Commercial ELISA Research Articles

Toxoplasma gondii Infection in the Eurasian Beaver (Castor fiber) in Switzerland: Seroprevalence, Genetic Characterization, and Clinicopathologic Relevance.

Toxoplasma gondii is a coccidian parasite able to infect all warm-blooded animals and humans. Rodents are one of the most important intermediate hosts for T. gondii, but little is known about infection in beavers and its clinical relevance. Toxoplasmosis was not considered an important waterborne disease until recently, but with increased outbreaks in humans and animals this perspective has changed. Serum samples from 247 Eurasian beavers (Castor fiber) collected from 2002 to 2022 were tested for antibodies to T. gondii by a commercial ELISA. Antibodies to T. gondii were found in 113 (45.8%) beavers. Higher weight and proximity to urban areas were found to be significant predictors for seropositivity. Additionally, T. gondii DNA was detected in 23/41 brain tissue samples by real-time PCR. Histopathologic examination of brain sections revealed inflammatory changes in 26/40 beavers, mainly characterized by encephalitis, meningitis, choroid plexitis, or a combination of them. In six of these cases the lesions were in direct association with parasitic stages. With an adapted nested PCR multilocus sequence typing and in silico restriction fragment length polymorphism analysis approach, three different T. gondii genotypes were detected in brain samples: the clonal Type II strain (ToxoDB 1), a Type II variant (ToxoDB 3), and a novel genotype exhibiting both Type II and I alleles in a further animal. Toxoplasma gondii infections in beavers have epidemiologic and clinical significance. The high seroprevalence indicates frequent contact with the parasite, and as competent intermediate hosts they may play an important role, contributing to maintaining the life cycle of T. gondii in semiaquatic habitats. In addition, although most beavers appear to develop subclinical to chronic disease courses, acute and fatal outcomes, mainly characterized by encephalitis and generalized infection, do also occur.

Development of recombinant capsid protein-based single serum dilution ELISA for sero-detection of porcine circovirus type 2 infection in pigs.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Measurement of 8-hydroxy-2'-deoxyguanosine in serum and cerebrospinal fluid of horses with neuroaxonal degeneration and other causes of proprioceptive ataxia.

Eight-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative damage evaluated in human neurodegenerative disease, has potential to correlate with postmortem diagnosis of neuroaxonal dystrophy/degenerative myeloencephalopathy (NAD/DM) in horses. We hypothesized that 8-OHdG will be higher in CSF and serum from NAD/DM horses compared with horses with other neurologic diseases (CVSM, EPM) and a control group of neurologically normal horses. We also hypothesized that 8-OHdG will be higher in CSF compared with serum from NAD/DM horses. Fifty client-owned horses with postmortem diagnoses: 20 NAD/DM, 10 CVSM, 10 EPM, and 10 control horses. Serum and CSF samples were obtained between November 2010 and March 2022. Case-control study using biobanked samples was performed and commercial competitive ELISA kit (Highly Sensitive 8-OHdG Check ELISA) utilized. Concentration of 8-OHdG was quantitated in both CSF and serum and compared between groups. No correlation was established between the measures of 8-OHdG in serum and CSF and group. CSF median [8-OHdG] for NAD/DM was 169.9 pg/mL (IQR25-75 : 67.18-210.6), CVSM 157.1 pg/mL (IQR25-75 : 132.1-229.1), EPM 131.4 pg/mL (IQR25-75 : 102.1-193.2), and control 149.8 pg/mL (IQR25-75 : 113.3-196.4). Serum median [8-OHdG] for NAD/DM was 130 pg/mL (IQR25-75 : 51.73-157.2), CVSM 125.8 pg/mL (IQR25-75 : 62.8-170.8), EPM 120.6 pg/mL (IQR25-75 : 87.23-229.7), and control 157.6 pg/mL (IQR25-75 : 97.15-245.6). Poisson regression analysis showed no difference established once confounding variables were considered. Eight-OHdG did not aid in antemortem diagnosis of NAD/DM in this cohort of horses. At the time of diagnosis horses with NAD/DM do not have ongoing oxidative stress.

