Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

PubMed

Näreoja, Tuomas; Rosenholm, Jessica M; Lamminmäki, Urpo; Hänninen, Pekka E

2017-05-01

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L -1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

PubMed

Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R T; Rodrigues, Sueli G; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

2006-11-01

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

On optimizing the blocking step of indirect enzyme-linked immunosorbent assay for Epstein-Barr virus serology.

PubMed

Lim, Chun Shen; Krishnan, Gopala; Sam, Choon Kook; Ng, Ching Ching

2013-01-16

Because blocking agent occupies most binding surface of a solid phase, its ability to prevent nonspecific binding determines the signal-to-noise ratio (SNR) and reliability of an enzyme-linked immunosorbent assay (ELISA). We demonstrate a stepwise approach to seek a compatible blocking buffer for indirect ELISA, via a case-control study (n=176) of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). Regardless of case-control status, we found that synthetic polymer blocking agents, mainly Ficoll and poly(vinyl alcohol) (PVA) were able to provide homogeneous backgrounds among samples, as opposed to commonly used blocking agents, notably nonfat dry milk (NFDM). The SNRs for NPC samples that correspond to blocking using PVA were approximately 3-fold, on average, higher than those blocking using NFDM. Both intra- and inter-assay precisions of PVA-based assays were <14%. A blocking agent of choice should have tolerable sample backgrounds from both cases and controls to ensure the reliability of an immunoassay. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk.

PubMed

Colazo, Marcos G; Ambrose, Divakar J; Kastelic, John P; Small, Julie A

2008-01-01

The objective of this study was to compare 2 enzyme immunoassays (EIAs) with a radioimmunoassay (RIA) as to sensitivity and accuracy in the measurement of the progesterone (P4) concentration in bovine plasma, skim milk, and whole milk. The 72 samples from 24 lactating dairy cows expected to have either a high P4 concentration (cows in diestrus or pregnant) or a low P4 concentration (cows in estrus or anestrus) were analyzed by RIA, solid-phase EIA (SPEIA), which included a solvent extraction step, or direct EIA (DEIA) without solvent extraction. The overall mean concentrations of P4 did not differ (P < 0.4) among the assays. However, for the cows that were in diestrus or pregnant, the mean P4 concentrations (and standard error) were higher (P < 0.03), regardless of sample type, with RIA than with SPEIA, at 7.3 (0.7) and 6.1 (0.6) ng/mL, respectively. When only the high-P4 samples analyzed by RIA were compared, the mean P4 concentration was higher (P < 0.001) in whole milk than in skim milk, at 9.8 (1.0) and 4.1 (0.7) ng/mL, respectively. Although the mean P4 concentrations in the low-P4 samples did not differ (P < 0.80) among assays, the proportions of cows with a P4 concentration > or = 1 ng/mL were 3%, 14%, and 44% for RIA, SPEIA, and DEIA, respectively (P < 0.01; DEIA > SPEIA > RIA).

Design and Fabrication of a PDMS Microchip Based Immunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Wang, Wanjun; Wang, Jun

2010-07-01

In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close amore » 200µm wide micro channel with flow rate up to 20µl/min.« less

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

PubMed

Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

2018-01-01

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

PubMed

Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

2013-08-01

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

PubMed Central

Mather, I H; Sullivan, C H; Madara, P J

1982-01-01

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk. Images Fig. 1. Fig. 2. Fig. 3. PMID:7046730

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine.

PubMed

Chuang, Jane C; Emon, Jeanette M Van; Durnford, Joyce; Thomas, Kent

2005-09-15

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within +/-20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within +/-20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30ng/mL by ELISA and 0.2ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10mL of urine converted into 1mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.

[Establishment of chemiluminescent enzyme immunoassay for detecting antibodies against foot-and-mouth disease virus serotype O in swine].

PubMed

Cui, Chen; Huang, Ligang; Li, Jing; Zou, Xingqi; Zhu, Yuanyuan; Xie, Lei; Zhao, Qizu; Yang, Limin; Liu, Wenjun

2016-11-25

Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

Kinase Activity Studied in Living Cells Using an Immunoassay

ERIC Educational Resources Information Center

Bavec, Aljos?a

2014-01-01

This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

PubMed

Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

2010-02-15

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

Identification, characterization, and quantitation of soluble HLA antigens in the circulation and peritoneal dialysate of renal patients.

