Enzyme-linked Sorbent Assay Research Articles

Prevention of Salmonella Infection in Poultry by Specific Egg-Derived Antibody

Salmonella enteritidis (SE) colonizes the intestinal tract of poultry and causes food born illness in humans. Reduction of (SE) colonization in the intestinal tract of poultry reduces potential carcass contamination during slaughter. The purpose of this study was to investigate the effect of SE-specific yolk immunoglobulin (IgY) on prevention of SE colonization in orally infected broiler chickens. Commercial Single Comb White Leghorn (SCWL) hens were hyperimmunized with SE whole cell antigens. The presence of anti-Salmonella antibody, IgY and IgG in egg yolk and serum respectively, was monitored by Enzyme Linked Sorbent Assay (ELISA). Two hundred forty male `Ross 308` day old chicks were randomly assigned to 8 groups and 3 replications of 10 birds were grown for 42 days of experiment. Eight experimental groups identified with, S, P, A, SP, SA, AP, SPA, C. Four birds from four challenged groups (S), were orally inoculated with 1 mL of bacterial suspension that contained 1×106 CFU mL-1 S. enteritidis at 3 day of age. The groups that supplemented with antibody (A) received 15 mL of yolk contained antibody mixed per 3.84 mL of drinking water on day 1 and continuing for duration of the experiment. The probiotic treated groups (P) were received probiotic, 0.1% of feed and 0.5% of feed, until day 21 and 56 respectively. One group as control (C) did not received any treatment of probiotic and antibody. A-treated and A-P treated groups had significantly lower fecal shedding (p<0.01) and lower concentration of SE cecal colonization (p<0.01). These groups also had a lower isolation of SE from the liver, spleen and ileum. The use of Salmonella enteritidis-specific IgY combined with probiotic had a beneficial effect in reducing the colonization of Salmonella in market-aged broiler under the condition of this study.

Plasma Membrane Residence of Hyaluronan Synthase Is Coupled to Its Enzymatic Activity

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

Expression of HYAL2 mRNA, hyaluronan and hyaluronidase in B-cell non-Hodgkin lymphoma: relationship with tumor aggressiveness.

Hyaluronidases and their substrate, hyaluronan (HA), were mainly explored in solid tumors but rarely in hematologic malignancies. While HA involvement was demonstrated in invasion and metastasis in most cases of solid tumors, the role of hyaluronidases in cancer progression remains controversial. One of the hyaluronidases, HYAL2, is suspected to be involved in the first step of HA degradation. In this work, HYAL2 mRNA, HA and total hyaluronidases expression were examined in lymphoma tissue extracts and correlated to the lymphoma subtype. Real-time RT-PCR was performed to evaluate HYAL2 mRNA. HA and hyaluronidase were assayed by enzyme-linked sorbent assay. Our results showed that HYAL2 mRNA expression was correlated to lymphoma diagnosis (p = 6 x 10(-3)) and was significantly lower in high-grade lymphoma, i.e., diffuse large B-cell diffuse lymphomas (DLBCLs). Several forms of hyaluronidase were detected by zymography and total hyaluronidase activity detected in tissue extracts was not significantly different according to tumor grade. HA levels also correlated to lymphoma subtype (p = 1 x 10(-5)) and were higher in DLBCLs. Moreover, HYAL2 mRNA and HA expressions were inversely correlated (p = 0.035). HYAL2 gene is localized on chromosome 3p21, which contains candidates tumor suppressor genes. Our results suggest that HYAL2 may have a prognostic significance in lymphomas and an antioncogenic activity. Conversely, HA overexpression in high-grade lymphomas is in favor of its involvement in tumor development and could provide a useful target for lymphoma therapy using HA-binding peptides.

Kinetics of Protein-Protein Interactions of Connexins: Use of Enzyme Linked Sorbent Assays

Kinetics of Protein-Protein Interactions of Connexins: Use of Enzyme Linked Sorbent Assays

Cleaved protein S (PS), total PS, free PS, and activated protein C cofactor activity as risk factors for venous thromboembolism.