Photothermal biosensing integrated with microfluidic paper-based analytical device for sensitive quantification of sarcosine

This article presents the development of a photothermal biosensing integrated with microfluidic paper-based analytical device (PT-μPAD) as a quantitative biosensor method for monitoring sarcosine in human control urine, plasma, and serum samples. The device utilizes gold nanoparticles (AuNPs) as both a peroxidase-like nanozyme and a photothermal substrate to enable sarcosine detection. In our PT-μPAD, hydrogen peroxide (H2O2) is generated through the oxidation of sarcosine by a sarcosine oxidase (SOx) enzyme. Subsequently, the H2O2 flows through the paper microchannels to the detection zone, where it etches the pre-deposited AuNPs, inducing a temperature change upon exposure by a 532 nm laser. The temperature variation is then measured using a portable and inexpensive infrared thermometer. Under optimized conditions, we obtained a linear range between 10.0 and 40.0 nmol L−1 (R2 = 0.9954) and a detection limit (LOD) of 32.0 pmol L−1. These values fall within the clinical range for sarcosine monitoring in prostate cancer diagnostics in humans. Moreover, our approach exhibits high selectivity without interfering effects. Recovery studies in various human control samples demonstrated a range of 99.05–102.11 % with the highest RSD of 2.25 %. The PT-μPAD was further validated for sarcosine determination in human control urine and compared with a commercial ELISA assay, revealing no significant difference between these two methods at a 95 % confidence level. Overall, our proposed sarcosine biosensor is well-suited for prostate cancer monitoring, given its affordability, sensitivity, and user-friendliness, even for unskilled individuals. Moreover, this strategy has promising prospects for broader applications, potentially detecting various biomarkers as a point-of-care (POC) diagnostic tool.

Vitamin D and its binding protein in patients with leiomyomas.

This study examined the levels of VitD, VitD binding protein (DBP), and free VitD in leiomyomas patients and their association with the quantity, dimensions, and site of fibroid growths. Additionally, we evaluated the potentiality of employing these factors as a biomarker tool for the diagnosis and assessment of uterine fibroid progression. This study involved the participation of 55 women with leiomyomas and 50 healthy women. We utilized commercial ELISA kits to measure the levels of total VitD and DBP in their serum. Additionally, we calculated the levels of free VitD and the ratio of VitD to DBP. Moreover, we determined the number, size, and location of the leiomyomas in the patients. There were no significant differences in the levels of total VitD between the groups. However, patients had significantly lower levels of free VitD and higher levels of DBP compared to the control group. The size of the largest leiomyomas showed a negative relationship with free VitD and a positive relationship with DBP. Receiver operating characteristic analyses, showed that the cut-off value for free VitD was 4.47 pg/mL, with a sensitivity of 75.6% and a specificity of 74.4%. The cut-off value for DBP was 256.2 μg/mL, with a sensitivity of 86% and a specificity of 70.3%. Free VitD and DBP potentially contribute to the development of leiomyomas and are linked to the size of these tumors. The measurement of serum levels of these factors could serve as additional biomarkers for the diagnosis of leiomyomas.

Evaluation of haptoglobin concentration in clinically healthy dairy cows: correlation between serum and milk levels

ABSTRACT The study aimed to analyse serum haptoglobin concentrations by calving number and lactation phase in clinically healthy dairy cattle and to determine the correlation between concentrations in serum and milk samples. For this purpose, serum samples were collected from 228 clinically healthy cows after milking from 14 randomly selected herds. Additionally, milk samples were collected from 80 out of the 228 cows from the milking prior to blood collection. Both serum and milk samples were analysed with a commercial ELISA test. The effect of the calving number and lactation phase on mean serum haptoglobin concentrations was examined by Kruskal–Wallis/Jonckheere–Terpstra tests. After that, in a multivariate approach, a mixed-effects linear model was also performed. In addition, intraclass correlation coefficients were used to evaluate the correlation between concentrations in serum and milk samples. Results indicated a downward trend in haptoglobin concentrations throughout lactation, that was statistically significant when comparing fresh cows with the remaining lactation phases. The higher haptoglobin concentrations in fresh cows could be the result of inflammatory illnesses without clinical disease, stress or variations within the physiological state. Although previous studies showed some discrepancies, a good correlation between serum and milk concentrations has been observed in the present paper.