PubMed Central

Gelder, F B; McDonald, J C; Landreneau, M D; McMillan, R M; Aultman, D F

1991-01-01

Human lymphocyte antigen (HLA) class I and class II antigens and beta 2 microglobulin (B2M) were identified in peritoneal dialysate (PD) and serum from patients with end-stage renal disease (ESRD) using monoclonal antibodies in an enzyme-linked immunoassay. The HLA class I and class II antigens each exhibited approximate molecular weights of 50,000 to 60,000 daltons by chromatography on Sepharose CL 6B. Class I antigens in serum and PD fluid were associated with B2M. Free B2M (Mr 11,500) also was detected in both sera and PD fluids. Unlike class I antigens, class II antigens were not found to have attached B2M. Class I and class II antigens eluted from 2-diethylaminoethanol ion exchange gradient columns at 0.07 mol/L (molar) phosphate buffer pH 7.2 and migrated with alpha 2-beta 1 mobility in agarose electrophoresis. Class I antigens were purified from ESRD patients' PD fluid by solid-phase immunoaffinity chromatography. Enzyme-linked immunoassay demonstrated that this purified protein was composed of a class I heavy chain and B2M. Class I allospecificity was confirmed by neutralization on known HLA typing antisera in a microcytotoxicity assay. Soluble HLA class I antigen preparations specifically inhibited blast transformation of responder lymphocytes in mixed lymphocyte culture reactions. Inhibition was dose dependent and ranged from 0% to 95%. The presence of soluble HLA antigens in body fluids may play an important part in the immunologic tolerance to self. This study demonstrates a ready source of large quantities of soluble HLA for detailed analysis. Images Fig. 1. PMID:2039290

Performance of a time-resolved fluorescence immunoassay for measuring varicella-zoster virus immunoglobulin G levels in adults and comparison with commercial enzyme immunoassays and Merck glycoprotein enzyme immunoassay.

PubMed

Maple, P A C; Gray, J; Breuer, J; Kafatos, G; Parker, S; Brown, D

2006-02-01

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species.

PubMed

Bienboire-Frosini, Cecile; Chabaud, Camille; Cozzi, Alessandro; Codecasa, Elisa; Pageat, Patrick

2017-01-01

The neurohormone oxytocin (OT) has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA) for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat). The Oxytocin EIA kit (EnzoLifeSciences) was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml). To validate the method, the following performance characteristics were evaluated using Validation Samples (VS) at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was verified. Although matrix effects assessments are lacking, our results indicate that OT plasma levels can reliably be measured in several domestic animal species by the method described here. Studies involving samples with low OT plasma concentrations should pay attention to reproducibility issues. This work opens new perspectives to reliably study peripheral OT in a substantial number of domestic animal species in various behavioral contexts.

Stabilization of penicillinase-hapten conjugate for enzyme immunoassay.

PubMed

Omidfar, K; Rasaee, Mohammad J; Zaraee, Ali B; Amir, M Pour; Rahbarizadeh, F

2002-01-01

The influence of various additives, such as organic solvents, polyhydric alcohols, salts, polymers, and cross-linker, on the stability and storage ability of penicillinase-morphine conjugate was studied in liquid and solid (freeze dried) states. The results of these experiments showed that using low concentrations of CaCl2 (0.1-0.2%) could stabilize enzyme activity in both states for more than seven months. The immunoreactivity of antigen toward the antibody did not change significantly. However, a cross-linker such as glutaraldehyde and various additives such as dimethylsulfoxide, glycerol, polyethylene glycol, gelatin, dextran, ammonium sulfate, lactose, and sucrose did not have any effect on stability. In addition, it was found that the presence of lactose and sucrose in the lyophilization procedure gives a significant amount of protection to the enzyme, which could last for a period of seven months and preserve almost 95% of the enzyme activity, as well as immunoreactivity of the tracer molecule.

IgE to penicillins with different specificities can be identified by a multiepitope macromolecule: Bihaptenic penicillin structures and IgE specificities.

