Commercial Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Reduction of Panton-Valentine Leukocidin Production in the Staphylococcal Strain USA300 After In Vitro Ascorbic Acid and Nicotinamide Treatment.

Background Panton-Valentine leukocidin (PVL) is one of the most important determinants of virulence in Staphylococcus aureus. It is associated with a propensity for complicating skin and soft tissue infections and necrotizing pneumonia. This study aims to quantitively examine the effect of ascorbic acid and nicotinamide on PVL production in the reference strain USA300. Methodology Sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantitively measure the production of PVL via the commercial LukS sandwich ELISA kit (IBT Bio-services, MD, USA). Results Incubating USA300 with subinhibitory concentrations of antioxidants resulted in a statistically significant eight-fold reduction in PVL production at 1.25 mg/mL and 30 mg/mL for ascorbic acid and nicotinamide, respectively. Although the mechanism by which antioxidants inhibit PVL production is yet to be elucidated, we suggest that it can be due to interrupting PVL gene expression. Conclusions Ascorbic acid and nicotinamide have the potential to be toxin-suppressing agents that may be effective in supporting the bactericidal effect of antibiotics to improve the outcome of PVL-associated infections; however, further extensive research is required.

Pro-inflammatory cytokines and reproductive hormone responses in bucks post-challenge with Mannheimia haemolytica A2 and its outer membrane protein.

The lipopolysaccharide (LPS) endotoxin and outer membrane protein (OMP) are among the virulence factors of Gram-negative bacteria responsible for inducing pathogenicity in the infected host. OMP and LPS occur on the outer membrane of M. haemolytica A2, the primary aetiological agent of pneumonic mannheimiosis in small ruminants. While the LPS is known to mediate Gram-negative bacterial infection by activating downstream inflammatory pathways, the potential role of OMP during inflammatory responses remained unclear. Hence, this study determined the effect of the OMP of M. haemolytica A2 on the serum concentration of pro-inflammatory cytokines and the male reproductive hormones (testosterone and Luteinizing Hormone). We randomly assigned twelve bucks to three groups (n = 4 bucks each): Group 1 was challenged with 2mL PBS buffer (pH 7.0) intranasally; Group 2 received 2mL of 1.2 X 109CFU/mL whole M. haemolytica A2 intranasally; and Group 3 received 2mL of OMP extract obtained from 1.2 X 109CFU/mL M. haemolytica A2 intramuscularly. Serum samples collected at pre-determined intervals were used for the quantitative determination of the pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) and reproductive hormones (testosterone and LH) using commercial sandwich enzyme-linked immunosorbent assay (ELISA). The serum concentration of IL1β was initially increased within the first-hour post-challenge in Groups 2 and 3, followed by a significant decrease in concentration at 21d and 35d (p < 0.05) in Group 3. Only mild fluctuations in IL-6 occurred in group 2, as opposed to the 1.7-fold rapid increase in TNFα within 2h post-challenge before decreasing at 6h. An increase in pro-inflammatory cytokines was accompanied by an acute febrile response of 39.5 ± 0.38°C (p < 0.05) at 2h and 40.1 ± 0.29°C (p < 0.05) at 4h in Group 2 and Group 3, respectively. Serum testosterone decreased significantly (p < 0.05) in both treatment groups but remained significantly (p > 0.05) lower than in Group 1 throughout the study. There was a moderate negative association between testosterone and IL1β (r = -0.473; p > 0.05) or TNFα (r = -0.527; p < 0.05) in Group 2. Serum LH also showed moderate negative associations with TNFα in Group 2 (r = -0.63; p < 0.05) and Group 3 (r = -0.54; p > 0.05). The results of this study demonstrated that M. haemolytica A2 and its OMP produced marked alterations in serum levels of pro-inflammatory cytokines and male reproductive hormones. The negative correlations between serum testosterone and inflammatory cytokines would suggest the potential role of OMP in causing male infertility by mediating innate inflammatory responses to suppress testosterone production in bucks.