Sıçanlarda 5-Fluorourasil ile Oluşturulmuş Gastrointestinal Mukozitte Berberinin Terapötik Etkisi

5-Fluorouracil (5-FU), an antineoplastic agent, causes intestinal mucositis (IM) that limits the drug efficacy. The aim of this study was to reveal the protective and/or therapeutic effects of berberine (BER), an isoquinoline quaternary alkaloid, on IM induced by 5-FU in rats. Forty Wistar albino male rats were randomly divided into five groups: Control, 5-FU, 5-FU+BER75 (75 mg/kg/day), 5-FU+BER150 (150 mg/kg/day) and 5-FU+BER300 (300 mg/kg/day). 5-FU was intraperitoneally (i.p) injected at a dose of 60 mg/kg on alternate days for 4 days, while other groups were orally received BER daily for 7 days. By the end of study, intracardiac blood samples were collected and intestinal tissues were removed for histological examination. TNF-α, IL-6 and NF-κB levels were measured in serum using commercial ELISA kits. Results showed that BER at increasing doses ameliorated body weight loss and histopathological damage. It was concluded that BER may be effective in the treatment and/or prophylaxis of the damage in the gastrointestinal system caused by 5-FU in a dose-dependent manner.

Investigating the Prevalence of Paratuberculosis in Hungarian Large-Scale Dairy Herds and the Success of Control Measures over Four Years.

Paratuberculosis (PTB) is a severe, slow-developing, untreatable disease of ruminants. Worldwide, the disease affects more than 50% of herds in the dairy industry, and causes substantial economic losses for dairy producers. Diagnostic tests show limited sensitivity, especially in the early stages of the disease. Our study aimed to investigate the seroprevalence of Mycobacterium avium ssp. paratuberculosis (MAP) in large-scale dairy herds in Hungary, in association with the self-reported presence or absence of screening and intervention measures against MAP transmission. We processed data from 42 large-scale Holstein Friesian farms in Hungary between 1 January 2018 and 31 December 2021. An average of 32,009 (min.: 31,702; max.: 32,207) animals were blood sampled yearly (127,372 in total during the four years), corresponding to 15% of the Hungarian dairy cattle population. All female cattle older than 2 years were blood sampled on the farms enroled in the study. The samples were tested using a commercial ELISA (IDEXX paratuberculosis screening Ab test). Farm managers were interviewed about their on-farm diagnostic and intervention approaches using a uniform questionnaire, including questions on the level of awareness, frequency of ELISA and PCR testing, and their strategies for culling adult animals and reducing transmission to newborn calves. By comparing the annual rate of change in seroprevalence and the amount of change observed during the four-year period, we concluded that test-and-cull strategies implemented in parallel with newborn calf management that aimed at preventing MAP transmission were superior to test-and-cull strategies alone; moreover, fortifying culling decision making via additional ELISA and PCR tests is superior to using a single ELISA result. For farms that carried out a complex program with both "test-and-cull" and proper newborn calf management, there was a proportional reduction in apparent seroprevalence at an average of 22.8% per year. Fifteen of the sampled farms had no measures in place to control paratuberculosis. On these farms, the seroprevalence increased by 12.1% per year on average.

Lateral Flow Immunochromatographic Assay for Competitive Detection of Crustacean Allergen Tropomyosin Using Phage-Displayed Shark Single-Domain Antibody.

The common food allergy crustacean tropomyosin (TM) poses a significant food safety challenge, which requires rapid and sensitive methods for screening TM in food. Herein, the variable new antigen receptor (VNAR) single-domain antibodies specific for the crustacean TM were isolated from a naïve phage-displayed shark VNAR library. Subsequently, a lateral flow immunochromatographic assay (LFIA) based on the gold nanoparticle-labeled phage-displayed shark VNAR (AuNPs@PSV) probe was developed for the detection of TM in food. The AuNPs@PSV-LFIA took 15 min for one test and had a visual limit of detection (vLOD) of 0.1 μg/mL and an instrumental LOD of 0.02 μg/mL. Good selectivity, accuracy, precision, and stability were confirmed for the AuNPs@PSV-LFIA. Moreover, the test results of 21 commercially available food products consisted of the allergen labels and were validated by a commercial ELISA kit. Therefore, this work demonstrated the great potential of VNAR for detecting TM in food by LFIA.