PubMed

Ariza, A; Barrionuevo, E; Mayorga, C; Montañez, M I; Perez-Inestrosa, E; Ruiz-Sánchez, A; Rodríguez-Guéant, R M; Fernández, T D; Guéant, J L; Torres, M J; Blanca, M

2014-04-01

Quantitation of specific IgE by immunoassay is a recommended in vitro test for the diagnosis of immediate hypersensitivity reactions to betalactams (BLs), particularly when skin test results are negative. IgE antibodies that recognize the common nuclear structure of all BLs or the specific side chain structure can be mainly distinguished by immunoassays. The aim of this study was to develop an immunoassay system to detect IgE antibodies with different specificities. Cellulose discs conjugated with benzylpenicillin (BP), amoxicillin (AX) or both drugs, with poly-l-lysine (PLL) as carrier molecule, were used as solid phases in the radioallergosorbent test (RAST). Direct and inhibition radioimmunoassay studies were made to verify the structures recognized by serum IgE antibodies from penicillin-allergic patients. Our results indicated that the addition of both haptens did not decrease the capacity to capture IgE when serum specific to either BP or AX was used, at least in terms of sensitivity. In addition, the inclusion of two haptens improved significantly the levels of IgE detection in patients who recognized both BP and AX. Therefore, the use of a solid phase with a carrier molecule conjugated with two determinants (AX and BP) is helpful to recognize IgE antibodies against either of these determinants and is useful for screening sera with different specificities. Copyright © 2014 Elsevier B.V. All rights reserved.

A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research.

PubMed

Donovan, R S; Datti, A; Baek, M G; Wu, Q; Sas, I J; Korczak, B; Berger, E G; Roy, R; Dennis, J W

1999-10-01

Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37 degrees C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well beta-counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 beta1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis(x) in O-glycans. A glycopolymer acceptor for beta1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5-6 fold increase in throughput compared to the corresponding solution-phase assay.

Soluble serum interleukin 2 receptor levels in leprosy patients

PubMed Central

Tung, K. S. K.; Umland, Edith; Matzner, P.; Nelson, K.; Schauf, Victoria; Rubin, L.; Wagner, D.; Scollard, D.; Vithayasai, Prakong; Vithayasai, Vicharn; Worobec, Sophie; Smith, T.; Suriyanond, Vinai

1987-01-01

Soluble interleukin 2 receptors (IL-2R) in sera of leprosy patients from Chiang Mai, Thailand, were quantified with a solid phase enzyme immunoassay using two monoclonal antibodies to the IL-2R. The IL-2R levels of untreated lepromatous, borderline lepromatous or midborderline patients and treated lepromatous and borderline lepromatous or treated borderline tuberculoid and tuberculoid patients were comparable to those of the Thai household or nonhousehold contacts; and they were significantly higher than the levels of USA control subjects. In contrast, IL-2R of untreated tuberculoid or borderline tuberculoid patients were significantly reduced. Patients with ongoing reversal reaction had very high circulating IL-2R, the levels of which correlated with fever and extent of skin lesions. Although erythrema nodosum leprosum patients also had elevated IL-2R levels, they were significantly below those of patients with reversal reaction. When treated with corticosteroid, precipitous reduction of IL-2R was noted in all patients with reversal reaction but not in patients with erythema nodosum leprosum. PMID:3115652

Development of an analytical scheme for simazine and 2,4-D in soil and water runoff from ornamental plant nursery plots.

PubMed

Sutherland, Devon J; Stearman, G Kim; Wells, Martha J M

2003-01-01

The transport and fate of pesticides applied to ornamental plant nursery crops are not well documented. Methodology for analysis of soil and water runoff samples concomitantly containing the herbicides simazine (1-chloro-4,6-bis(ethylamino)-s-triazine) and 2,4-D ((2,4-dichlorophenoxy)acetic acid) was developed in this research to investigate the potential for runoff and leaching from ornamental nursery plots. Solid-phase extraction was used prior to analysis by gas chromatography and liquid chromatography. Chromatographic results were compared with determination by enzyme-linked immunoassay analysis. The significant analytical contributions of this research include (1) the development of a scheme using chromatographic mode sequencing for the fractionation of simazine and 2,4-D, (2) optimization of the homogeneous derivatization of 2,4-D using the methylating agent boron trifluoride in methanol as an alternative to in situ generation of diazomethane, and (3) the practical application of these techniques to field samples.

Capture-ELISA for serum IgM antibody to respiratory syncytial virus.

PubMed Central

Cevenini, R.; Donati, M.; Bertini, S.; Moroni, A.; Sambri, V.

