Sandwich Enzyme-linked Immunoassay Research Articles

A Proof-of-Concept Sandwich Enzyme-Linked Immunoassay Development for Small Molecules.

A sandwich immunoassay theoretically exhibits higher sensitivity and specificity compared to a competitive counterpart; however, it is extremely difficult to obtain a pair of antibodies that can bind to a small molecule simultaneously, which is always thought to be a single epitope. In the present study, abamectin (ABM) was selected to prove the effect of hapten design and antibody recognition properties on the development of a sandwich immunoassay for small molecules. First, the epitopes of ABM were roughly located, and epitope distances were determined. Then, two haptens were designed by introducing spacer arms at the C4″-OH and C5-OH of ABM, respectively, aiming to provide the longest epitope distances. A total of seven rabbit polyclonal antibodies (pAbs) and 21 mouse monoclonal antibodies (mAbs) with various recognition properties were obtained. Extensive combinatorial associations of antibody pairs for simultaneously binding to ABM were performed, and only two mAb-mAb pairs were observed to achieve a sandwich immunoassay for ABM with a total success rate of 0.27%. The best mAb pair for sandwich immunoassay was confirmed by surface plasmon resonance, used to develop a sandwich immunoassay, and then evaluated by cross-reactivities and molecular docking with structurally similar analogues and abamectin. Altogether, the study provided a theoretical foundation as well as practical experience and demonstrated the importance of careful hapten design and extensive antibody screening to successfully establish the sandwich immunoassay for small molecules.

Kruppel benzeri faktör 2 polimorfizmlerinin koroner arter hastalığı riski ile ilişkisi

Purpose: Coronary artery disease is defined as complications that develop in proportion to the prevalence of ischemia due to occlusion of the coronary arteries and the resulting cell death. The development of atherosclerosis is significantly influenced by endothelial cell dysfunction. Kruppel-like factor 2, a transcription factor, has been shown to regulate critical biological events in endothelial biology, such as vascular tone, migration, proliferation, vasoreactivity, and angiogenesis. In our study, it was aimed to clarify the relationship between coronary artery disease and Kruppel-like factor 2 protein levels and C1239A polymorphisms.
 Materials and Methods: 191 individuals who underwent coronary angiography at Mersin University Faculty of Medicine, Department of Cardiology, Health, Research and Application Center were included in the study. Measurements of serum fasting blood glucose, HDL cholesterol, total cholesterol, and triglyceride levels were performed on the AU5800 (Beckman Coulter, United States) autoanalyzer. Serum LDL levels were calculated using the Friedwald equation. Serum Kruppel-like factor 2 protein levels were measured by sandwich enzyme-linked immunoassay method on the Multiskan GO (Thermo Scientific, Finland) device. Kruppel-like factor 2 C1239A variations were detected on the Applied Biosystem VIIA™ 7 Real-Time PCR (Life Technologies Co., United States) device by TaqMan® single nucleotide polymorphism (SNP) genotyping method.
 Results: Men had a 3.8-fold higher risk of CAD than women. (Odd’s ratio 3.83, 95% Confidence interval 1.98-7.39; p