Ultrasensitive Point-of-Care Detection of Protein Markers Using an Aptamer-CRISPR/Cas12a-Regulated Liquid Crystal Sensor (ALICS).

Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.

Development of a fluorescence immunochromatography method for quantitative measurement of matrix metalloproteinase-9

ObjectiveAbnormal serum matrix metalloproteinase-9 (MMP-9) levels are closely related to the occurrence and development of many diseases. This study aimed to establish a fluorescence immunochromatography (FIC) method using the lanthanide fluorescent element europium(III) (Eu3+) for the quantitative measurement of MMP-9 in serum. Design & MethodsThe FIC method for quantifying MMP-9 was optimized and established, and the FIC test strips (FICTS) were assembled and subsequently evaluated for sensitivity, specificity and precision. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive serum samples, and a commercial ELISA was used for comparison. ResultsWe successfully established an FIC method and prepared FICTS. The analytical sensitivity of the FICTS was 0.92 ng/mL, with a linearity range of 0–1000 ng/mL. The cross-reactivity of the 7 common serum interferents was less than 1.56%. All recoveries of the intra-array and inter-array samples ranged from 102.50% to 110.99%, and all CVs were less than 5%. The reference interval of the FICTS was >161.15 ng/mL. The clinical sensitivity was 96.00%, and the specificity was 97.5%. The results of 270 clinical serum samples were highly coincident with the clinical diagnostic results. Pearson correlation analysis and Bland‒Altman plots indicated that the FICTS and commercial ELISA results were consistent with the quantitative MMP-9 concentration. ConclusionsThe designed FIC method and test strips may be suitable for point-of-care quantitative measurement of MMP-9, which provides a new method for screening for atherosclerosis, xerophthalmia, etc.

Evaluation of interleukin-6 in gingival crevicular fluid in patients with periodontitis

Introduction: Periodontal diseases are disease processes involving the periodontium, a term that describes the supportive apparatus surrounding a tooth. Periodontitis is when the periodontal condition has progressed beyond gingivitis into a chronic, destructive, and irreversible inflammatory disease state. The bacteria can then penetrate deeper into the tissues and surrounding periodontium, triggering a host response in an attempt to defend against the invading bacteria. Many inflammatory cytokines regulate periodontitis pathophysiology through a cross-talk between tissue and immune cells. Interleukin (IL)-6 is a vital cytokine regulating host response to bacterial infection. Aim: The current study aimed to quantify IL-6 levels in gingival crevicular fluid (GCF) and assess the correlation with periodontitis. Materials and Methods: There were 22 study subjects: 11 study subjects (with periodontitis) and 11 control subjects (with healthy periodontium). The commercial human IL-6 ELISA kit (Elabscience) was used to measure the level of IL-6 as ng IL-6/ml of GCF. Results and Discussion: The control group had a mean ± standard deviation level of IL-6 of 0.56 ± 0.29, whereas the study group had 1.58 ± 0.23. The difference was statistically significant (P < 0.05). Conclusion: IL-6 assessment in GCF has an excellent prognostic role in periodontitis.

Evaluation of Hormonal Changes in Obese Individuals with Constipation Problems

Aim: Constipation is frequently seen in obese individuals. It is still not fully known whether the constipation in these individuals is related only to excess weight or due to other reasons. It is limited that the hormones secreted from the gut can play an important role in constipation. In this study, we think that hormones such as intestinal peptide YY (PYY), SS, GLP-1, GLP-2 and Oxyntomodulin, which are known to have effects on bowel movements, may have effects on constipation. In this study, it will be tried to determine how the changes in the levels of the mentioned hormones with constipation in individuals who are obese and have constipation problems. Materials and Methods: Twenty-two obese and 22 obese and constipated individuals aged 20-64 years with VKI>30 who would sign the voluntary consent form were included in the study. Individuals with chronic diseases such as diabetes and hypertension in addition to obesity, those who received psychiatric treatment and those who used alcohol were not accepted. The demographic information of the individuals was determined by the face-to-face survey method. GLP-1, GLP-2, somatostatin, peptide yy, serotonin, oxyntomodulin levels were determined with the help of commercial ELISA kit in fasting blood samples taken from obese and obese+constipated individuals in the morning. Results: Peptide yy, SS, Oxyntomodulin levels did not differ in obese and constipated individuals (p>0.05). GLP-1, GLP-2, Serotonin levels were found to be significantly higher in obese individuals than in constipated individuals (p<0.01). Conclusion: In our study, high levels of GLP-1, GLP-2, and Serotonin, which have conflicting results in the literature, and positive correlations between these hormones can be considered as important findings in our study.