1986-01-01

A four-component solid-phase capture enzyme immunoassay was set up to test for serum IgM antibody to respiratory syncytial (RS) virus and was compared with immunofluorescence assay (IFA). A total of 128 young children with acute respiratory infections were studied. Thirty-six were shown to be RS virus-positive by the detection of RS virus in nasopharyngeal secretions and 92 were RS virus-negative. A serum specimen was collected after admission to the hospital (days 0-4) and a further specimen was obtained during days 10-14. Out of 36 RS virus-positive patients, 28 (77.7%) were found to be positive for IgM by both capture-ELISA and IFA. Out of 92 RS virus-negative patients 5 (5.4%) were IgM-positive. Four false-positive results were obtained by IFA due to the presence of rheumatoid factor. The capture-ELISA was shown to be a reliable technique in detecting specific IgM antibody to RS virus. PMID:3540115

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

PubMed

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

NASA Astrophysics Data System (ADS)

Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

2014-02-01

Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals.

PubMed

Danišová, Olga; Halánová, Monika; Valenčáková, Alexandra; Luptáková, Lenka

The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN ® Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA ® QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

PubMed

Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi

2017-01-01

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

False-negative syphilis treponemal enzyme immunoassay results in an HIV-infected case-patient.

PubMed

Katz, Alan R; Komeya, Alan Y; Tomas, Juval E

2017-06-01

We present a case report of a false-negative syphilis treponemal enzyme immunoassay test result in an HIV-infected male. While treponemal tests are widely considered to be more sensitive and specific than non-treponemal tests, our findings point to potential challenges using the reverse sequence syphilis screening algorithm.

Effects of Dehydroepiandrosterone Supplementation during Stressful Military Training: A Randomized, Controlled, Double-Blind Field Study

DTIC Science & Technology

2011-01-01

DHEAS. All samples were assayed for salivary DHEA in duplicate using a highly sensitive enzyme immunoassay (Salimetrics, LLC). The test used 50ml of...p , 0.0001, n ¼ 39). Similarly, samples were assayed for salivary DHEAS in duplicate using a highly sensitive enzyme immuno- assay (Salimetrics, LLC...assayed for salivary testosterone. This was performed in duplicate using a highly sensitive enzyme immunoassay (Salimetrics, LLC). The test used 25ml of

Challenges in the Laboratory Diagnosis and Management of Dengue Infections.

PubMed

Bhat, Vivek G; Chavan, Preeti; Ojha, Shashank; Nair, Pravin K

2015-01-01

Dengue fever is considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. An accurate and efficient diagnosis of dengue plays an important role in case confirmation. The virus may be isolated during the viremic phase (within day 5 of illness), from serum, plasma and peripheral blood mononuclear cells. Enzyme linked immunoassay (ELISA) has demonstrated the presence of high levels of dengue NS1 antigen and tests may be performed by enzyme-immunoassays (EIAs) or immune-chromatographic (ICT) methods. These assays are specific with respect to different flaviviruses. Conventional and real time RT PCR, nested PCR, multiplex PCR and Nucleic acid sequence based amplification (NASBA) have been described as sensitive and relatively rapid method of detecting the virus during the early viremic phase. Other tests used include assay of anti-dengue specific IgM and IgG ELISA. Currently no curative treatment in terms of anti-viral drugs is available for dengue and patients are managed with rest and aggressive supportive therapy. Management may be done at home or in the hospital depending on the severity of the illness. Hospital management includes fluid therapy, blood component transfusion and other modalities of treatments like steroids, recombinant factor VII and management of complications. Various vaccines are in trial stages and may become available in the near future.

Development and Validation of an Immunoassay for Quantification of Topoisomerase I in Solid Tumor Tissues

PubMed Central

Pfister, Thomas D.; Hollingshead, Melinda; Kinders, Robert J.; Zhang, Yiping; Evrard, Yvonne A.; Ji, Jiuping; Khin, Sonny A.; Borgel, Suzanne; Stotler, Howard; Carter, John; Divelbiss, Raymond; Kummar, Shivaani; Pommier, Yves; Parchment, Ralph E.; Tomaszewski, Joseph E.; Doroshow, James H.

2012-01-01

Background Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. Methodology/Principal Findings We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. Conclusions/Significance Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors. PMID:23284638

A simple, rapid atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry method for the determination of 25-hydroxyvitamin D2 and D3.

PubMed

Garg, Uttam; Munar, Ada; Frazee, Clinton; Scott, David

2012-09-01

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods. © 2012 Wiley Periodicals, Inc.

Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.

PubMed

Oliver-Calixte, Nyoté J; Uba, Franklin I; Battle, Katrina N; Weerakoon-Ratnayake, Kumuditha M; Soper, Steven A

2014-05-06

The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 10(3) nucleotides s(-1) for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis-Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.