Glucose induced ApoB100/ApoAI ratio changes in cultured HepG2 cells in vitro

Backgroundː Numerous in vivo human cohort studies have suggested that the apolipoprotein B100/apolipoprotein AI (ApoB100/ApoAI) ratio might be a risk factor in coronary heart disease. The aim of this study was to measure ApoB100/ApoAI ratio changes in cell secretions by incubating HepG2 cells with various amounts of glucose in vitro. Methods ː HepG2 cells were cultured in low-, normal- or high-glucose Dulbecco's Modified Eagle Medium (DMEM) (1, 4.5 and 10g/L, respectively). Levels of ApoAI and ApoB100 were measured with commercial sandwich enzyme-linked immunosorbent assay kits (cat#: H0123 and H0124) from ShangHai MEIXUAN Biological Science and Technology Ltd (Shanghai, China). Experiments were repeated six times for each assay. Resultsː The results showed that ApoB100/ApoAI ratio have positive correlations with the glucose concentration increase. Conclusionsː A higher concentration of glucose induced an undesirable ApoB100/ApoAI ratio change, which suggests a new regulatory pathway in lipoprotein catabolism and provides a cell model for further mechanism study. This finding may lead to novel therapeutic ways for diagnosis and treatment for coronary artery disease.

Diagnostic Performance of Erythropoietin and Erythropoietin Receptors Levels in Children with Attention Deficit Hyperactivity Disorder.

ObjectiveAttention deficit hyperactivity disorder (ADHD) is a heterogeneous, highly heritable, a common childhood neurobehavioural disorder resulting from complex gene-gene and gene-environment interactions. The erythropoietin (Epo)/erythropoietin receptors (EpoR) system turned out to have additional important functions in nonhematopoietic tissue. In this study, we aimed to investigate the levels of Epo and and EpoR, and also their diagnostic values in children with ADHD.MethodsA total of 70 children were included in the study, 35 drug-naive patients with ADHD (age 6−12 years; male/female 20/15) and 35 healthy controls (age 6−12 years; male/female 22/13). Serum Epo and EpoR levels was determined using a commercial sandwich enzyme-linked immunosorbent assay kit.ResultsThe results indicated that the levels of Epo decreased in patients with ADHD compared to control (p < 0.05). On the other hand, EpoR levels increased in these patients (p < 0.05). Furthermore, the ratio of Epo/EpoR was significantly lower in ADHD patients than controls (p < 0.05). Receiver operator characteristic curve analysis showed high diagnostic performance for Epo and EpoR, areas under curve were 0.980 and 1.000, respectively.ConclusionThis is the first report to investigate the association between serum Epo and EpoR levels in ADHD patients. Our results indicated that Epo may play a role in the etiology of ADHD, and Epo therapy may be beneficial in these disorders if given in addition to the routine treatment of children with ADHD. Furthermore, our results reveal possible diagnostic value of Epo and EpoR.

Glucose induced hepatic lipase expression and ApoB100/ApoAI ratio changes in cultured HepG2 cells in vitro

Backgroundː Hepatic lipase (HL) plays a very important role in lipoprotein catabolism. The aim of this study was to measure both HL activity and ApoB100/ApoAI ratio changes in cell secretions by incubating HepG2 cells with various amounts of glucose. Methodsː HepG2 cells were cultured in low-, normal- or high-glucose Dulbecco's Modified Eagle Medium (DMEM) (1, 4.5 and 10g/L, respectively). HL activities were determined using the Hepatic Lipase Detection Kit (cat. no. A067) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Levels of ApoAI and ApoB100 were measured with commercial sandwich enzyme-linked immunosorbent assay kits (cat#: H0123 and H0124) from ShangHai MEIXUAN Biological Science and Technology Ltd (Shanghai, China). Experiments were repeated six times for each assay. Resultsː Pearson’s correlation coefficient results showed that ApoB100 and ApoB100/ApoAI ratio have positive and significant correlations with HL activity, and ApoAI has a negative and significant correlation with HL activity. Conclusionsː Glucose may increase or decrease ApoB100/ApoAI ratio through upregulation or downregulation of hepatic lipase activity, which suggests a new regulatory pathway in lipoprotein catabolism. This finding may lead to novel therapeutic ways for diagnosis and treatment for coronary artery disease.

Detection of follistatin-based inhibitors of the TGF-β signaling pathways in serum/plasma by means of LC-HRMS/MS and Western blotting.