21 A comparison of assays measuring haptoglobin in equine plasma

Heightened inflammation is an important indicator of many diseases that affect the equine athlete. Haptoglobin, an acute phase protein present in blood, is commonly used as a biomarker for systemic inflammation. Recent literature provides a range for plasma haptoglobin in horses of 0.29–2.26mg/mL. When inflammation or infection occurs, the acute phase response of the immune system is activated, and haptoglobin concentrations can rise to 9 times that of healthy concentrations. A variety of assays have been developed to measure haptoglobin concentration, but there is disagreement in the scientific literature regarding which assay measures haptoglobin concentrations most accurately. The objective of this study was to compare 2 commonly used haptoglobin assays: The TriDelta “PHASE” Haptoglobin Assay (TD) and the ALPCO Horse Haptoglobin Assay (AP). The TD assay is based on the peroxidase activity of the haptoglobin-hemoglobin complex at lower pHs. The AP assay is a double antibody sandwich enzyme-linked immunoassay. Blood samples were collected from 8 Quarter Horse long yearlings (16–18 mo) split into a nonexercised group and exercised group. These horses and the experimental design were the focus of a different study. A total of 80 samples were collected over the 10-week study. The plasma was separated and stored in a –80°C freezer until analysis. Samples were measured in triplicate for eachassay. Results were compared using Pearson correlation analysis, linear regression, and Bland-Altman analysis, with significance set at P < 0.05. There was a good correlation (0.76) between the 2 assays. The linear regression (TD = 1.5*AP+1.9, r2 = 0.58) and the Bland-Altman (bias 2.7, 95% LOA 0.31 to 5.06) analyses indicated that the TD assay results in a higher haptoglobin measurement, with the difference between the assays increasing at higher concentrations. The TD inter-assay and intra-assay coefficient of variations were 11.4% and 4.4%, respectively. The TD measurements ranged from 1.2 to 8.8 mg/mL with a median of 3.9 mg/mL. The AP inter-assay and intra-assay coefficient of variations were 10.5% and 5.4%, respectively. The AP measurements ranged from 0.1 to 5.6 mg/mL with a median of 1.2 mg/mL. In conclusion, TD haptoglobin measurements were consistently higher than those quantified using the AP assay. The linear relationship indicates that calibration would facilitate some comparison between studies using the different assays; however, there is still significant unexplained variation. With no accepted gold standard for haptoglobin measurement, a more complete validation of one or both commonly used assays is required.

Efficacy of auditory integration therapy (AIT) on plasma syntaxin1A (STX1A) levels and amelioration of behavioral, social, and sensory symptoms in children with autism spectrum disorder (ASD)

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder. Previous research reported the beneficial effects of Auditory Integration Training (AIT) on a considerable range of behavior and learning problems. Limited studies examined the association between AIT and biological biomarkers in autistic subjects. Therefore, this study aims to examine the effect of auditory integrative training on the plasma syntaxin1A protein (STX1A) level and also to assess its impact on behavioral, social, and sensory symptoms in autistic children, using a sandwich enzyme-linked immunoassay (ELISA). Total scores of the Childhood Autism Rating Scale (CARS), Social Responsiveness Scale (SRS), and Short Sensory Profile (SSP) were calculated before one month and three months after AIT for all participants. Results show that the plasma level of STX1A was significantly increased immediately, one month, and three months after AIT (P&lt;0.05). Moreover, Pearson correlation (r) values between STX1A levels before and after AIT shows strong and positive significant correlations between STX1A levels before AIT and immediately after AIT (r=0.594, p=0.01) and one month after AIT (r=0.819, p=0.01). Additionally, our results revealed that behavioral, social, and sensory symptoms were significantly improved in terms of disease severity three months after AIT (p&lt;0.05). The study supports the usefulness of AIT as a therapeutic intervention to improve some measures of ASD such as symptoms. It may also induce the up-regulation of STX1A in plasma in ASD subjects. However, Additional research, on a larger size population, is necessary to evaluate the AIT effect on behavioral and social changes in ASD children, and the up-regulation of STX1A.

Bevacizumab suppressed degenerative changes in articular cartilage explants from patients with osteoarthritis of the knee