Total Triiodothyronine by Fluorescence Polarization Immunoassay (FPIA),

DTIC Science & Technology

Graves ’ disease . Traditionally, radioimmunoassays (RIA) have been employed for the determination of total T3. Enzyme immunoassays (EIA) and fluorescence immunoassays (FIA) have been developed for many of the analytes that formerly were measured using RIA. One variation of this new generation of immunoassays is fluorescence polarization. A fluorescence polarization immunoassay (FPIA) method for total T3 has been automated by adaptation to the TDx (Abbott, Chicago, IL) clinical analyzer. The TDx total T3 assay has been evaluated as a replacement for an RIA total T3

Enzyme-catalysed deposition of ultrathin silver shells on gold nanorods: a universal and highly efficient signal amplification strategy for translating immunoassay into a litmus-type test.

PubMed

Yang, Xinjian; Gao, Zhiqiang

2015-04-25

On the basis of enzyme-catalysed reduction of silver ions and consequent deposition of ultrathin silver shells on gold nanorods, a highly efficient signal amplification method for immunoassay is developed. For a model analyte prostate-specific antigen, a 10(4)-fold improvement over conventional enzyme-linked immunosorbent assay is accomplished by leveraging on the cumulative nature of the enzymatic reaction and the sensitive response of plasnomic gold nanorods to the deposition the silver shells.

[Automated analyzer of enzyme immunoassay].

PubMed

Osawa, S

1995-09-01

Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

Silver nanoparticles deposited on graphene oxide for ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.

PubMed

Yang, Lin; Zhen, Shu Jun; Li, Yuan Fang; Huang, Cheng Zhi

2018-06-14

Graphene oxide (GO) exhibits distinctive Raman scattering features for its high frequency D (disordered) and tangential modes (G-band), which are characteristically sharp at 1580 cm-1 and 1350 cm-1, respectively, but are too weak for sensitive quantitation purposes. By depositing silver nanoparticles on the surface of GO in this contribution, both D and G bands of GO become enhanced. The enzyme label of this method controls the dissolution of silver nanoparticles on the surface of GO through hydrogen peroxide which is produced by the oxidation of the enzyme substrate. With the dissolution of the silver nanoparticles a greatly decreased SERS signal of GO was obtained. This strategy involves dual signal amplification of the enzyme and nanocomposites to improve the detection sensitivity. As a proof of concept, prostate specific antigen (PSA), a biomarker for prostate cancer, is successfully detected as a target by forming a sandwich structure in immunoassay. The SERS immunoassay possesses excellent analytical performance in the range 0.5 pg mL-1 to 500 pg mL-1 with a limit of detection of 0.23 pg mL-1, making the detection of PSA serum samples from prostate cancer patients satisfactory, demonstrating that the sensitive enzyme-assisted dissolved AgNPs SERS immunoassay of PSA has potential applications in clinical diagnosis.

Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Wang, Jun; Li, Zhaohui

2011-09-20

In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstratemore » the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 ul sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.« less

Evaluation and reporting of enzyme immunoassay determinations of antibody to herpes simplex virus in sera and cerebrospinal fluid.

PubMed Central

Cremer, N E; Cossen, C K; Hanson, C V; Shell, G R

1982-01-01

Several methods for evaluating and reporting enzyme immunoassay (EIA) determinations of antibody to herpes simplex virus derived from one dilution of single serum samples were studied. An EIA ratio method for serological evidence of current infection from paired serum samples was also evaluated. Optical density (OD) of the reaction at a 1:100 serum dilution and estimated titers obtained by reference of the OD of the serum dilution to a standard curve were compared to the corresponding plotted EIA titer obtained by titration to endpoint. Neither the OD per se nor the estimated titer was completely predictive of the plotted titer (correlation coefficient [r] of 0.824 and 0.817, respectively), and they provided only a semiquantitative measurement of antibody concentration. For an antibody status report, however, OD would be sufficient if related to the cutoff value as an EIA index (OD of sample divided by cutoff OD for positive specimens). The OD of the EIA reaction at a single dilution (1:5) of cerebrospinal fluid, on the other hand, correlated quite well with the titer obtained by titration (r = 0.950). For serological diagnosis of current infection, the OD ratio of convalescence-phase/acute-phase sera was determined at several dilutions. A ratio of greater than or equal to 1.54 was calculated as a reliable index for a significant rise in antibody concentration and compatible with current infection. By determining the convalescent-phase/acute-phase serum ratio at two dilutions, 1:100 and 1:1,000, the EIA ratio method appeared to be a sensitive as or more sensitive than, complement fixation in diagnosing current infection. PMID:6284791