Cytokines of the transforming growth factor beta (TGF-β) superfamily such as myostatin and activin A are considered as key regulators of skeletal muscle mass. In vivo, their activity is controlled by different binding proteins such as follistatin (FST), whose interaction with the circulating growth factors prevents activation of the activin type II receptors. FST-based protein therapeutics are therefore not only promising drug candidates for the treatment of muscular diseases but also potential performance-enhancing agents in sports. Within this study, two complementary detection assays for FST-based inhibitors of the TGF-β signaling pathways in doping control serum and plasma samples were developed by using both monomeric FST and dimeric FST-Fc fusion proteins as model compounds. The initial testing procedure is based on immunoaffinity purification, tryptic digestion, and LC-HRMS/MS, offering high specificity by targeting tryptic signature peptides of FST. As the glycoprotein is also produced endogenously, the confirmation method employs immunoaffinity purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blotting in order to detect the intact proteins and differentiate synthetic FST-Fc constructs from naturally occurring FST isoforms. Both assays were found to be highly specific with an estimated detection limit of 10 ng/ml. Moreover, a commercial sandwich enzyme-linked immunosorbent assay was used to determine endogenous FST values. The detected FST serum levels of healthy volunteers were found below 5 ng/ml, which is in accordance with reference values from the literature and below the doping control detection methods' limit of detection (LOD). The presented assays expand the range of available tests for emerging doping agents, and the initial testing procedure can readily be modified to include further protein drugs.

SUN-199 URINARY ANGIOTENSINOGEN TO CREATININE RATIO (UAGT/CR) IS NOT A PREDICTIVE BIOMARKER FOR RENAL PROGRESSION IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE: KNOW-CKD STUDY

Decreased renal blood flow by cystic compression and subsequent hypertension has been suggested as one of the main mechanisms for renal progression in autosomal dominant polycystic kidney disease (ADPKD) and intrarenal renin-angiotensin-aldosterone system (RAAS) is known to play the major role in this process. Urinary angiotensinogen to creatinine ratio (UAGT/Cr) was suggested as a novel biomarker to reflect intrarenal RAAS activity. Previous study demonstrated that ADPKD patients have 5- to 6- fold higher level of UAGT/Cr compared to non-ADPKD CKD patients and the level of UAGT/Cr was associated with decreased renal function. However, whether UAGT/Cr can predict renal function decline or kidney growth has not been elucidated. This study was performed to evaluate UAGT/Cr in a prospective ADPKD cohort and demonstrate its usefulness as a predictive biomarker in ADPKD patients. From April 2011 to February 2016, a total of 364 ADPKD patients were enrolled in the prospective cohort called KoreaN Cohort Study for Outcomes in Patients With Chronic Kidney Disease (KNOW-CKD). UAGT was measured by commercial sandwich enzyme-linked immunosorbent assay (ELISA) (Immuno-Biological Laboratories, Co., Ltd., Gunma, Japan) and adjusted by urinary creatinine (Cr) content. Serum Cr was measured by IDMS-traceable method at a central laboratory and glomerular filtration rate (GFR) was estimated by CKD-EPI(Cr) equation. Annual GFR decline rate was calculated from the difference between final and initial Cr divided by follow-up years. Total kidney volume (TKV) was measured upon enrollment and every two years afterwards. HtTKV was calculated by TKV divided by the height of the patient. The annual eGFR decline rate and HtTKV growth rate was compared among UAGT/Cr quartile groups. The variables that do not follow normal distribution was natural log-transformed before analysis. The mean age was 47 years and male were 184 (50.5%). Most of the patients were taking angiotensin converting enzyme inhibitors or angiotensin receptor blockers (85.9%). Baseline estimated GFR was 68.1±33.3 mL/min/1.73m2 and HtTKV was 740.7±541.6 cc/m. Mean UAGT.Cr was 16.3±45.3 μg/g. Ln(UAGT/Cr) was negatively correlated with estimated GFR (r2=0.03, p=0.001) and positively correlated with Ln(HtTKV) (r2=0.03, p=0.004). UAGT/Cr was significantly increased as CKD stage progressed (8.1±20.9 in stage 1 vs. 8.9±16.4 in stage 2 vs. 12.9±16.2 in stage 3 vs. 28.6±74.1 in stage 4 vs. 125.2±138.4 in stage 5, p<0.001). A total of 246 patients with relatively preserved GFR (>30 mL/min/1.73m2) were evaluated for the effect of UAGT/Cr upon prospective eGFR decline rate and HtTKV growth rate. The mean follow-up duration was 45.3±14.8 months. The highest quartile UAGT/Cr group showed higher systolic and diastolic blood pressure (131.9±13.6 and 83.0±10.9 mmHg, p<0.05), lower GFR at baseline (63.9±20.9 mL/min/1.73m2, p<0.001) and higher HtTKV (816.5±499.2 cc/m, p=0.079). However, neither annual GFR decline rate or HtTKV growth rate was statistically different among UAGT/Cr quartile groups. Ln(UAGT/Cr) was not associated with either annual GFR decline rate (r2=0.01, p=0.876) or Ln(HtTKV growth rate) (r2=0.036, p=0.761). UAGT/Cr is a marker of concurrent renal function and HtTKV. However, UAGT/Cr cannot predict renal progression in ADPKD.