BackgroundThis study was designed to test the hypothesis that blockade of vascular endothelial growth factor (VEGF) suppresses degenerative changes in articular cartilage from patients with osteoarthritis (OA).MethodsArticular cartilage from eight OA patients was subjected to explant culture for 2 days in the presence or absence of 10 ng/ml recombinant interleukin (IL)-1β. The blocking effect of VEGF was examined by the addition of 10 or 100 ng/ml of bevacizumab. The culture media were harvested, and markers for cartilage degradation were measured by sandwich enzyme-linked immunoassay. Total RNA was isolated from cartilage tissues, and gene expressions associated with the anabolic response were examined by the quantitative real-time polymerase chain reaction.ResultsBevacizumab significantly reduced concentrations of matrix metalloproteinase (MMP)-2, MMP-3, and cartilage oligomeric matrix protein in the culture media with and without IL-1β. Significant suppressive effects of bevacizumab on MMP-9 and MMP-13 were shown only in the presence of IL-1β. Gene expression of Col2a1 was significantly increased by the addition of bevacizumab in the absence of IL-1β.ConclusionBevacizumab inhibits catabolic reactions and stimulates anabolic function in articular cartilage derived from OA patients directly, suggesting a protective effect on articular cartilage from OA progression.

A Nitrocellulose Paper-Based Multi-Well Plate for Point-of-Care ELISA.

Low-cost diagnostic tools for point-of-care immunoassays, such as the paper-based enzyme-linked immunoassay (ELISA), have become increasingly important, especially so in the recent COVID-19 pandemic. ELISA is the gold-standard antibody/antigen sensing method. This paper reports an easy-to-fabricate nitrocellulose (NC) paper plate, coupled with a desktop scanner for ELISA, which provides a higher protein immobilization efficiency than the conventional cellulose paper-based ELISA platforms. The experiments were performed using spiked samples for the direct ELISA of rabbit IgG with a limit of detection (LOD) of 1.016 μg/mL, in a measurement range of 10 ng/mL to 1 mg/mL, and for the sandwich ELISA of sperm protein (SP-10) with an LOD of 88.8 ng/mL, in a measurement range of 1 ng/mL to 100 μg/mL. The described fabrication method, based on laser-cutting, is a highly flexible one-step laser micromachining process, which enables the rapid production of low-cost NC paper-based multi-well plates with different sizes for the ELISA measurements.

Characterization and application of a series of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has a significant global social and economic impact, and the emergence of new and more destructive mutant strains highlights the need for accurate virus detection. Here, 90 monoclonal antibodies (MAbs) that exclusively reacted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) were generated. These MAbs did not cross-react with NPs of common human coronaviruses (HCoVs, i.e., 229E, OC43, HKU1, and NL63) and Middle East Respiratory Syndrome Coronavirus. Subsequently, overlapped peptides in individual fragments (N1-N4) of NP were synthesized. N1-3 (25-GSNQNGERSGARSKQ-39), N3-1 (217-AALALLLLDRLNQL-230), and N4-8 (393-TLLPAADLDDFSKQL-407) were identified as major epitopes using enzyme-linked immunoassay (ELISA) and recognized by 47, 1, and 18 MAbs, respectively. The 24 remaining MAbs exhibited no reactivity with all synthetic peptides. Among MAb-epitope pairs, only MAbs targeting epitope N1-3 displayed no cross-reaction with NPs of SARS-CoV-1 and other SARS-related CoVs. All Omicron variants contained a three-amino acid deletion (31ERS33) in the N1-3 region. Thus, MAbs targeting N1-3 failed to recognize these variants. Furthermore, a double-antibody sandwich ELISA for antigen detection was established using the optimal MAbs. Overall, a series of MAbs targeting SARS-CoV-2 NP was prepared, characterized with epitope mapping, and applied for the detection of SARS-CoV-2 antigens, and some novel B-cell epitopes of the viral NP were identified.

Increased plasma asprosin levels are associated with overeating and loss of control in drug-free bulimia nervosa.

