Standard Sandwich Enzyme-linked Immunosorbent Assay Research Articles

The concentration of brain homogenates with the Amicon Ultra Centrifugal filters

Accurately measuring the brain concentration of a neurotherapeutic is critical in determining its pharmacokinetic profile in vivo. Biologics are potential therapeutics for neurologic diseases and biologics fused to an antibody targeting a transcytosis receptor at the Blood-Brain Barrier, designated as antibody-biologic fusion proteins, are Blood-Brain Barrier penetrating neurotherapeutics. The use of sandwich immunosorbent assays to measure concentrations of antibody-biologic fusion proteins in brain homogenates has become increasingly popular. The raw brain homogenate contains many proteins and other macromolecules that can cause a matrix effect, potentially interfering with the limit of detection of such assays and reduce the overall sample signal. Further, the low sample loading volumes while running these assays can reduce the sample signal. Our aim was therefore to optimize the existing tissue sample preparation and processing to concentrate the sample to elevate the signal of the analyte. Here, we present a protocol for concentrating and increasing the signal of transferrin receptor antibody-biologic fusion proteins in mouse brain homogenates using the Amicon Ultra Centrifugal filters.• The presented method uses the Amicon Ultra Centrifugal filters to concentrate mouse brain tissue homogenates.• The concentrated brain tissue homogenates are then assayed using standard sandwich enzyme-linked immunosorbent assay (ELISA) protocols.• This method improves upon the traditional brain homogenization procedure and ELISA measurements for antibody-biologic fusion proteins by effectively concentrating brain tissue homogenates.

Study of association of varicose veins and inflammation by inflammatory markers.

In varicose veins, increased levels of inflammatory markers are indicators of endothelial damage and increased procoagulant activity. These findings support the assumption that the constitution of blood in varicose veins differs from that of systemic blood. The purpose of the study was a correlative study of blood constituents in varicose veins and peripheral veins (normal vein) in same individual with varicose vein which was done by comparing the level of concentration of interleukin-6, fibrinogen, haemoglobin from blood of varicose veins and normal peripheral vein (antecubital vein). Using citrated plasma samples withdrawn from arms and legs of same patient and plasma obtained by centrifugation of citrated venous blood at 5000 r/min for 10 min was used for correlation. Serum concentration of interleukin-6 and fibrinogen were determined by human enzyme-linked immunosorbent assay Kit for both interleukin-6 and fibrinogen, which is based on the standard sandwich enzyme-linked immunosorbent assay technology. This assay employs a monoclonal antibody specific for human interleukin-6 coated on a 96-well plate. Expressed as median (interquartile range) in pg/mL, leg samples from patient having varicose vein has significantly increased interleukin-6 in cases as compared to controls (p value of <0.001). Leg samples from patient having varicose vein has significantly increased fibrinogen concentration than their arm samples (p value of <0.001). Concentration of haemoglobin significantly increased in leg samples as compared to blood withdrawn from arms (p value of 0.012). Blood withdrawn from the site of varicose vein appears to have significantly increased concentration of interleukin-6, fibrinogen and haemoglobin when compared to same patient's antecubital blood sample supporting the hypothesis that inflammation is increased in tissues drained by varicose vein.

Correlation between laryngopharyngeal reflux and otitis media with effusion in adults patients

Objective:Using the pepsin A concentration to investigate the relationship among gastroesophageal Reflux/laryngopharyngeal reflux and otitis media with effusion in adults patients.Method: Adults with OME underwent myringotomy with tube placement or eardrum puncturing suction were collected in the study group and the patients with coclear implatation were collected as control group. A standard sandwich Enzyme-linked Immunosorbent Assay (ELISA) method was applied to measure pepsin A concentration in the middle ear fluid.RSI and GERD-Q were used for the patients in study group.Result: Pepsin A concentration was (23.72±26.64; 2.00-84.47 ng/ml) in study group.The positive rates of pepsin A in the patients and samples were 21.69%(18/83) and 17.27%(19/110) respectively.In the control group, no pepsin A was detected in 26 samples. In the study group, 5 (6.02%) were diagnozid with GERD and 19 out of 83 patients (22.89%,19/83) were diagnosed with LPRD.LPRD, 8 out of 18 patients (44.44%) in the pepsin A positive group, was much higher than that in the pepsin A negative group (16.92%)(P<0.01).Conclusion: Laryngopharyngeal reflux was an important pathogeny of OME in adults patients, although those patients maybe have no any symptoms of GERD. The fat maybe be one of a indirect risk factor of OME in adults.