Serum and follicular fluid fetuin-B levels are correlated with fertilization rates in conventional IVF cycles.

To investigate the relationship between serum/follicular fluid fetuin-B levels and fertilization outcomes in conventional IVF cycles. A prospective cohort study of conventional IVF treatments including 78 cycles with low fertilization rates (two pronuclei [2PN] rate < 30%; LF group) and 104 cycles performed during the same period with 2PN rate > 70% (high fertilization group, HF). To calculate the required sample size, a two-sample t test power analysis was applied to data from our pilot study, using PASS 11.0 software. Fetuin-B was measured using a commercial sandwich enzyme-linked immunosorbent assay. Serum fetuin-B and follicular fluid fetuin-B were positively correlated (r = 0.703, P < 0.001). Compared to the HF group, the LF group had significantly lower levels of fetuin-B, both in serum (5.81 ± 1.53 vs. 7.19 ± 1.42, P < 0.001) and follicular fluid (5.06 ± 1.29 vs. 6.16 ± 1.52, P < 0.001). The serum fetuin-B level from cycles with polypronuclear (PPN) zygotes was significantly lower when compared to cycles without PPN zygotes (6.82 ± 1.65 vs. 6.10 ± 1.43, P = 0.006). However, serum fetuin-B level was not correlated with preimplantation embryo development or clinical pregnancy. Serum fetuin-B level is correlated with fertilization rate in conventional IVF and it may be used as a predictive marker of fertilization in IVF treatment.

Evaluation of estrogen and G protein-coupled estrogen receptor 1 (GPER) levels in drug-naïve patients with attention deficit hyperactivity disorder (ADHD).

Estrogen has a crucial role in the regulation of reproductive and neuroendocrine function and exerts its effects through two classes of receptors, nuclear and membrane estrogen receptors (mERs). G protein-coupled estrogen receptor 1 (GPER) is a member of mERs, and despite limited research on the levels of GPER in patients with psychiatric diseases, a role of GPER in such conditions has been suggested. Here we evaluated serum estrogen and GPER levels in children with attention deficit hyperactivity disorder (ADHD) in relation to their age- and gender-matched healthy controls. A total of 82 children were included in the study, 47 drug- naïve patients with ADHD (age: 6-12 years; male/female: 34/13) and 35 healthy controls (age: 6-12 years; male/female: 19/16). The subgroups according to ADHD types were inattentive, hyperactive/impulsive, and combined. Serum estrogen was measured using an immunoassay system, while serum GPER was determined using a commercial sandwich enzyme-linked immunosorbent assay kit. Estrogen levels in children with ADHD were similar as in control group, while GPER levels were significantly lower in ADHD group compared to controls (p < 0.05). Logistic regression analysis showed a significant association between GPER levels and ADHD (p < 0.05), and no association between estrogen levels and ADHD (p > 0.05). No significant differences were found in GPER and estrogen levels between ADHD subgroups (p > 0.05). To the best of our knowledge, this study is the first to investigate estrogen and GPER levels in ADHD. Our preliminary findings suggest a relationship between serum GPER levels and ADHD, and this should be further investigated.