Abnormalities in appetite hormones have been implicated in bulimia nervosa (BN). Orexigenic hormone asprosin has been reported to be associated with food intake and weight gain, but no relevant studies have yet been reported in BN. This study investigated asprosin concentrations and their association with eating disorder symptoms in patients with BN. This study recruited a total of 26 BN patients and 23 healthy controls (HC). Symptom severity for eating disorders, depression, and anxiety was determined by the Eating Disorder Examination Questionnaire 6.0, Beck Depression Inventory, Version 2, and Beck Anxiety Inventory, respectively. In addition, the study employed sandwich enzyme-linked immunoassay technology to determine plasma asprosin and glucose concentrations in all participants. The results revealed that plasma asprosin concentrations were significantly higher in BN patients than in HC (P = 0.037), but the difference disappeared after adjusting for the covariate BMI (F = 2.685, P = 0.108). Correlation analysis showed that asprosin concentration was positively correlated with overeating (r = 0.451, P = 0.021) and eating loss of control (r = 0.483, P = 0.012) in BN patients. Linear regression analysis indicated that an increase in asprosin concentration was associated with an increase in the times of overeating (F = 6.303, P = 0.019, R2 = 0.208). Multiple linear regression showed that increases in asprosin concentration and BDI-II total score could explain the frequent eating loss of control (F = 5.766, P = 0.009, R2 = 0.334). The present study is the first report of plasma asprosin concentration in BN patients and found that overeating and eating loss of control increased with the increase of asprosin concentration. Additionally, asprosin level and degree of depression may explain the frequency of loss of control. Level III: Evidence obtained from case-control studies.

Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer–antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.

Determination of some hematological profiles, inflammatory markers, and their relation with insulin resistance in anemic Type 2 diabetes mellitus patients

Background: Extreme alterations in hematological values and inflammatory biomarkers affecting dependent variables in clinical tests are associated with type-2 diabetes mellitus and anemia. Objective: To investigate some hematological and inflammatory risk factors and their relation in developinginsulin resistance in anemic type-2 diabetes mellitus. Patients and Methods: This case-control study was conducted in 190 patients and healthy subjects in the hospital of Layla Qasim Center in Erbil city and ethical clearance was obtained from the university ethical review committee. The included subjects were divided into three groups: non-diabetic (control) group, non-anemic type-2 diabetes mellitus, and anemic type-2 diabetes mellitus. Blood samples were analyzed for hematological parameters by Coulter counter analyzer, and biochemical tests included glycated hemoglobin, fasting blood glucose, insulin, and some inflammatory biomarkers by spectrophotometry and standard sandwich enzyme-linked immunoassay. Harvested data were analyzed by the statistical package for social science version 23. Results: The results showed remarkable alterations in glycated hemoglobin, red and white blood corpuscles, and inflammatory parameters in anemic type-2 diabetes mellitus compared to non-anemic type-2 diabetes mellitus. Inflammatory parameters including interleukin-1 and tumor necrosis factor-alpha exhibited a strong positive correlation with anemic diabetes than non-anemic diabetes. Conclusion: We concluded that some hematological and biochemical parameters were positively related to developing anemic type-2 diabetes mellitus and can be helpful as diagnosing tools for predicting the progression of anemic type-2 diabetes mellitus. Keywords: Anemia; Insulin resistance; Inflammatory biomarkers; Type 2 diabetes mellitus

Outcome and Determinants of Neutropenic Enterocolitis in Pediatric Cancer Patients.

Neutropenic enterocolitis (NEC) is a dreaded complication of chemotherapy. There is scant literature regarding incidence, clinical features, and determinants. The understanding of gut dysbiosis in NEC and pediatric cancer is evolving. Pediatric cancer patients with neutropenia and gastrointestinal symptoms were evaluated for NEC with contrast-enhanced computed tomography abdomen. Clinical, imaging, and laboratory features were analyzed. Fecal samples were analyzed for fecal calprotectin by sandwich enzyme-linked immunoassay and gut microbiota by conventional culture and compared with healthy controls and children without NEC. NEC was diagnosed in 44 children based on clinical and imaging features with incidence of 7.4% (4 had recurrent episodes). Common manifestations included fever (98%), pain abdomen (88%), and diarrhea (83%). Hypoalbuminemia was observed in 78% of patients. Large bowel involvement (94%) with diffuse bowel involvement (63%) and pancolitis (64%) were common. Fecal calprotectin was significantly elevated in NEC group than non-NEC group and healthy controls (median: 87, 53, and 42 µg/g, respectively). A higher degree of gut dysbiosis was observed in children with NEC with higher isolation of Bacteroides and infrequent isolation of Lactobacilli. Mortality rate of 23% was observed. Only the presence of free fluid predicted higher mortality. Though levels of fecal calprotectin and gut dysbiosis were higher in NEC, they did not increase mortality. Isolation of Bacteroides and absence of Lactobacilli predicted a longer duration of intravenous alimentation. NEC caused significant morbidity and mortality in pediatric cancer patients. Gut dysbiosis was significantly higher in NEC group suggesting a role in pathogenesis and influencing outcome. This highlights the role of targeted interventions towards gut dysbiosis like prebiotics and probiotics.