Elevated lipocalin-2 level in aqueous humor of patients with central retinal vein occlusion.

To assess the concentrations of lipocalin-2 (LCN2) in the serum and the aqueous humor of patients with central retinal vein occlusion (CRVO). The concentrations of LCN2 in the serum and aqueous humor of 16 cataract patients and 16 patients with CRVO with macular edema were compared. Collection of aqueous samples was conducted in the operating theater under sterile conditions and just prior to intravitreal ranibizumab injection or cataract surgery. LCN2 levels in serum and aqueous humor samples were measured using a commercial kit (human lipocalin-2/NGAL PicoKine ELISA Kit, MyBioSource Inc., USA; Catalog No: MBS175829) based on standard sandwich enzyme-linked immunosorbent assay technology. The concentrations of LCN2 in the aqueous humors of the CRVO group were higher than those of the control group (p = 0.021). There was no significant difference in serum LCN2 level between the two groups (p = 0.463). Concentrations of LCN2 in aqueous humor are increased in CRVO. LCN2 may be part of a pro-catabolic phenotype, and it may play an important role in the dreaded complications of CRVO, such as macular edema, macular ischemia, and neovascularization, which lead to blindness.

Detection of Plasmodium Aldolase Using a Smartphone and Microfluidic Enzyme Linked Immunosorbent Assay.

Background Malaria control efforts are limited in rural areas. A low-cost system to monitor response without the use of electricity is needed. Plasmodium aldolase is a malaria biomarker measured using enzyme linked immunosorbent assay (ELISA) techniques. A three-part system using ELISA was developed consisting of a microfluidic chip, hand crank centrifuge, and a smartphone. Methods A circular microfluidic chip was fabricated using clear acrylic and a CO2 laser. A series of passive valves released reagents at precise times based upon centrifugal force. Color change was measured via smartphone camera using an application programmed in Java. The microchip was compared to a standard 96-well sandwich ELISA. Results Results from standard ELISA were compared to microchip at varying concentrations (1–10 ng/mL). Over 15 different microfluidic patterns were tested, and a final prototype of the chip was created. The prototype microchip was compared to standard sandwich ELISA (n = 20) using samples of recombinant aldolase. Color readings of standard ELISA and microfluidic microchip showed similar results. Conclusion A low-cost microfluidic system could detect and follow therapeutic outcomes in rural areas and identify resistant strains.

The growth endocrine axis and inflammatory responses after laparoscopic cholecystectomy.

It is well known that conventional surgery leads to detrimental immune and catabolic responses, thus there is growing interest in the effect of minimally invasive techniques on postoperative endocrine and immune function. The aim of this prospective study was to evaluate the growth hormone (GH)/insulin-like growth factor-1 (IGF-1)/IGF binding protein-3 (IGFBP-3) axis and acute phase (interleukin-6, IL-6, and C-reactive protein, CRP) responses in patients who underwent laparoscopic cholecystectomy. Twenty-nine patients (16 women, 13 men; age: 58+8 years) with a history of uncomplicated symptomatic cholelithiasis participated in the study. Blood samples were collected prior to and at 24 hrs and 48 hrs after laparoscopic cholecystectomy. Serum concentrations of GH, IGF-1, IGFBP-3, and IL-6 were determined by standard sandwich enzyme-linked immunosorbent assay (ELISA), while CRP was measured by nephelometry. ANOVA with repeated measures and Tukey's post-hoc test were used to evaluate changes in serum measurements. The laparoscopic cholecystectomy resulted in a significant postoperative increase in circulating levels of IL-6 (p=0.031), which is the main cytokine responsible for inducing the acute inflammatory response, and of the acute phase protein CRP (p=0.005). A significant increase in GH levels at 24 hrs (p=0.034) and decrease of IGF-1 on both postoperative days were also found (p=0.045, 0.044), while no changes were documented in IGFBP-3 levels. Significant correlations were revealed between postoperative levels of the acute phase proteins and growth axis hormones (p<0.05 - 0.001). Our findings suggest that laparoscopic cholecystectomy induces acute phase endocrine and immune responses. These changes may represent a state of systemic inflammation and GH resistance, compatible with possible cytokine-induced anti-anabolic or catabolic effects even after this minimally invasive cholecystectomy.