Seroprevalence of Mycobacterium avium subsp. paratuberculosis in Swiss dairy herds and risk factors for a positive herd status and within-herd prevalence.

Bovine paratuberculosis (PTB) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). Control of PTB is important given its negative economic consequences and the potential zoonotic role of MAP in Crohn's disease in humans. To determine the seroprevalence of MAP in Swiss dairy herds and to identify risk factors associated with seropositive herd status and high within-herd seroprevalence, 10,063 serum samples collected from cattle over 12 months of age in 171 Swiss dairy farms were analyzed using a commercial ELISA test. Eight herds were excluded due to non-interpretable ELISA results. Risk factors associated with seropositive herd status and high within-herd seroprevalence were investigated with regression models using results from a questionnaire on management practices possibly associated with the introduction or spread of MAP in the remaining 163 herds. Univariable logistic regression was performed, carrying forward for multivariable regression analysis when p < 0.2. The calculated between-herd true seroprevalence was 3.6% (95% CI, 0.96-8.4%). Due to the low within-herd seroprevalence, it was not possible to calculate the true seroprevalence at animal level; the apparent within-herd seroprevalence ranged from 2.3 to 5.5% with a median of 3.6% in nine positive farms. Herd size (p = 0.037) and the common grazing of lactating cows with cows from other herds (p = 0.014) were associated with seropositive herd status, while heifers sharing alpine pasture with dairy cattle from other herds were associated with a decreased probability of the herd to test seropositive (p = 0.042). Reliable identification of significant risk factors associated with MAP spread and high seroprevalence of PTB within seropositive herds was not possible due to low observed seroprevalence within herds and low sensitivity of the ELISA test. These results highlight the limitation of serology for MAP diagnosis in small herds with low infection prevalence.

Predictive Value of Interleukin 6 and Interleukin 8 in Response to Treatment of Hepatitis C Virus.

Chronic hepatitis C (CHC) infection is a major public health problem in many low- and middle-income countries. The study aimed to find out how interleukin IL-6 and IL-8 levels in the blood affect the virological response to directacting antivirals (DAAs) and to find useful clinical or immunological markers for the response to HCV treatment. CHC patients from a real Egyptian population (n = 4,300), who were treated during the Egyptian national initiative to eliminate HCV at the Sherbin Central Hospital, Dakahlia Governorate, Ministry of Health, Egypt, were enrolled in our study. They were all patients who did not obtain a sustained virological response (SVR) (n = 75; non-responder; the response rate was 98.26%), and a total of 100 patients were randomly selected from patients who obtained SVR (responder) and were age- and gender-matched (p > 0.05) with non-responder patients. Serum levels of IL-6 and IL-8 were measured by commercial ELISA kits. Non-responder patients were associated with significantly high levels of ALT, AST, ALP, and total bilirubin. Non-responders had significantly (p < 0.05) higher baseline IL-6 (16.7 ± 4.92 pg/mL) and IL-8 (37.81 ± 10.55 pg/mL) levels compared to responders (12.68 ± 2.06, 29.06 ± 5.94 pg/mL, respectively). There was a substantial (p < 0.05) association between the combination of two cytokines and a high likelihood of treatment failure, as indicated by all parameters examined, with the highest correlation values seen. The present study showed that increased IL-6 and IL-8 were associated with HCV treatment failure. Also, IL8 was associated with hepatic fibrosis.