Serum GP88 as a predictive biomarker for hepatocellular carcinoma in patients with viral hepatitis C after direct-acting antiviral agents.

Progranulin (GP88) is an 88-kDa glycoprotein growth factor with important biological effects in tumorigenesis and tumour survival. We investigated the usefulness of measuring serum GP88 concentrations as a predictive biomarker for hepatocellular carcinoma in patients with viral hepatitis C after treatment with direct-acting antiviral agents. We measured the serum GP88 concentrations by using a sandwich enzyme-linked immunoassay from 67 healthy control subjects and 29 patients (20 patients who did not develop hepatocellular carcinoma and 9 patients who developed hepatocellular carcinoma after treatment) with viral hepatitis C after treatment with asunaprevir and daclatasvir. The serum GP88 concentrations of patients with chronic hepatitis C prior to antiviral treatment were significantly higher than those of healthy control subjects. After antiviral treatment, the serum GP88 concentrations of patients who eventually developed hepatocellular carcinoma were significantly higher than those who did not develop hepatocellular carcinoma. The changes in the serum GP88 concentrations before and after treatment in patients who developed hepatocellular carcinoma were significantly lower than those in patients who did not develop hepatocellular carcinoma. The cumulative incidence of hepatocellular carcinoma was significantly higher in either patients with high serum GP88 concentrations after treatment or those with small changes of serum GP88 concentrations pre- and post-treatment. Sustained high concentrations of serum GP88 in patients treated with direct-acting antiviral agents are correlated with the risk of developing hepatocellular carcinoma.

Serum epidermal growth factor-like domain 7 serves as a novel diagnostic marker for early hepatocellular carcinoma

BackgroundEpidermal growth factor-like domain 7 (Egfl7), a recently identified secreted protein, was significantly increased in patients with HCC by our previous studies. However, its efficacy in the diagnosis of early HCC remains unknown. In this study, we therefore evaluate the efficacy of serum Egfl7 for early HCC diagnosis and compare it with alpha-fetoprotein (AFP).MethodsSerum Egfl7 levels in testing cohort (1081 participants) and validation cohort (476 participants) were measured by a sandwich enzyme-linked immunoassay (ELISA). The cut-off value of Egfl7 was determined by Youden’s index and the efficacies of Egfl7 and AFP in diagnosing early HCC were estimated by receiver operating characteristic (ROC).ResultsSerum Egfl7 was significantly elevated in patients with early HCC than all non-HCC controls in whatever Testing Cohort or Validation Cohort. In the Testing Cohort, ROC curves showed the optimum cut-off value of Egfl7 was 2610 ng/mL and Egfl7 showed a significantly higher sensitivity than AFP in discriminating early HCC from healthy individuals (77.4% vs. 65.3%, P = 0.0013) but the area under ROC (AUROC) and accuracy of Egfl7 and AFP were similar (0.860 vs. 0.868, P = 0.704; 80.2% vs. 83.8%, P = 0.184). In distinguishing patients with early HCC from patients with chronic liver disease (CLD), the AUROC, sensitivity, specificity and accuracy of Egfl7 were 0.800, 75.2, 71.7 and 73.5%, which were all significantly higher than AFP (0.675, 61.8, 62.0 and 61.9% in order). Egfl7 also showed a significant higher sensitivity and accuracy than AFP (76.6% vs. 64.0%, P = 0.0031; 79.9% vs. 66.1%, P < 0.0001) in differentiating early HCC patients from non-HCC individuals. Additionally, 70.8% of early HCC patients with negative AFP could be diagnosed by Egfl7 and the combined use of Egfl7 and AFP increased the sensitivity to 91.0%. These results were confirmed by a validation cohort.ConclusionEgfl7 is a valuable serum marker in the diagnosis of early HCC and could complement the efficacy of AFP, especially in distinguishing early HCC from CLD and identifying patients with AFP-negative early HCC.