Performance analysis of rapid diagnostic tests on atypical bovine spongiform encephalopathy

The preferred method to determine the prevalence of bovine spongiform encephalopathy (BSE) in a country is to use immunology-based rapid-tests. Though these tests are validated to detect C-type BSE disease-associated prion (PrP(sc)), test-specific properties may influence their ability to detect H- and/or L-type BSE PrP(sc), where both are atypical from C-type PrP(sc). Molecular characterization shows atypical BSE PrP(sc) to have a different sensitivity to proteinase activity and different affinities for certain prion-specific antibodies. It is important to understand how atypical BSE PrP(sc) may affect the performance of rapid-tests, which are typically dependent on the use of specific proteases and antibodies. The current study used experimentally generated C-, H-, and L-type BSE PrP(sc) to evaluate 3 tests used in various national BSE surveillance programs: an immunochromatographic assay, a standard sandwich enzyme-linked immunosorbent assay (stndELISA), and a PrP(sc)-conformation-specific ELISA (confELISA). Although BSE PrP(sc) type had some effects on rapid-test performance, analytical sensitivity for atypical BSE PrP(sc) on all 3 platforms was not significantly compromised. When testing for atypical BSE PrP(sc), the 3 tests were able to meet the same requirements that the European Food Safety Authority set when evaluating the tests for C-type BSE PrP(sc).

Detection of weak receptor–ligand interactions using IgM and J‐chain‐based fusion proteins

Detection of weak receptor–ligand interactions using <scp>I</scp>g<scp>M</scp> and <scp>J</scp>‐chain‐based fusion proteins

Serum tumor antigen REG4 as a diagnostic biomarker in pancreatic ductal adenocarcinoma

Serum biomarkers for the early detection of pancreatic cancer are not currently available. We evaluated the usefulness of a novel serum marker, REG4, in the diagnosis of pancreatic cancer, as compared to carbohydrate antigen (CA) 19-9. We collected pretherapeutic sera from 92 patients with pancreatic cancer, as well as sera from 28 patients with other pancreatic tumors, 11 patients with pancreatitis, and 69 healthy controls. Serum levels of REG4 were measured using a standard sandwich enzyme-linked immunosorbent assay (ELISA). Compared with healthy controls, serum levels of REG4 were higher in pancreatic cancer patients (P < 0.001), and in patients with pancreatitis (P < 0.001). Receiver operating characteristic (ROC) analysis indicated that serum REG4 performed better than serum CA19-9 for distinguishing patients with pancreatic cancer from healthy controls [areas under the curve (AUC) for REG4 and CA19-9 were 0.922 and 0.884, respectively]. When we validated the study, the sensitivity of REG4 for pancreatic cancer was 94.9%, specificity was 64.0%, and accuracy was 77.5% for the REG4 cutoff value of 3.49 ng/ml. No correlation was seen between serum REG4 and CA19-9 levels, with the sensitivity, specificity, and accuracy of the combined markers reaching 100.0, 60.0, and 77.5%, respectively. No significant differences were seen among any stages of pancreatic cancer. In surgical specimens, immunohistochemical analysis found a correlation between serum REG4 levels and REG4 expression in pancreatic cancers. REG4 is expressed in pancreatic cancer, and serum levels of REG4 offer a useful indicator for distinguishing between patients with pancreatic cancer and healthy subjects. Serum REG4 has potential for use as a screening serum marker for pancreatic cancers, including early-stage cancers.

A Microtiter Assay for Quantifying Protein-Protein Interactions Associated with Cell-Cell Adhesion

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein beta-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of beta-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z' factor of 0.74. The quantitative nature of the E-cadherin:beta-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:beta-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and beta-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: beta-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression.

Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Serum REG4 protein in pancreatic cancer as a tumor marker: A prospective study

15063 Background: Pancreatic cancer (PC) shows the worst mortality rate in common malignancies, with 5-year survival rate of 4%. The only way to cure the disease is surgical resection of early stage PC. Establishment of a screening strategy to detect early stage PC is eagerly expected. REG4, a member of the regenerating islet-derived (REG) family, are secreted proteins that play a role in tissue regeneration and inflammation in digestive organs. We reported overexpression of REG4 in PC cells and serum, and preliminary data of the serum REG4 level of pancreatic disease patients including PC patients. We conducted a prospective study to evaluate the role of serum REG4 in PC. Methods: The series included 57 patients diagnosed pathologically as PC between November 2004 and December 2005. Serum REG4 was quantified by standard sandwich ELISA (Enzyme Linked Immunosorbent Assay) using original kit (MBL116: provided by Medical and Biological Laboratories Co., LTD, Japan) before treatment. The upper limit of the test was set at 3.52ng/ml and was based on studies of serum from 48 healthy control subjects. Results: With a specificity of 100%, the diagnostic sensitivity and accuracy were 63.2% and 80.0%, respectively. The ROC (receiver operating characteristic) analysis showed that area under the curve was 0.91. REG4 levels were a significant differences between PC and control (p&lt;0.001), between each T stage and control (T1,T2, T3 or T4 v control), and between each TMN stage and control (stage 1, stage 2, stage 3 or stage 4 v control), but were not a statistical significance with T stage (T1 v T2 v T3 v T4), M stage (M0 v M1) or TNM stage (stage 1 v stage 2 v stage 3 v stage 4) in PC patients. The diagnostic sensitivity of carcinoembryonic antigen (CEA&gt;5.0ng/ml) and carbohydrate antigen19–9 (CA19–9&gt;50U/ml) was 56.5% and 68.4%, respectively. No significant correlation was demonstrated between REG4 and CA19–9 (coefficient of correlation [rs]=0.45). Conclusions: This study shows the potential of serum REG4 as a screening test for PC, especially for early PC. REG4 is considered to be a more useful marker in combination with CA19- 9. No significant financial relationships to disclose.

Serum Relaxin in the Newborn Is Not a Marker of Neonatal Hip Instability

It has been suggested that intrauterine exposure to high levels of relaxin causes hip instability in newborns. The aim of this work was to evaluate whether the serum relaxin concentration in umbilical vein blood is associated with hip instability in the newborn. Blood was collected by cordocentesis from 2,185 newborns, and serum relaxin levels were obtained by using a standard sandwich enzyme-linked immunosorbent assay (ELISA). Movement of the femoral head in the hip joint was determined for all 2,185 children by the anterior-dynamic ultrasound method. Hereby their status on neonatal hip instability was determined. Fifteen cases (12 girls, three boys) were found, and 106 controls were selected. Only six newborns had unstable hips to a degree requiring treatment. Ten newborns had Ortolani-positive hips. Only three specimens of the 121 samples measured had serum relaxin concentrations above the detection limit of 10 pg/ml. None of these were cases. This study does not show an association of serum relaxin with neonatal hip instability. It is suggested that detectable serum relaxin levels are found in samples from the umbilical cord only when these are contaminated with maternal blood.

Faecal tumour necrosis factor-alpha in individuals with HIV-related diarrhoea.

HIV-related gastrointestinal infection is associated with diarrhoea, weight loss, mucosal inflammation and increased intestinal permeability. As tumour necrosis factor (TNF)-alpha may mediate these features this cytokine was measured in the faeces of HIV-seropositive individuals with diarrhoea to assess its role in the pathogenesis of HIV-related gastrointestinal disease and the association with specific intestinal pathogens. Prospective study. Two hundred and four HIV-seropositive individuals provided stool samples that were analysed for faecal TNF-alpha (FTNF-alpha) using a standard sandwich enzyme-linked immunosorbent assay. Stool from patients with bacterial, cytomegalovirus (CMV) and microsporidial diarrhoea had significantly elevated FTNF-alpha compared with those who had pathogen-negative diarrhoea (P < 0.05). FTNF-alpha was not raised in cryptosporidiosis, pathogen-negative or solid stool. In subjects with diarrhoea of more than 2 weeks duration and three stool samples negative for enteric pathogens, FTNF-alpha greater than 15 U/ml has a sensitivity of 88% and a specificity of 66% for the diagnosis of diarrhoea-related CMV enteritis. TNF-alpha production may have a role in the pathogenesis of bacterial, microsporidial and CMV-related diarrhoea in HIV-seropositive individuals. Thus, anti-TNF-alpha agents may have a therapeutic role in the management of these conditions. FTNF-alpha greater than 15 U/ml in apparently pathogen-negative diarrhoea may suggest endoscopic gastrointestinal biopsy to diagnose CMV enteritis.