Surveillance for Enteric Pathogens in a Case-Control Study of Acute Diarrhea in Western Kenya

DTIC Science & Technology

2012-11-01

isolates was ascertained using the MicroScan WalkAway 40 Plus. An enzyme immunoassay kit was used to detect rotavirus , and ova and parasite...examination was conducted by micros- copy and an enzyme immunoassay. Results: Rotavirus (10.2% and 10.5%) and Shigella (11% and 8%) were isolated significantly...Keywords: Gastroenteritis, Protozoa, Rotavirus , Shigella, Escherichia coli, Antimicrobial resistance Introduction Diarrheal disease continues to be a

Evaluation of automated enzyme immunoassays for five anticonvulsants and theophylline adapted to a centrifugal analyzer.

PubMed

Urquhart, N; Godolphin, W; Campbell, D J

1979-05-01

We report a clinical evaluation of the enzyme immunoassay (EMIT) performed with the GEMSAEC centrifugal analyzer as compared to gas-liquid and liquid chromatography for anticonvulsant drugs and theophylline, respectively. A good correlation was obtained for all drugs, although some difficulties were experienced with one lot of reagent for ethosuximide. The analyzer has an economic advantage if many samples are being analyzed for few drugs in each sample.

Occurrence of nonspecific reactions among stool specimens tested by the Abbott TestPack rotavirus enzyme immunoassay.

PubMed Central

Lipson, S M; Leonardi, G P; Salo, R J; Schutzbank, T E; Kaplan, M H

1990-01-01

Sixty-five stool specimens obtained from children suffering from gastroenteritis were tested for the presence of antigen to rotavirus by the Abbott TestPack Rotavirus (TestPack) enzyme immunoassay kit. The Kallestad Pathfinder enzyme immunoassay, polyacrylamide gel electrophoresis, immune electron microscopy, and virus isolation were utilized as reference assays. Fifty-four specimens were in accord by TestPack and Kallestad Pathfinder. Among 11 discordant specimens positive with TestPack but negative by Kallestad Pathfinder, rotavirus was not identified by polyacrylamide gel electrophoresis, immune electron microscopy, or isolation in primary African green monkey kidney cell cultures. TestPack displayed a performance specificity of 83%. The inordinately high number of stool specimens reported as false-positive by TestPack precludes the incorporation of this antigen detection kit into our routine regimen of diagnostic virologic testing. Images PMID:2166074

Determination of atrazine in rainfall and surface water by enzyme immunoassay

USGS Publications Warehouse

Dankwardt, Andrea; Wüst, Susanne; Elling, Wolfram; Thurman, E. Michael; Hock, Bertold

1994-01-01

Rainwater and surface water from four sites in Germany (Bavaria and Lower Saxony) were analyzed for atrazine by enzyme immunoassay from June 1990 until October 1992. The limit of quantification of the immunoassay was 0.02 μg/L with a middle of the test at 0.2 μg/L. About 60 % of the samples contained measurable amounts of atrazine. Seasonal trends were observed, with the highest concentration in the summer months of up to 4 μg/L for rainwater and up to 15 μg/L for surface waters. The highest concentrations were found in agricultural areas, while in the investigated national parks up to 0.56 μg/L could be detected in rain water. This points to long-range atmospheric transport from agricultural areas to pristine national parks. Samples from forest stands usually showed higher atrazine concentrations than samples from open fields. Deposition rates of 10 – 50 μg/m2 · yr were observed in the national parks and 10–180 μg/m2 · yr at the agricultural sites. Comparison of results obtained by enzyme immunoassay and GC/MS showed a good correlation of r = 0.95.

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

NASA Astrophysics Data System (ADS)

Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

1999-04-01

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

PubMed Central

Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

2011-01-01

A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

Detection of antibodies to proteases used in laundry detergents by the radioallergosorbent test.