Although hereditary protein S (PS) deficiency is clearly associated with venous thromboembolism (VTE), the importance of low PS concentrations as a risk factor for VTE in other patients is still a matter of debate. To clarify this issue, we designed a case-control study to evaluate the role of different molecular forms of plasma PS. We quantified plasma cleaved, total, and free PS and activated protein C (APC) cofactor activity in 87 VTE patients and 174 controls matched for age, sex, and hormonal treatment. Free PS was measured by ELISA or by enzyme-linked ligand sorbent assay (ELSA). Cleaved and total PS were measured by ELISA. In controls, the mean (SD) concentration of circulating cleaved PS was 39 (14) nmol/L, corresponding to 10% (3.5%) of total PS. Concentrations of cleaved PS and total PS were not significantly different in patients with VTE compared with controls. However, in our population, low free PS measured by ELISA or ELSA, as well as APC cofactor activity values were significantly associated with VTE with odds ratios (95% confidence intervals) of 2.9 (1.3-6.3), 2.5 (1.1-5.6), and 2.9 (1.3-6.4), respectively, in multivariate analyses. Phenotypic low PS detected by APC cofactor activity assay or by an assay specific for free PS should be considered a risk factor for VTE.

Kinetics of protein-protein interactions of connexins: use of enzyme linked sorbent assays.

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.

Hyaluronidase in sera of tumour-bearing nude mice

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l−1 (mean±SD 432±70 mU l−1, median 440 mU l−1). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.

Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres

C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. Thus, generic systems for CRP detection, based solely on the interaction between CRP and phosphocholine (PC), have been developed. PC–bovine serum albumin (PC–BSA) conjugates were produced and either labeled with horseradish peroxidase to facilitate CRP detection in a CRP enzyme-linked sorbent assay (ELSA) or coupled to carboxy-modified microspheres to facilitate the nonenzymatic, turbidimetric detection of CRP. The CRP-ELSA is a competitive assay, where the total assay time is 45 min, the assay sensitivity is 1.06 mg/L CRP, and the dynamic range of the assay is 0–500 mg/L. When PC–BSA conjugate is covalently coupled to carboxylated microspheres, agglutination occurs in the presence of CRP, the extent of which depends on the quantity of CRP present in the sample. Total assay time is 5 min with a dynamic range of 25–500 mg/L. Both assay formats are capable of accurately detecting human CRP and the CRP-ELSA can detect canine CRP as a disease state indicator.

PH-Dependent Intramolecular Binding and Structure Involving Cx43 Cytoplasmic Domains

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119-144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced alpha-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.

Importance of Hyaluronan Length in a Hyaladherin-Based Assay for Hyaluronan

Specific hyaladherin-based assays have been set up to measure the concentration of hyaluronan in biological fluids. Hyaluronectin (HN; a hyaladherin extracted from ovine brain) binds to hyaluronan (HA) that must be 10 units (HA10) or more long. It was therefore of interest to determine whether HN would continue to bind to HA10 in full-length HA since conformational changes might mask potential binding sites. We used the enzyme-linked sorbent assay (ELSA) to assay HA and hyaluronan-derived oligosaccharides, with different standard HAs, and the results were compared to results obtained with the carbazole technique. Oligosaccharide length was calculated from the ratio glucuronic acid/reducing N-acetylglucosamine in fractions of hyaluronidase-digested macromolecular hyaluronan prepared by chromatography; the size of the HA12 oligosaccharide was confirmed by matrix-assisted laser desorption ionization mass spectrometry. During the digestion of macromolecular HA with hyaluronidase, the binding of HN to HA first increased and then decreased as shown using the ELSA. The concentration of HA fragments of HA60 and below was overestimated when intact macromolecular HA was used as the reference for the ELSA, while the concentration of HA100 and above was underestimated when HA10 was used as the reference. The binding of HN to HA20, HA40, and HA60 saccharides was consistent with binding to multiples of HA10 sites. In conclusion, the level of HN binding is determined by the conformation of HA, which may mask binding sites. Hence, calibration HA used in the ELSA must be adapted to the size of HA to assay.

Hyaluronan digestion and synthesis in an experimental model of metastatic tumour.

To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse. H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan. Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography. Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique. Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH. Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium. The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion. These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.