The Interplay Between Innate Immunity (TLR-4) and sCD40L in the Context of an Animal Model of Colitis-associated Cancer.

Several studies have found elevated soluble CD40 Ligand (sCD40L) in the serum of patients with malignancies as well as those with inflammatory bowel disease (IBD). Our goal was to determine the possible causal role of sCD40L in colitis-associated colorectal cancer (CAC) by using the well-established azoxymethane/dextran sulfate sodium (AOM/DSS) protocol. Twelve wild type (WT) and twelve TLR4 knock out (KO) female C57BL6 mice were divided into 4 experimental groups. Six WT and six TLR4 KO mice were treated with a single intraperitoneal dose (10 mg/kg of body weight) of AOM followed by three 7-day cycles of oral 2.5% DSS. The other two groups included 6 WT and 6 TLR4 KO mice that received only water and served as the control groups. The mice were sacrificed after 84 days. All mice in the AOM/DSS WT group developed CAC while all mice from the AOM/DSS TLR4 KO group were protected from CAC. We measured the serum and pathologic tissue levels of sCD40L with quantitative sandwich enzyme-linked immunoassay (ELISA) and found that serum sCD40L was significantly higher in wild-type mice that developed CAC compared to their healthy counterparts (wild-type and TLR-4 KO controls). In comparison, serum sCD40L levels were comparable between TLR-4 KO mice, which are protected from developing CAC, and their healthy counterparts (wild-type and TLR-4 KO controls). Of note, tissue levels of sCD40L were not affected by the development of CAC. Our findings point to the presence of an axis between TLR-4 and sCD40L, which may lead to decreased immunosurveillance and the subsequent development of colitis-associated cancer.

Usefulness of metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in clinical characterisation of children with newly diagnosed Crohn's disease.

The aim of this study was to determine the relation of non-invasive markers representing gut mucosal damage (metalloproteinase-9 (MMP-9)) and remodelling (tissue inhibitor of metalloproteinase-1 (TIMP-1)) with Crohn's disease (CD) phenotype, disease activity scores (clinical and endoscopic) and radiological evaluation of the ileum in newly diagnosed children. Serum and faecal MMP-9 and TIMP-1 concentrations were determined with the sandwich enzyme-linked immunoassay technique. The performance of each marker with reference to the Paris classification, disease activity scores and magnetic resonance enterography results was assessed using non-parametric tests. A total of 32 children with CD demonstrated higher levels of serum and faecal MMP-9 and TIMP-1 compared with a control group including children without gastrointestinal inflammatory disease (all P < 0.05). Only the serum MMP-9 concentration was significantly higher in children with L3 (ileocolonic) compared with children with L1 (distal ileum). The serum TIMP-1 level was significantly higher in patients with an magnetic resonance enterography-detected ileum involvement longer than 51 mm and in children with severe disease activity compared with other patients. The serum MMP-9 level was lower in patients with stenosis combined with prestenotic dilation compared with children without stenosis. Increased serum and faecal MMP-9 and TIMP-1 concentrations are some reliable markers of inflammation in newly diagnosed children with CD, but without facilitating clear phenotyping of the disease.