Evaluation of urinary C-reactive protein as an early detection biomarker for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide. Up to now, no specific screening or diagnostic tests are available for early PDAC detection. As a result, most patients are diagnosed with advanced or metastatic disease, which leads to a poor prognosis. In this study, we aimed to evaluate the diagnostic value of urinary CRP (uCRP) alone and in combination with our previously established urine biomarker panel (REG1B, LYVE1 and TFF1) for early detection of PDAC. A total of 534 urine samples from multiple centres were analysed: 93 from healthy individuals, 265 from patients with benign hepatobiliary diseases and 176 from PDAC patients. The uCRP and the urinary biomarker panel were assessed using commercial ELISA assays, while plasma CA19-9 and blood CRP (bCRP) were measured using Roche Cobas platform. Multiple logistic regression and nonparametric Kruskal-Wallis test were used for statistical analysis. An internal validation approach was applied, and the validated AUC estimators were reported to ensure accuracy. A significant difference was observed in the medians of uCRP between healthy and benign controls and PDAC sample groups (p < 0.001). uCRP levels were not dependent on gender and age, as well as cancer stage. When uCRP was combined with the urinary biomarker panel, it achieved AUCs of 0.878 (95% CI: 0.802-0.931), 0.798 (95% CI: 0.738-0.859) and 0.813 (95% CI: 0.758-0.869) in healthy vs PDAC, benign vs PDAC and healthy and benign vs PDAC sample groups, respectively. However, adding plasma CA19-9 to the urinary biomarker panel yielded a better performance, with AUCs of 0.978 (95% CI: 0.959-0.996), 0.911 (95% CI: 0.873-0.949) and 0.919 (95% CI: 0.883-0.955) in the healthy vs PDAC, benign vs PDAC and healthy and benign vs PDAC comparisons, respectively. In conclusion, we show that measuring CRP in urine is a feasible analytical method, and that uCRP could potentially be a promising biomarker in various diseases including other cancer types.

Expression of Acidic Fibrillar Protein and Neuroglobin in Thrombolytic Patients in Ischemic Stroke.

Glial fibrillary acidic protein (GFAP) and neuroglobin (NGB) are important biomarkers of cerebral hypoxia. For this reason, an attempt was made to assess their concentrations in various time intervals and their impact on the severity of neurological symptoms and functional prognosis of thrombolytic ischemic stroke patients. The study involved 94 patients reporting to the emergency department of the Collegium Medicum University Hospital in Bydgoszcz within < 4.5 hours of the onset of stroke symptoms. GFAP and neuroglobin levels were measured in plasma at indicated times using a commercial ELISA kit. Based on the data gathered, statistically significant differences were found between the concentration of biomarkers in stroke patients and the control group. The concentrations of both biomarkers, GFAP and NGB, were elevated in patients after ischemic stroke and the changes in their concentrations in the subsequent stages of stroke may suggest their prognostic value strictly dependent on time. NGB was determined on the 7th day, and mRS - after a year (0.35). GFAP measured after 24 h and on day 7 could be a promising biomarker of functional outcome after one year (cut-off point ≤ 0.231 ng/mL, sensitivity 75.0%, specificity 61.2%, cut off point ≤ 0.235 ng/mL, sensitivity 75.0%, specificity 73.9%, respectively) and the severity of the patient's neurological condition. At GFAP concentrations above 0.25 ng/mL, measured within 24 hours, a sharp increase in mortality was observed in stroke patients. In the case of NGB, at the time of stroke occurrence (14 ng/mL) and after 24 hours (10-60 ng/mL). Differences in the concentrations of these biomarkers have been demonstrated in different stroke subtypes. NGB and GFAP are important biomarkers of ischemic brain injury and may also participate in predicting neurological outcomes.

A connection between levels of soluble Fas and Fas ligand in the aqueous humor and the parameters of structural and functional damage of glaucoma patients