PubMed

Dor, P J; Agarwal, M K; Gleich, M C; Welsh, P W; Dunnette, S L; Adolphson, C R; Gleich, G J

1986-11-01

Two proteases, Esperase and Alcalase, derived from Bacillus licheniformis and B. subtilis, respectively, are used in laundry products. In testing for the prevalence of IgE antibodies to these enzymes in sera among 300 laundry product workers, we experienced two problems in the establishment of a reliable RAST for these antigens. The first problem was the propensity of the allergen, Esperase, to undergo autolysis, suggesting that solid-phase Esperase might also lose reactivity through degradation. Treatment of Esperase with phenylmethylsulfonyl fluoride stabilized the enzyme and permitted the synthesis of a stable solid-phase antigen. The second problem was the finding that sera reactive with Esperase in the RAST were also reactive with Savinase, an enzyme from B. licheniformis to which the workers were not exposed. Immunochemical analyses of the three enzymes with specific rabbit antisera by gel diffusion and by two-site immunoradiometric assay demonstrated that they were not cross contaminated to any appreciable extent. RAST inhibition demonstrated that solid-phase Esperase possessed unique allergenic determinants in that the reactivity of IgE antibodies was inhibited by low concentrations of Esperase and only by very high concentrations of Alcalase and Savinase. In contrast, the reactivity of solid-phase Alcalase was occasionally inhibited equally well by Esperase and Alcalase. Most strikingly, the reaction of IgE antibodies with solid-phase Savinase was always inhibited by comparable quantities of Esperase, Alcalase, and Savinase. Thus, the establishment of the RAST for these proteases appears to require the use of phenylmethylsulfonyl fluoride to retard autolysis, and the results must be interpreted with caution because IgE antibodies in certain sera demonstrate cross-reactivity with Alcalase and Savinase.

IMMUNOASSAY HUMAN EXPOSURE STUDIES

EPA Science Inventory

The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

SITE EVALUATION OF FIELD PORTABLE PENTACHLOROPHENOL IMMUNOASSAYS

EPA Science Inventory

Four pentachlorophenol (PCP) enzyme immunoassays for environmental analysis have been evaluated through the U.S. EPA Superfund Innovative Technology Evaluation (SITE) program. Three assays were formatted for on-site field use and one assay could be used in a field laboratory sett...

Molecularly imprinted polymer nanoparticle-based assay (MINA): application for fumonisin B1 determination.

PubMed

Munawar, Hasim; Smolinska-Kempisty, Katarzyna; Cruz, Alvaro Garcia; Canfarotta, Francesco; Piletska, Elena; Karim, Khalku; Piletsky, Sergey A

2018-06-20

The enzyme-linked immunosorbent assay (ELISA) has been used as a standard tool for monitoring food and animal feed contamination from the carcinogenic fumonisin B1 (FB1). Unfortunately, ELISA is not always efficient due to the instability of the antibody and enzyme components in the immunoassay, the presence of natural enzyme inhibitors in the samples and the high levels of non-specific protein binding. Additionally, the production of antibodies for ELISA can be time-consuming and costly, due to the involvement of animals in the manufacturing process. To overcome these limiting factors, a molecularly imprinted nanoparticle based assay (MINA) has been developed, where the molecularly imprinted nanoparticles (nanoMIPs) replace the primary antibody used in a competitive ELISA. Herein, computational modelling was used to design the nanoMIPs by selecting monomers that specifically interact with FB1. The affinity of the monomers to FB1 was verified by measuring their binding in affinity chromatography experiments. The nanoMIPs were produced by solid phase synthesis and the results showed that nanoMIPs had a hydrodynamic diameter of around 249 ± 29 nm. The assay tested in model samples is highly selective and does not show cross-reactivity with other mycotoxins such as fumonisin B2 (FB2), aflatoxin B1 (AFB1), citrinin (CTT), zearalenone (ZEA), and deoxynivalenol (DON). The MINA allows the detection of FB1 in the concentration range of 10 pM-10 nM with a detection limit of 1.9 pM and a recovery of 108.13-113.76%.

Diagnostic performance of the EMIT-tox benzodiazepine immunoassay, FPIA serum benzodiazepine immunoassay, and radioreceptor assay in suspected acute poisoning.