The Expression of MIR17HG Protein as a Potential Therapeutic Target in Meningioma

MIR17 host gene (MIR17HG) is a potential therapeutic target for some cancer types. The aim of this study was to assess MIR17HG protein levels in patients with meningioma who had not been reported previously in the literature and comparing with normal meninges tissues. MIR17HG protein levels were measured in 46 samples including 25 meningioma tissues procured during surgery and 21 normal meninges tissues obtained within 4hours of death during autopsy procedures. Each sample was stored at -80°C until the evaluation of MIR17HG protein using a sandwich enzyme-linked immunoassay principle. Results were compared between the groups. MIR17HG protein levels were significantly higher in meningioma tissues compared with controls and difference was statistically significant (P= 0.012). Both World Health Organization grade I and grade II meningiomas had higher MIR17HG protein levels compared with controls and differences were statistically significant (P=0.026 for grade I and P= 0.042 for grade II). Receiver operating characteristic curve analysis was performed to determine the cutoff of MIR17HG protein value in differentiating meningioma and control groups. At the cutoff value for MIR17HG protein of >0.0998 ng/mL, the sensitivity was 73.91%, 71.43%, and 77.78% and area under the curve was 0.756, 0.753, and 0.761 for meningioma group, grade I, and grade II subgroups, respectively, and specificity was 69.23% for each group. MIR17HG protein expression was found to have a higher level in meningiomas than in normal meninges tissues in our study. Considering the recurrence and irresectability for some meningiomas, which require further treatment, MIR17HG may be a new target for treatment in meningiomas and our study will shed light on further studies.

Aqueous two-phase system antibody confinement enables cost-effective analysis of protein analytes by sandwich enzyme-linked immunosorbent assay with minimal optical crosstalk.

An aqueous two-phase system formed from polyethylene glycol and dextran was used to uniformly coat the bottom surfaces of the wells of standard 96-well assay plates with capture and detection antibodies to improve the performance and cost-effectiveness of sandwich enzyme-linked immunosorbent assay (ELISA). Using this approach, limits of detection and linear dynamic range values comparable to those obtained for conventional sandwich ELISA were obtained using considerably lower antibody quantities due to the much lower reagent volumes required when antibodies are applied in a dextran solution beneath a polyethylene glycol overlay. Confinement of the antibody reagents to the bottom surfaces of the wells within the dextran phase also dramatically decreased the optical crosstalk present between neighboring wells when using transparent microplates. Adaptation of the conventional single sandwich ELISA for aqueous two-phase system antibody confinement was demonstrated by analysis of standard curves for C-reactive protein, transforming growth factor beta 1, and the chemokine CXCL10.

Effect of Coagulation Factor Concentrates on Markers of Endothelial Cell Damage in Experimental Hemorrhagic Shock.

Plasma-based resuscitation showed protective effects on the endothelial glycocalyx compared with crystalloid resuscitation. There is paucity of data regarding the effect of coagulation factor concentrates (CFC) on the glycocalyx in hemorrhagic shock (HS). We hypothesized that colloid-based resuscitation supplemented with CFCs offers a therapeutic value to treat endothelial damage following HS. Eighty-four rats were subjected to pressure-controlled (mean arterial pressure (MAP) 30-35 mm Hg) and lab-guided (targeted cutoff: lactate >2.2. mmol/L and base deficit > 5.5 mmol/L) HS. Animals were resuscitated with fresh frozen plasma (FFP), human albumin (HA) or Ringer's lactate (RL) and RL or HA supplemented with fibrinogen concentrate (FC) or prothrombin complex concentrate (PCC). Serum epinephrine and the following markers of endothelial damage were assessed at baseline and at the end-of-observation (120 min after shock was terminated): syndecan-1, heparan sulfate, and soluble vascular endothelial growth factor receptor 1 (sVEGFR 1). Resuscitation with FFP had no effect on sVEGFR1 compared with crystalloid-based resuscitation (FFP: 19.3 ng/mL vs. RL: 15.9 ng/mL; RL+FC: 19.7 ng/mL; RL+PCC: 18.9 ng/mL; n.s.). At the end-of-observation, syndecan-1 was similar among all groups. Interestingly, HA+FC treated animals displayed the highest syndecan-1 concentration (12.07 ng/mL). Resuscitation with FFP restored heparan sulfate back to baseline (baseline: 36 ng/mL vs. end-of-observation: 36 ng/mL). The current study revealed that plasma-based resuscitation normalized circulating heparan sulfate but not syndecan-1. Co-administration of CFC had no further effect on glycocalyx shedding suggesting a lack of its therapeutic potential. VExperimental in vivo study.