Introduction/Objective. Fas ligant (FasL) induces apoptosis when interacting with the Fas-receptor. The aim was to determine the concentration of soluble Fas (sFas) and FasL (sFasL) in the aqueous humor of open-angle glaucoma patients, and establish a connection between these markers of apoptosis and the parameters of structural and functional glaucoma damage. Methods. This study examined 88 aqueous humor samples; 35 primary open-angle glaucoma with elevated intraocular pressure (POAG-HTG) patients, 24 open angle pseudoexfoliative glaucoma patients (XFG) and 29 patients with senile cataract (CAT). The concentration of sFas and sFasL was determined by commercial ELISA tests in the aqueous humor. Results. The conducted study showed that aqueous humor sFas concentration was the highest in XFG (720.14 ? 167.39 pg/ml), and slightly lower in POAG-HTG (713.43 ? 162.69 pg/ml), than in cataract patients (632.46 ? 217.11 pg/ml), without statistical significance. There was a significant negative correlation of sFas concentration and RNFL Inf thickness in POAG-HTG (p &lt; 0.05). The concentration of sFasL was the lowest in POAG-HTG (9.28 ? 0.551 pg/ml), higher in XFG (9.45 ? 0.61 pg/ml; p = 0.0566), and the highest in the cataract group (9.48 ? 0.73 pg/ml). A negative correlation of sFasL and MD in the POAG-HTG, and a negative correlation with RNFL Sup in the XFG were significant. Conclusion. sFasL has an active role in the regulation of the inflammatory process in glaucoma. sFas and sFasL, as markers of apoptosis, are associated with the parameters of structural, RNFL thinning, and functional glaucoma damage, namely visual field defects.

Evaluation of the ability of commercial enzyme-linked immunosorbent assays to measure mouse tissue factor

BackgroundTissue factor (TF) is the primary cellular initiator of the blood coagulation cascade. Increased levels of TF expression on circulating monocytes or on extracellular vesicles (EVs) are associated with thrombosis in a variety of diseases, including sepsis and COVID-19. ObjectivesHere, we aimed to evaluate the ability of 4 commercial TF enzyme-linked immunosorbent assays (ELISAs) to measure mouse TF in cells and plasma. MethodsWe used 4 commercial mouse TF ELISAs (SimpleStep, R&D Systems, MyBioSource [sandwich], and MyBioSource [competitive]). We used recombinant mouse TF (rmTF; 16-1000 pg/mL), cell lysates from a TF-expressing mouse pancreatic cancer cell line, and plasma and EVs isolated from plasma from mice injected with vehicle or bacterial lipopolysaccharide (LPS). ResultsThe 2 MyBioSource kits failed to detect rmTF or TF in cell lysates. The SimpleStep and R&D kits detected rmTF in buffer or spiked into plasma in a concentration-dependent manner. These kits also detected TF in cell lysates from a mouse pancreatic cancer cell line. A higher signal was observed with the SimpleStep kit compared to the R&D kit. However, the SimpleStep and R&D kits failed to detect TF in plasma or EVs from LPS-treated mice. ConclusionOur results indicate that some commercial ELISAs can be used to measure mouse TF levels in cell lysates but they cannot detect TF in plasma or EVs from endotoxemic mice.

Ultrasensitive fluorescence immunoassay of pepsinogen I based on enzyme-triggered decomposition of AuNCs/MnO2.

Gastric cancer is a prevalent malignant tumor of the gastrointestinal tract accompanied by a high mortality rate; therefore, early gastric cancer screening is critical for improving patient survival. In this study, we present a facile fluorescence immunoassay for highly sensitive screening of pepsinogen I (PG I) based on a one-pot biomimetic mineralization process for the synthesis of gold nanocluster-anchored manganese dioxide (AuNCs/MnO2) nanosheets. MnO2 first quenches the fluorescence of AuNCs through the Förster resonance energy transfer effect, whereas the introduction of ascorbic acid (AA) leads to the decomposition of MnO2 and rapidly recovers the fluorescence of AuNCs. Based on the above principles and phenomena, we developed a sensitive fluorescence immunoassay for the in situ generation of AA to detect PG I. Specifically, in the presence of PG I, the sandwich-type immunoreactivity-enriched alkaline phosphatase-labeled secondary antibody catalyzes the production of AA from the substrate, which enhances the fluorescence intensity. Under optimized conditions, the fluorescence intensity increased linearly with the concentration of PG I (0.05 to 200 ng mL-1) with a limit of detection (LOD) of 0.013 ng mL-1 (S/N = 3). The designed sensing platform has good stability (more than one year) and excellent anti-interference capability and demonstrates satisfactory accuracy for detection in real samples compared to commercial ELISA kits.