PubMed

Verstraete, A G; Belpaire, F M; Leroux-Roels, G G

1998-01-01

We evaluated the diagnostic performance of the EMIT-tox serum benzodiazepine assay adapted to a Hitachi 717 analyzer (EMIT), the Abbott ADx serum benzodiazepine fluorescence polarization immunoassay (FPIA), and a radioreceptor assay (RRA) in 113 patients with suspected acute poisoning. The reference method was high-performance liquid chromatography with ultraviolet detection after solid-phase extraction. For the discrimination between negative and positive samples, the areas under the receiver-operating characteristic (ROC) curves were 0.976, 0.991, and 0.991 for EMIT (cutoff, 50-ng/mL diazepam), FPIA (cutoff, 12-ng/mL nordiazepam), and RRA (cutoff, 50-ng/mL diazepam), respectively. For the discrimination between non-toxic and toxic concentrations, the areas under the ROC curves were 0.896, 0.893, and 0.933, respectively. EMIT (with the cutoff lowered to 50 ng/mL), FPIA, and RRA can be reliably used to screen for the presence of benzodiazepines in serum, but in many cases they cannot discriminate between toxic and nontoxic concentrations.

Direct analysis of airborne mite allergen (Der f1) in the residential atmosphere by chemifluorescent immunoassay using bioaerosol sampler.

PubMed

Miyajima, Kumiko; Suzuki, Yurika; Miki, Daisuke; Arai, Moeka; Arakawa, Takahiro; Shimomura, Hiroji; Shiba, Kiyoko; Mitsubayashi, Kohji

2014-06-01

Dermatophagoides farinae allergen (Der f1) is one of the most important indoor allergens associated with allergic diseases in humans. Mite allergen Der f1 is usually associated with particles of high molecular weight; thus, Der f1 is generally present in settled dust. However, a small quantity of Der f1 can be aerosolized and become an airborne component. Until now, a reliable method of detecting airborne Der f1 has not been developed. The aim of this study was to develop a fiber-optic chemifluorescent immunoassay for the detection of airborne Der f1. In this method, the Der f1 concentration measured on the basis of the intensity of fluorescence amplified by an enzymatic reaction between the labeled enzyme by a detection antibody and a fluorescent substrate. The measured Der f1 concentration was in the range from 0.49 to 250 ng/ml and a similar range was found by enzyme-linked immunosorbent assay (ELISA). This method was proved to be highly sensitive to Der f1 compared with other airborne allergens. For the implementation of airborne allergen measurement in a residential environment, a bioaerosol sampler was constructed. The airborne allergen generated by a nebulizer was conveyed to a newly sampler we developed for collecting airborne Der f1. The sampler was composed of polymethyl methacrylate (PMMA) cells for gas/liquid phases and some porous membranes which were sandwiched in between the two phases. Der f1 in air was collected by the sampler and measured using the fiber-optic immunoassay system. The concentration of Der f1 in aerosolized standards was in the range from 0.125 to 2.0 mg/m(3) and the collection rate of the device was approximately 0.2%. © 2013 Elsevier B.V. All rights reserved.

Cytomegalovirus Antibody in Cerebrospinal Fluid of Schizophrenic Patients Detected by Enzyme Immunoassay

NASA Astrophysics Data System (ADS)

Fuller Torrey, E.; Yolken, Robert H.; Winfrey, C. Jack

1982-05-01

By means of enzyme immunoassay techniques to detect the presence of antibody to cytomegalovirus, the cerebrospinal fluid of 178 patients with schizophrenia, 17 patients with bipolar disorders, and 11 other psychiatric patients was compared with that of 79 neurological patients and 41 normal control subjects. The cerebrospinal fluid of 20 of the schizophrenic patients and 3 of the patients with bipolar disorders showed significant increases in immunoglobulin M antibody to cytomegalovirus; no difference was found in patients on or off psychotropic medications.

Development of ochratoxin a in cereal by chemiluminescence enzyme immunoassay

NASA Astrophysics Data System (ADS)

Li, Min; Liu, Renrong; Zhu, Lixin; Chen, Zhenzhen

2017-11-01

A rapid, simple and sensitive chemiluminescence enzyme immunoassay method (CLEIA) was established to detect ochratoxin A (OTA) in cereal. Optimal conditions including antibody dilution ratio and enzyme conjugate, ionic strength, pH value and organic solvent. Established indirect competition inhibition curve to determine the linear working range, detection limit and recovery rate. Results: The 50% inhibitory concentration and the detection limit of the CLEIA were78.8pg/mL and 14.86 pg/mL, respectively, with a linear range of 0.015-0.4ng/mL. At 1∼4μpg/kg fortified levels in wheat, mean recoveries ranged from 67.47% to100.35%.

Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies

NASA Astrophysics Data System (ADS)

Pandey, