)-vaccinated, and QuantiFERON-TB-negative healthy adults in South Korea. Adults (n = 107) with no signs or symptoms of tuberculosis were randomly assigned to receive three intramuscular injections of 2μg ID93 + 5μg GLA-SE, 10μg ID93 + 5μg GLA-SE, or 0.9% normal saline placebo on days 0, 28, and 56. For safety assessment, data on solicited adverse events (AEs), unsolicited AEs, serious AEs (SAEs), and special interest AEs were collected. Antigen-specific antibody responses were measured using serum enzyme-linked immunosorbent assay. T-cell immune responses were measured using enzyme-linked immunospot and intracellular cytokine staining. No SAEs, deaths, or AEs leading to treatment discontinuation were found. The solicited local and systemic AEs observed were consistent with those previously reported. Compared with adults administered with the placebo, those administered with three intramuscular vaccine injections exhibited significantly higher antigen-specific antibody levels and Type 1T-helper cellular immune responses. The ID93 + GLA-SE vaccine induced antigen-specific cellular and humoral immune responses, with an acceptable safety profile in previously healthy, BCG-vaccinated, Mycobacteriumtuberculosis-uninfected adult healthcare workers. This clinical trial was retrospectively registered on 16 January 2019 at Clinicaltrials.gov (NCT03806686).

Higher serum β2-microglobulin is a predictive biomarker for cognitive impairment in spinal cord injury.

Recent studies have suggested that high levels of β2-microglobulin are linked to cognitive deterioration; however, it is unclear how this connects to spinal cord injury (SCI). This study sought to determine whether there was any association between cognitive decline and serum β2-microglobulin levels in patients with SCI. A total of 96 patients with SCI and 56 healthy volunteers were enrolled as study participants. At the time of enrollment, specific baseline data including age, gender, triglycerides (TG), low-density lipoprotein (LDL), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), smoking, and alcohol use were recorded. Each participant was assessed by a qualified physician using the Montreal cognitive assessment (MoCA) scale. Serum β2-microglobulin levels were measured using an enzyme-linked immunosorbent assay (ELISA) reagent for β2-microglobulin. A total of 152 participants were enrolled, with 56 in the control group and 96 in the SCI group. There were no significant baseline data differences between the two groups (p > 0.05). The control group had a MoCA score of 27.4 ± 1.1 and the SCI group had a score of 24.3 ± 1.5, with the difference being significant (p < 0.05). The serum ELISA results revealed that the levels of β2-microglobulin in the SCI group were considerably higher (p < 0.05) than those in the control group (2.08 ± 0.17 g/mL compared to 1.57 ± 0.11 g/mL). The serum β2-microglobulin level was used to categorize the patients with SCI into four groups. As serum β2-microglobulin levels increased, the MoCA score reduced (p < 0.05). After adjustment of baseline data, further regression analysis showed that serum β2-microglobulin level remained an independent risk factor for post-SCI cognitive impairment. Patients with SCI had higher serum levels of β2-microglobulin, which may be a biomarker for cognitive decline following SCI.

Evaluation of the role of serum DcR3 levels in the early clinical prognosis of patients with Crimean-Congo hemorrhagic fever

BackgroundCrimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease that is transmitted by Hyalomma ticks and is endemic in many parts of the world. This study aimed to determine the relationship between early serum Decoy receptor-3 (DcR3) level and clinical severity in patients with CCHF. MethodsThe study included 88 patients hospitalized for CCHF between April and August 2022 and a control group of 40 healthy individuals. The patients were divided according to clinical course as those with mild/moderate (group 1, n = 55) and severe (group 2, n = 33) CCHF. DcR3 levels were measured by enzyme-linked immunosorbent assay of serum obtained at the time of diagnosis. ResultsFever, hemorrhage, nausea, headache, diarrhea, and hypoxia were significantly more common among patients with severe CCHF than patients with mild/moderate CCHF (p < 0.001, <0.001, 0.02, 0.01, <0.001, and < 0.001, respectively). Group 2 had higher serum DcR3 levels than both group 1 and the control group (p < 0.001 for both). Serum DcR3 levels were also significantly higher in group 1 than in the control group (p < 0.001). Using 98.4 ng/mL as the cut-off value, serum DcR3 had 99% sensitivity and 88% specificity in differentiating patients with severe CCHF from those with mild/moderate CCHF. ConclusionDuring the high season in our endemic region, CCHF can present with a severe clinical course independent of age and comorbidities, unlike other infectious diseases. Elevated DcR3 observed early in the disease may allow additional immunomodulatory therapies to be tried in addition to antiviral therapy in CCHF, for which treatment options are limited.