Development of a 96-Well Enzyme-Linked Solid-Phase Assay for β-Glucanase and Xylanase

An enzyme-linked sorbent assay (ELSA) for estimating beta-glucanase activity was developed on the basis of the use of biotinylated beta-glucan as a solid-phase substrate. The assay involves the coating of titer plate wells with biotinylated beta-glucan, the partial hydrolysis of this substrate with beta-glucanase, the reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and quantitation of the remaining beta-glucan using alkaline phosphatase. The activity of the bound indicator enzyme, alkaline phosphatase, is proportionally related to the beta-glucanase activity in the sample. The ELSA is simple, can be readily adapted to the routine assay of a large number of samples (as many as 200 per person/day), and has good precision (CV = 4.0-6.4%) and high sensitivity (detects as low as 0. 001 mU of beta-glucanase/assay). A similar assay was developed for xylanase using biotinylated arabinoxylan. The ELSA provides a simple and sensitive estimate of beta-glucanase and xylanase activity.

Preliminary Data on the Age-Dependent Decrease in Basic Fibroblast Growth Factor and Platelet-Derived Growth Factor in the Human Vein Wall and in Their Influence on Cell Proliferation

The roles of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in vein disease and aging were investigated. Smooth muscle cells from human saphenous veins were cultured. The age dependence of bFGF and PDGF activation of the smooth muscle cell proliferation was determined, and the bFGF and PDGF contents in vein wall homogenates were measured by an enzyme-linked sorbent assay. There were morphological alterations in the cells with more polygonal and polynucleated cells in cultures from aged donors, similar to those observed in vitro in aged cell cultures. Some cultures did not reach confluency after the tenth passage, suggesting early decay of the cultures from diseased veins. bFGF and PDGF stimulated the proliferation of the vein smooth muscle cells, but only in cultures treated with hyaluronidase. This stimulation decreased with the age of the donor. The amount of the two growth factors in human vein walls decreased with donor age. The amount of bFGF decreased faster (slope: 3.3138 ng/mg DNA/year) than that of PDGF (slope: 1.021 ng/mg DNA/year). This results in an age-dependent change in the bFGF/PDGF ratio from 4 mol/mol at the age of 20 years to 1 mol/mol at the age of 80. These growth factors also modulate the synthesis of extracellular matrix components. The continuous change in the bFGF/PDGF ratio may alter the composition of the extracellular matrix of the vein wall during aging and thus its susceptibility to varicose disease.

Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides

Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in the intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for intracellular tTGase activity, using the incorporation of a biotinylated primary amine, 5-{[(N-biotinoylamino)hexanoyl]amino}pentylamine(biotin-x-cadaveri ne; BTC), into endogenous protein substrates of tTGase. This incorporation process was inhibited by competitive inhibitors of tTGase, cystamine and monodansylcadaverine, in a dose-dependent manner. Over a 30 min period tTGase and its protein substrates did not leak out of the cell, and no incorporation of BTC occurred in unpermeabilized cells, indicating the reaction to be intracellular. In the presence of 10 nM or 10 muM CA2+, when nucleotides ATP and GTP were added at concentrations mimicking cytosolic levels, tTGase activity was decreased virtually to zero. Only at 100 muM Ca2+, when nucleotides were low or absent was tTGase activity observed. Under these conditions a variety of proteins was labelled by the enzyme, with the major labelling found in a protein of molecular mass around 51 kDa when analysed by SDS/PAGE/Western blotting.

Protein-boronic acid conjugates and their binding to low-molecular-mass cis-diols and glycated hemoglobin.

Different methods for covalent linkage of phenylboronic acid (PBA) to structural proteins and enzymes are presented. Protein-PBA conjugates, free in solution or immobilised on magnetizable polymer particles, were tested for their binding of D-sorbitol, D-mannose and glycohemoglobin (GHb). Similarly, alkaline phosphatase-PBA conjugates were used in an attempted enzyme-linked sorbent assay for the detection of GHb. Affinity chromatography on immobilised D-mannose and gel chromatographic studies of protein-PBA complexes with [14C]sorbitol, clearly illustrated a low affinity of the interaction studied. Glycated hemoglobin could not be detected using the enzyme-linked sorbent assay approach. However, GHb was found to be specifically retained on columns filled with protein-PBA-coated particles as affinity matrix, enabling the glycation level of blood samples to be determined.

Enzyme-Linked Hyaluronectin: A Unique Reagent for Hyaluronan Assay and Tissue Location and for Hyaluronidase Activity Detection

Several techniques for assaying and localizing hyaluronan (HA), all based on the affinity to hyaluronan of proteins isolated from cartilage, chondrosarcoma, or brain, have been proposed. We show here that a unique reagent, alkaline phosphatase-linked hyaluronectin, can be used to assay hyaluronan in biological fluids or tissue extracts (enzyme-linked sorbent assay method) and to characterize it in cells or tissue sections in two steps: reagent incubation and staining. Results of assays in biological fluids or tissue extracts showed a good correlation with results of the previously described technique using antibodies to detect hyaluronectin bound to a plastic microtest plate (B. Delpech et al., 1985, Anal. Biochem. 149, 555-565) for both low concentrations (<1 mg/liter, r = 0.973, P < 0.001) and high concentrations (>1 mg/liter, r = 0.953, P < 0.001). The interassay variations were 8.5% when the assay was performed at 4°C and 18.5% at 37°C. The intraassay variations under those conditions were, respectively, 14.4 and 6.5%. Tissue HA could be detected easily with the reagent, as shown in fetal tissues and in tumors. Specificity of the reaction was controlled either by blocking the reagent with an excess of hyaluronan (which was not possible with other glycosaminoglycans) or by destroying tissue hyaluronan with streptomyces hyaluronidase. Alkaline phosphatase-linked hyaluronectin was also used to assay hyaluronidase activity in several biological fluids. One-hour incubation of hyaluronidase preparations on HA-coated plates made it possible to detect as low as 1 mU bovine testis hyaluronidase and 0.1 mTRU streptomyces hyaluronidase. Four-hour incubation made it possible to detect activity in a 1/12,500 dilution of human serum.

Antiretroviral therapy in sporadic adult amyotrophic lateral sclerosis.

Clinical and experimental findings in idiopathic amyothrophic lateral sclerosis (ALS) would be compatible with a retroviral involvement. In 35 adult patients with non-familial ALS we observed elevated circulating immune complexes, a decrease in IgG3 isotype and enzyme-linked sorbent assay (ELISA) serum antibodies against human spuma retrovirus (HSRV), confirmed by specific human foamy virus immunoblots. All 35 were negative for IgM or relevant IgG anti-ganglioside antibodies. We treated 12 HIV-negative, immune-complex-positive ALS patients with 500 mg d-1 zidovudine p.o. over 2-10 months and found reductions of serum creatine kinase and circulating immune complexes from two days to two weeks after the beginning of medication.

Enzyme‐linked sorbent assay to quantify d‐biotin in blood

AbstractAn enzyme‐linked sorbent assay (ELSA) is described to determine quantitatively d‐biotin in blood. This assay utilises the strong binding of streptavidin (as opposed to avidin which gave high background reactions). A competition reaction in a microtitration plate is set up between biotin bound to the plate with biotin in the sample and streptavidin conjugated to horseradish peroxidase using tetramethylbenzidine as a marker. This ELSA can be applied to samples with a d‐biotin concentration of down to 20 ng litre−1. There was a good correlation coefficient of 0·981 between the ELSA and the standard microbiological method for d‐biotin.

Increased Production of Transforming Growth Factor‐α in Psoriatic Epidermis

To elucidate the involvement of transforming growth factor-alpha (TGF-alpha) in the pathogenesis of psoriasis, we measured TGF-alpha levels in the extracts from six normal epidermis and four psoriatic involved epidermis samples by enzyme-linked sorbent assay using monoclonal antibody specific for TGF-alpha. The amount of TGF-alpha in the extracts of normal epidermis was 1.45 +/- 1.06 ng/g of wet tissue, while the amount in psoriatic involved epidermis was 6.71 +/- 0.75 ng/g of wet tissue. The TGF-alpha level in psoriatic involved epidermis was thus 4.62 fold higher than that of normal epidermis (P less than 0.001). TGF-alpha binds to epidermal growth factor receptors and functions as an autocrine growth factor for epidermal keratinocytes. Therefore, the increased levels of TGF-alpha may be involved in the induction or the maintenance of hyperproliferation of psoriatic epidermal keratinocytes.

Quantification of biotin in serum by competition with solid-phase biotin for binding to peroxidase-avidin conjugate.

We have developed a simple, fast, accurate, and reproducible method--an Enzyme-Linked Ligand Sorbent Assay (ELLSA)--for quantifying biotin without the use of isotopes and immunological reagents. The analytical principle is based on the competition between biotin of the sample and immobilized biotin, i.e., biotinylated bovine serum albumin coated onto immunoplates, for binding to horseradish peroxidase (EC 1.11.1.7)-avidin conjugate. The amount of conjugate bound to immobilized biotin is subsequently measured.