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

IL-33 gene variants and protein expression in pediatric Tunisian asthmatic patients

Interleukin-33 (IL-33) is one of the last discovered members of the human IL-1 family. It is involved in the pathogenesis of many inflammatory diseases. This study investigates the relationship between IL33 gene variants and serum protein levels with the development of childhood asthma. We analyzed in this case-control study the distribution of two IL33 polymorphisms, rs7044343 and rs1342326, within 200 Tunisian children, using predefined Taqman genotyping assays. IL-33 serum levels were assessed by commercial sandwich Enzyme-linked immunosorbent assay (ELISA). The presence of rs1342326 polymorphism was significantly associated with a lower risk of asthma development. The CC [OR=0.20, CI (0.08–0.50)] and AC [OR=0.24, CI (0.11–0.49)] genotypes, as well as the C-allele [OR=0.40; CI: 0.26–0.61, P=0.00001] were associated significantly with a decreased asthma risk. However, the C-allele was more frequent in severe asthma patients than in milder ones. No association was found between rs7044343 variant and asthma. The level of IL-33 in sera was significantly increased in asthmatic children [1.48±0.47pg/mL] compared to controls [0.70±0.18pg/mL; P<0.001]. Furthermore, this increase of IL-33 was associated with the presence of rs1342326 C allele. The IL33 rs1342326 polymorphism was associated with a lower childhood asthma risk in the Tunisian population and a higher IL-33 protein expression.

A rapid and highly sensitive immunoassay format for human lipocalin-2 using multiwalled carbon nanotubes

A sensitive and rapid sandwich immunoassay (IA) was developed for human lipocalin-2 (LCN2) by functionalizing a KOH-treated polystyrene microtiter plate with multiwalled carbon nanotubes (MWCNTs) dispersed in 3-aminoproyltriethoxysilane (APTES). The significantly increased surface area due to the presence of MWCNTs led to a high immobilization density of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activated anti-LCN2 capture antibodies (Ab). The anti-LCN2 Ab-bound MTPs were stable for 6 weeks when stored in 0.1M PBS, pH 7.4 at 4°C. The IA detects LCN2 from 0.6 to 5120pgmL−1 with a limit of detection (LOD) and limit of quantification (LOQ) of 0.9pgmL−1 and 6pgmL−1, respectively. The assay offered a ~50-fold lower LOD and ~3-fold faster IA, compared to a commercial sandwich enzyme-linked immunosorbent assay kit.

Association of high fetuin-B concentrations in serum with fertilization rate in IVF: a cross-sectional pilot study

Is serum fetuin-B associated with the fertilization rate in in vitro fertilization (IVF)? Serum fetuin-B increased during IVF cycles when oocytes could be fertilized while remained unchanged in fertilization failure. Fetuin-B deficiency in mice causes premature zona pellucida hardening mediated by the zona protease ovastacin. Thus fetuin-B deficiency renders females infertile. We determined the human serum fetuin-B reference range, studying longitudinally, over the course of one month, five male and seven female volunteers without hormone treatment and four female volunteers on varying hormonal contraception. We sampled blood and determined serum fetuin-B, luteinizing hormone (LH), estradiol (E2) and progesterone (P4). In addition, we determined serum fetuin-B and estradiol in eight women undergoing intracytoplasmatic sperm injection (ICSI, nine ICSI cycles) and 19 women undergoing IVF (21 IVF cycles) after ovarian stimulation with recombinant human follicular stimulating hormone (rFSH) and/or a combined medication of FSH and LH. At least three blood samples were analyzed in each cycle. We compared serum fetuin-B and follicular fluid fetuin-B in nine patients by measuring follicular fetuin-B in pooled follicular fluid, and in fluid obtained from individual follicles. Samples were drawn from January 2012 to March 2014. All volunteers and patients gave informed consent. Fetuin-B was measured employing a commercial sandwich enzyme-linked immunosorbent assay. Serum fetuin-B was determined as duplicates in 5 male (34 ± 14.6 years) and 11 female volunteers (29.4 ± 4.1 years) as well as in female volunteers on hormonal contraception (30.0 ± 6.5 years). The duplicate standard deviation was 4.0 ± 2.3%. The contraceptive drugs were mono or combined preparations containing 0-0.03 mg ethinyl estradiol, and 0.15-3.0 mg of various progestins. In addition, serum fetuin-B was determined as triplicates in 27 female patients undergoing conventional IVF (19) or ICSI (8). The triplicate standard deviation was 3.3 ± 1.8%. IVF was declared as 'successful', if at least one oocyte was fertilized, and 'unsuccessful', if no oocyte could be fertilized. Patient age was 34.4 ± 4.4 years in successful IVF, and 35.4 ± 3.3 years in unsuccessful IVF. Serum and follicular fluid of patients undergoing controlled ovarian hyperstimulation were analyzed. Serum was drawn at the day of follicle aspiration. Serum fetuin-B and follicular fluid fetuin-B were not significantly different in six out of nine patients suggesting, in principle, free exchange of fetuin-B between serum and follicular fluid. Thus serum fetuin-B may be used as a proxy of follicular fluid fetuin-B. Serum fetuin-B increased during successful IVF cycles (n = 15, P < 0.0001), but did not change in unsuccessful IVF cycles (n = 6, P = 0.118) despite increased estradiol levels (P = 0.0019 and P = 0.0254, respectively). The female volunteers self-reported their respective hormone medication. Medication was verified by serum estradiol, LH and progesterone measurements. For oocyte harvesting, the vaginal wall was punctured once only to minimize co-morbidity. Low grade cross-contamination of individual follicular fluid aspirates and contamination of the follicular fluid with small amounts of blood were inevitable. Samples were routinely checked for the presence of hemoglobin that would suggest blood contamination. Only samples containing <250 erythrocyte equivalents/µl were used for analysis. Serum fetuin-B may be used as a marker to predict the fertilization success in IVF. Fetuin-B levels attained during IVF stimulation may help to make an informed decision whether oocytes should be fertilized by IVF or by ICSI to overcome the zona pellucida as a barrier. The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. J.F., E.D., J.N., B.R. and W.J.-D. declare that they are named inventors on the RWTH Aachen University patent application EP 13157317.2, 'Use of fetuin-B for culture of oocytes', applied for by RWTH Aachen University.