Pro-inflammatory cytokines in patients with acute coronary syndrome

The pathogenesis of acute coronary syndrome is based on the processes of atherosclerosis of coronary arteries and atherothrombosis, accompanied by the destruction of non-stable atherosclerotic plaque coating and, in the future, by thrombosis. Myocardial ischemia initiates local and systemic inflammatory responses with the release of pro-inflammatory cytokines, which trigger a cascade of reactions, contributing to the more profound damage to cardiomyocytes. That is why it is extremely important to determine the activity of proinflammatory cytokines in patients with acute coronary syndrome.The aim of the work. To investigate the indices of pro-inflammatory cytokines in patients with acute coronary syndrome.Materials and Methods. 33 patients with acute coronary syndrome, namely with unstable angina pectoris, who underwent inpatient treatment in the cardiology department of the Lviv hospital for emergency medicine, were examined. The patients were 12 (36%) males and 21 (64%) females, with an average age of 68.5±2 years. The patients received treatment according to modern protocols for medical care for patients with acute coronary syndrome without ST-segment elevation and recommendations of the European Heart Association. The control group consisted of 20 practically healthy individuals of the same age and status. The patients were subjected to general clinical and instrumental examination methods, proinflammatory interleukins in serum enzyme-linked immunosorbent assay (on admission for hospital treatment).Results of the research. It was found that in patients with acute coronary syndrome, pro-inflammatory cytokines (interleukin-1β, interleukin-6, interleukin-17, tumor necrosis factor-alpha) exceed the values of almost healthy individuals by almost 2 times (p˂0.01).Conclusion. In patients with acute coronary syndrome, there is a significant (p˂0.01) increase in pro-inflammatory cytokines, which indicates the direct involvement of inflammation in the pathogenesis of the pathological process and requires appropriate correction of treatment tactics.

Performance of the JF5-Based Galactomannoprotein EIA Compared to the Lateral Flow Device and the Galactomannan EIA in Serum and Bronchoalveolar Lavage Fluid.

Early diagnosis of invasive aspergillosis is an important factor to improve survival but remains challenging. The detection of Aspergillus antigens is included in the consensus case definitions of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group as a criterion of "probable" invasive aspergillosis. JF5, a mouse IgG3 monoclonal antibody detecting an Aspergillus mannoprotein, has already been implemented as a lateral flow device (LFD). Now, also a JF5-based enzyme-linked immunosorbent assay (EIA) is commercialized (Aspergillus specific galactomannoprotein [GP] EIA, Euroimmun Medizinische Labordiagnostika AG). In this study, we analyzed the diagnostic performance of GP in 63 bronchoalveolar lavage fluid (BALf) samples and 224 serum samples and compared it to performance of the galactomannan (GM) (Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France)) and the JF5-based LFD (AspLFD; OLM Diagnostics, Newcastle Upon Tyne, United Kingdom). The diagnostic performance of GP and GM correlated well with both having high specificity. With an optimized cutoff threshold for positivity of 0.4-deviating from the 0.5 threshold recommended by the manufacturer-sensitivity of GP in serum is not significantly different than that of GM. However, in BALf sensitivity of GP is significantly less than for GM.