Sex-specific alteration to α2-antiplasmin incorporation in patients with type 2 diabetes

BackgroundType 2 diabetes mellitus (T2DM) is associated with hypofibrinolysis and increased factor XIII-mediated α2-antiplasmin incorporation into the fibrin clot. It is unclear whether there are sex-related differences in α2-antiplasmin incorporation in relation to impaired clot lysis in T2DM. AimWe investigated α2-antiplasmin incorporation into fibrin clots as a determinant of clot lysability in patients of both sexes with T2DM. MethodsIn a group of 113 T2DM patients, 54 (47.8%) of which were women, we investigated α2-antiplasmin incorporation using an in-house sandwich enzyme-linked immunoassay and plasma clot lysis by turbidimetry, along with fibrinogen and thrombin generation using calibrated automated thrombogram and factor XIII activity. ResultsFemale patients had 15.2% greater α2-antiplasmin incorporation into the fibrin clot (p = 0.008) and slightly higher plasma α2-antiplasmin concentration (p = 0.005) along with 8.4% longer time to 50% lysis (Lys50MA, p = 0.012) compared with men. Female patients had enhanced thrombin generation represented by shorter lag phase (p = 0.042), shorter time to peak (p = 0.033), and higher endogenous thrombin potential (p = 0.003) compared with men, while factor XIII activity was comparable between sexes (p = 0.085). On multivariate regression, patient sex and glycated hemoglobin (HbA1c) level were the predictors of α2-antiplasmin incorporation in the entire patient group, while α2-antiplasmin incorporation was associated with Lys50MA, as were fibrinogen, male sex and body-mass index. ConclusionsThis study suggests that a more compromised fibrinolysis in diabetic women when compared with men could be in part mediated by increased α2-antiplasmin incorporation into the fibrin.

Toxicity Study of a Textile Effluent Treated with Electrohydraulic Discharge and Coagulant/Flocculants

Exposure to complex organic substances present in textile wastewater has been considered a threat to human health and aquatic organisms. Development of appropriate treatment mechanisms, as well as sensitive monitoring assays, is considered important in order to safeguard and protect the delicate natural equilibrium in the environment. In this study, combined coagulation/flocculation and electrohydraulic discharge (EHD) system were explored for treatment of textile wastewater. Pre- and post-treatment samples were used to evaluate process efficiencies. Process efficiencies were evaluated using physicochemical characteristics, and cytotoxicity and inflammatory activities induced in macrophage RAW264.7 cell line. The RAW264.7 cell line was evaluated as an alternative to animals and human blood culture models, whose routine applications are hindered by stern ethical requirements. The toxicity of effluent was evaluated using WST-1 assay. The inflammatory activities were evaluated in RAW264.7 cell culture supernatant using nitric oxide (NO) and interleukin 6 (IL-6) as biomarkers of inflammation. The levels of NO and IL-6 were determined using the Griess reaction assay and double-antibody sandwich enzyme-linked immunoassay (DAS ELISA), respectively. Overall, the results of this study show that combined approaches and not the single EHD system are sufficient for complete removal of chemical oxygen demand (COD) and total organic carbon (TOC), toxicity and inflammatory activities in textile wastewater. The study shows that induction of NO and IL-6 secretions in macrophage RAW264.7 cells is a very sensitive model system to monitor the efficiency of textile effluent treatment processes.