The correlation between tenascin-C expression, and formation of intestinal stricture.

OBJECTIVE:A strong correlation exists between tenascin-C induction, and acute inflammation. Generally increased tenascin-C concentrations are correlated with various inflammatory, and infectious diseases. In patients with diagnosis of Inflammatory Bowel Disease (IBD) presence of tenascin-C in colonic mucosa demonstrates tissue repair, and its mucosal concentrations are correlated with local disease activity Therefore plasma levels of tenascin-C have been demonstrated to be a helpful indicator of the activity of inflammatory bowel diseases. In this study, firstly in the literature, we aimed to display the correlation between tenascin-C expression, and formation of intestinal stricture.METHODS:A total of 43 patients (male, n=19; 44.2%; and female, n=24, 55.8%) aged between 19, and 63 years, with clinically, endoscopically, radiologically, and histopathologically confirmed definitive diagnosis of Crohn’s disease who were examined, diagnosed, and treated in the Gastroenterology Clinic of Haydarpasa Numune Training and Research Hospital between January 2011, and April 2012 were investigated. Serum tenascin-C levels were measured using commercial sandwich enzyme-linked immunosorbent assay Human Tenascin-C Purified Protein kit (Chemicon, Millipore(R), USA). Study groups were categorized based on the type of the disease as inflammatory (n=17; 39.5%), obstructive (27.2%), and fistule formation (n=10; 23.3%) Crohn’s disease. For statistical analysis SPSS (Statistical Package for Social Sciences) Statistics 15 program was used.RESULTS:Median tenascin- C value in the obstructive group (6.57 ng/mL; range, 4.26-21.87 ng/mL) was statistically significantly higher than that detected in the inflammatory (1.74 ng/mL; range,1.29-3.16 ng/mL), and fistulizing (1.44 ng/mL; range, 0.74-2.47 ng/mL) groups (p=0.002).CONCLUSION:Intestinal fibroblasts have an important role in the stricture formation process in CD. Transforming growth factor (TGF)-b1 cytokine is in the center of this process. A strong correlation exists between tenascin-C induction, and acute inflammation. As a known fact, serum tenascin-C levels can be used in the determination of activity of IBD. Starting from this point, serum tenascin-C levels can be useful in the categorization of the Crohn’s disease without the need for invasive methods. In the future, studies with larger patient series investigating use of serum tenascin-C in the prediction of stricturing Crohn’s disease should be conducted.

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

Serum functional dickkopf-1 levels are inversely correlated with radiographic severity of ankylosing spondylitis.

Dickkopf-1 (DKK-1) has been proved dysfunctional in ankylosing spondylitis (AS), and decreased serum functional DKK-1 levels have been identified to be decreased in AS patients. This study aims to examine the relation of binding (functional) DKK-1 levels in serum with the radiographic severity of AS. Total Dkk1 levels were assessed by commercial sandwich enzyme-linked immunosorbent assay and functional DKK-1 levels of serum were measured using functional enzyme-linked immunosorbent assay method in 84 patients with AS and 79 healthy controls. The radiographic progression of AS was classified according to the radiographic events of modified New York Criteria for sacroiliac joints evaluation and modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) system for spine assessment. No significant difference was seen between AS patients and healthy controls for total Dkk1 levels (p = 0.273). Decreased levels of functional DKK-1 in serum were found in AS patients compared with healthy controls 17.24 (1.24-11.15) vs. 9.15 (5.33-13.35) pg/mL, p < 0.001]. Functional DKK-1 levels in serum of AS patients were significantly associated with the disease radiographic severity evaluated by modified New York grading criteria (r = -0.236, p = 0.04) and mSASSS system (r = -0.389, p = 0.025). Serum functional DKK-1 levels showed an independent and negative correlation with radiographic severity of the disease in patients with AS. Functional DKK-1 in serum might serve as a potential biomarker for reflecting the progression of ankylosing spondylitis.

Quenching effect on gold nano-patterned cardiac troponin I chip by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy (TIRFM) was investigated for enhanced detection sensitivity of cardiac troponin I (cTnI) on a gold nano-patterned protein chip. A 4×5 nanoarray patterning of a gold chip based on a single-molecule sandwich immunoassay was prepared with a spot diameter of 500nm on glass substrates formed via electron-beam nanolithography. Quantitative trace analysis was possible as a function of increasing total internal reflection fluorescence intensity in the range of 84aM–350pM at prism-type and 35aM–350pM at objective-type TIRFM. Although using same gold nano-patterned cTnI chips, however, the objective-type TIRFM showed enhanced detection sensitivities 2.4 times better than the prism-type due to weaker gold quenching. This result is also about 7000 times lower in the detection limit compared with those obtained by conventional commercial sandwich enzyme-linked immunosorbent assay techniques.

Soluble Fas in pemphigus vulgaris.

The Fas/Fas ligand (FasL) system has been recognized as an important pathway for apoptosis induction in cells and tissues. It has recently been shown that skin lesions of pemphigus vulgaris are associated with Fas mediated apoptosis. The aim of this study was to evaluate the level of serum soluble Fas of ten newly diagnosed patients with pemphigus vulgaris. Sera were collected from ten patients with pemphigus vulgaris. Commercial sandwich enzyme-linked immunosorbent assay (ELISA) for quantitative detection of soluble Fas was applied. Patients with mucosal skin involvement had higher median values in contrast to patients with cutaneous involvement. Elevation of soluble Fas in our study may give insights for the pathogenesis of pemphigus vulgaris. Suppression of this underlying mechanism may be an important target for novel therapies and relapse prevention.

Detection of hazelnuts and almonds using commercial ELISA test kits

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.

Usefulness of a commercial enzyme-linked immunosorbent assay kit for Candida mannan antigen for detecting Candida in oral rinse solutions

The aim was to test the performance of a commercial enzyme-linked immunosorbent assay (ELISA) kit for Candida mannan antigen for detecting Candida in oral rinse solutions. Forty-eight oral rinse solutions (38 from patients and 10 from healthy volunteers) were available. Mannan antigen was measured using a commercial sandwich ELISA kit, Unimedi Candida. The result of the mannan assay was compared with the result of conventional detection and identification by culture. The result of the mannan assay revealed that 31 of 38 clinical and 3 of 10 healthy volunteer samples were positive for Candida. Using the culture as a gold standard, the overall sensitivity and specificity of the mannan antigen detection were 90.9% and 46.2%, respectively. The results of this study suggested that mannan antigen detection might be a possible and sensitive technique for the detection of oral Candida. The conditions of the ELISA-based assay should be optimized for oral rinse solutions.