A study on the safety and therapeutic effect of Xiaoyaosan on depressive disorder related dry eye disease in a murine animal model

ABSTRACT This study was done to observe the safety and effect of Xiaoyaosan (XYS) on the stimulated depressive disorder (DD) related dry eye disease (DED) in mice. Normal control (NC) group, Vehicle group, and drug treatment groups, including Sertraline Hydrochloride (SH), XYS low-dose (XYS-LD), medium-dose (XYS-MD), and high-dose (XYS-HD), were established. The drug quality of XYS was assessed by high-performance liquid chromatography (HPLC). XYS toxicity in kidney and liver was assessed with Hematoxylin & Eosin (H&E) staining. Serum enzyme-linked immunosorbent assay (ELISA) and body weights were used to evaluate the depression status of mice. Tear production, corneal sensitivity, Oregon Green Dye (OGD) staining, and corneal confocal microscopy were used to assess ocular surface changes. H&E staining was also used to assess pathological cornea and lacrimal gland changes. HPLC results showed that XYS complied with Chinese drug quality standards. The drug treatment groups observed no drug toxicity reactions in the liver and kidney. SH and XYS groups improved DD-related serological indices as compared with Vehicle. Body weight was enhanced in mice with XYS groups compared to Vehicle and SH. Mice with XYS treatments showed increased tear production and corneal sensitivity, decreased corneal OGD staining scores, improved morphology, and decreased inflammatory cell infiltration in the cornea and lacrimal gland. In conclusion, XYS had no drug toxicity, improved serological indices, and ocular surface pathological changes in DD-related DED mice. XYS is safe and may have a therapeutic effect on DD-related DED.

Decline of humoral immune responses after natural SARS-CoV-2 infection can be efficiently reversed by vaccination.

Our aim was to analyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody level kinetics after coronavirus disease 2019 (COVID-19) infection and determine the efficiency of vaccination on SARS-CoV-2-specific antibody levels. The study included 50 SARS-CoV-2 infected and 70 uninfected cases. Levels of SARS-CoV-2-specific IgG nucleocapsid protein (IgG-NP), IgG spike protein (IgG-SP), IgM nucleocapsid protein (IgM-NP), and IgA spike protein (IgA-SP) antibodies were evaluated by an enzyme-linked immunosorbent assay in sera obtained at baseline, 1st, 3rd, and 6th month follow-up visits for infected cases and at postvaccination visits for all cases. In symptomatic cases (n = 50), IgG-SP levels were decreased in 6 months compared with baseline, while IgA-SP levels were significantly increased. IgG-NP levels were significantly decreased in symptomatic cases at the 6-month visit. After vaccination, IgG-SP levels were increased in symptomatic cases compared with prevaccination levels. Among subjects vaccinated with CoronaVac (the Sinovac COVID-19 vaccine), infected cases had approximately double the IgG-SP level of uninfected cases. SARS-CoV-2-specific antibody levels were higher at the baseline in symptomatic cases. Nevertheless, all infected cases showed significantly reduced IgG-SP levels at the 6th month. Vaccination effectively increased IgG-SP levels.

Validation of pregnancy associated glycoproteins-based ELISA kits to determine early pregnancy status in lactating Nili-Ravi buffaloes.

The objective of the current study was to evaluate pregnancy-associated glycoproteins (PAGs) based enzyme-linked immunosorbent assay (ELISA) kits utilizing whole blood, serum, or milk samples for diagnosis of early pregnancy status in lactating Nili-Ravi buffaloes. Dairy buffaloes (n =174) of mixed parity, 4-6 years of age, having mean (± standard deviation) days in milk 165 ± 87, and body condition score of 3.26 ± 0.34 were randomly enrolled in this study. Buffaloes were exposed to penile deviated bulls with 12 h interval for estrus detection during peak breeding season and eventually bred naturally at their respective standing estrus (day 0). Blood and milk samples were collected at days 24, 28, and 35 post-breeding to run a rapid visual pregnancy test® (RVPT) as a buffalo-side test or ELISA-based assay in the laboratory to detect early pregnancy status. Transrectal B-mode ultrasonography was performed to diagnose pregnancy at day 35 post-breeding and used as a gold standard to validate results of RVPT or ELISA-based tests. The RVPT is a visual readout test for pregnancy detection and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (77.9 vs. 89.7 vs. 93.3%), and accuracy (84.5 vs. 90.1 vs. 94.2%) at days 24, 28, and 35 post-breeding, respectively. The PAGs were assayed using ELISA kits in serum and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (84.2 vs. 87.7 vs. 93.9%), and accuracy (82.1 vs. 88.4 vs. 93.7%) at days 24, 28, and 35 post-breeding, respectively. Similarly, PAGs were also analysed using ELISA kits in milk samples and had sensitivity (77.6 vs 89.5 vs 95.0%), specificity (89.1 vs 91.9, vs 93.9%), and accuracy (85.1 vs 91.1 vs 94.3%) at days 24, 28, and 35 post-breeding, respectively. Overall, the Kappa values in this study exceeded 0.85 at day 35 post-breeding using RVPT or ELISA-based test kits in serum or milk samples, indicating a high level of agreement between PAGs detection method and gold standard for pregnancy diagnosis. The pregnancy outcomes based on ELISA-based PAGs detection at day ≥28 post-breeding had a high negative predictive value, indicating that the probability of incorrectly administering prostaglandins to pregnant buffaloes would be low if these tests were implemented on a commercial dairy herd. Taken together, it is concluded that PAGs-based determination of pregnancy using RVPT or ELISA in blood, serum, or milk samples can be used effectively for pregnancy diagnosis at ≥28 days post-breeding with more than 90% accuracy in Nili-Ravi buffaloes.

Ectopic peritoneal paragonimiasis mimicking tuberculous peritonitis: A care report

BACKGROUNDThe most common site of paragonimiasis is in the lungs. The migratory route passes through the duodenal wall, peritoneum, and diaphragm to the lungs; thus, the thoracic cavity and central nervous system, as well as the liver, intestine, and abdominal cavity may be involved. Here, we present a case of intraperitoneal paragonimiasis without other organ involvement, mimicking tuberculous peritonitis.CASE SUMMARYA 57-year-old man presented with recurrent abdominal pain for 4 wk. Physical examination revealed tenderness in the right lower quadrant. Laboratory findings showed complete blood counts within the normal range without eosinophilia. Multiple reactive lymph nodes and diffuse peritoneal infiltration were noted on abdominal computed tomography (CT). There were no abnormalities on chest CT or colonoscopy. Intraoperative findings of diagnostic laparoscopy for the differential diagnosis of tuberculous peritonitis and peritoneal carcinomatosis included multiple small whitish nodules and an abscess in the peritoneum. Pathological reports confirmed the presence of numerous eggs of Paragonimus westermani (P. westermani). A postoperative serum enzyme-linked immunosorbent assay revealed P. westermani positivity. Persistent and repetitive history-taking led him to retrospectively recall the consumption of freshwater crab. After 3 d of treatment with praziquantel (1800 mg; 25 mg/kg), he recovered from all symptoms.CONCLUSIONIn patients who require diagnostic laparoscopy for the differential diagnosis of tuberculous peritonitis and peritoneal carcinomatosis, repetitive history-taking and preoperative serologic antibody tests against Paragonimus may be helpful in diagnosing intraperitoneal paragonimiasis without other organ involvement.

Characteristics of antibody production to individual cytomegalovirus proteins in children with keratitis and uveitis

The clinical diagnosis of cytomegalovirus (CMV) eye pathology in children needs laboratory confirmation. The disadvantages of determining antibodies to CMV in serum in enzyme-linked immunosorbent assay (ELISA) include the presence of false positive and false negative results.&#x0D; AIM: To determine the features of the synthesis of antibodies to the proteins of the tegument pp65, pp150, pp28 and the DNA-binding protein pp52 of cytomegalovirus, as well as to compare the diagnostic effectiveness of two laboratory methods of serodiagnostics of CMVI in children with uveitis and keratitis of different genesis: enzyme immunoassay and linear immunolysis.&#x0D; MATERIAL AND METHODS: A total of 30 children (age 516 years) with uveitis (n=14) and keratitis (n=16) were included in the study. The IgM and IgG antibodies to the immediate early (IE) and late antigens of the virus (serological markers of primary, chronic, and reactivation of chronic infection) were determined in the ELISA. The IgG antibodies to individual recombinant CMV antigens containing only immunodominant protein fragments of viral antigens were studied in the line immunoassay (LIA): the main nonstructural IE protein, DNA-binding phosphoprotein p52, and phosphoproteins of the tegument p150, p65, and p28. The results of LIA were evaluated visually.&#x0D; RESULTS: CMV infection (CMVI) in children with uveitis in ELISA was almost 2 times higher than that with keratitis (10/14%71% vs. 6/16%37.5%, p 0.05). Of the four positive results for detection of antibodies to IE antigen in ELISA, one was confirmed in LIA. In general, the discrepancy between the results of the determination of IgG antibodies to IE antigen in ELISA and LIA was noted in 13% of serum samples. LIA showed the increased frequency (p 0.05) and intensity (p 0.05) of antibody formation to viral antigens p65 and p52 in uveitis compared with keratitis, which confirmed the important role of CMVI in the pathogenesis of uveitis.&#x0D; CONCLUSION: Both methods revealed a higher level of CMVI in children with uveitis than those with keratitis. IgG antibodies to IE-antigen serological markers of the reactivation of chronic CMVI have clinical importance because they serve as the basis for the appointment of antiviral therapy. Discrepancies between the results of ELISA and LIA in the detection of antibodies to IE antigen indicate the expediency of confirming the results of ELISA in LIA.&#x0D; Thus, laboratory examination of sera in ELISA with subsequent analysis of antibodies to individual recombinant CMV antigens in LIA is an effective, highly sensitive, and highly specific method to verify CMVI and determine its activity. The most informative action is the determination of IgG antibodies to recombinant CMV antigens: IE antigens, p65, and p52.

Dexamethasone attenuated thoracic aortic aneurysm and dissection in vascular smooth muscle cell Tgfbr2-disrupted mice with CCL8 suppression.

What is the central question of this study? What is the relationship of chemokine (C-C motif) ligand 8 (CCL8) to thoracic aortic aneurysm and dissection (TAAD) formation in postnatal mice with vascular smooth muscle cell (VSMC) Tgfbr2 disruption, and is dexamethasone a potential treatment? What is the main finding and its importance? CCL8 was associated with the formation of TAAD in VSMC Tgfbr2-disrupted mice. Dexamethasone reduced TAAD formation and inhibited mitogen-activated protein kinase (p-p38) and nuclear factor-κB (p-p65) signalling pathways. CCL8 might be an important promoter of aortic inflammation. Dexamethasone provided potential therapeutic effects in TAAD treatment. Aortic inflammation plays a vital role in initiation and progression of thoracic aortic aneurysm and dissection (TAAD). Disturbance of the transforming growth factor-β (TGF-β) signalling pathway is believed to be one of the pathogenic mechanisms of TAAD. Initially, Myh11-CreERT2 .Tgfbr2f/f male mice were used to build a TAAD mouse model, and bioinformatic analyses revealed enriched inflammatory signal pathways and upregulated chemokine (C-C motif) ligand 8 (CCL8). So we hypothesized that vascular smooth muscle cell (VSMC) Tgfbr2 disruption in postnatal mice results in aortic inflammation associated with CCL8 secretion. Real-time quantitative PCR and serum enzyme-linked immunosorbent assay (ELISA) results confirmed that CCL8 expression began to increase after VSMC Tgfbr2 disruption. Next, we cultured mouse thoracic aortas ex vivo, and observed that the protein expression of CCL8 in culture supernatants was increased by ELISA. Subsequently, the co-localization of CCL8 with α-smooth muscle actin or CD68 was found to be significantly increased by immunofluorescence. Then, dexamethasone (DEX) was used to treat TAAD in VSMC Tgfbr2-disrupted mice; the results of histochemical, immunofluorescence and immunohistochemical staining indicated that DEX therapy reduced CCL8 secretion, inflammatory cell recruitment, aortic medial thickening, elastic fibre fragmentation, extracellular matrix degradation and contractile apparatus impairment, and thereby ameliorated TAAD formation. Western blotting showed that mitogen-activated protein kinase and nuclear factor-κB signalling pathways in aorta were overactivated after VSMC Tgfbr2 disruption, but inhibited by DEX therapy. Altogether, CCL8 might be an important promoter in TAAD formation of VSMC Tgfbr2-disrupted mice, and DEX provided potential therapeutic effects in TAAD treatment.

Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3–97.2%) compared to 91.7% (95% CI 77.5–98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3–100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.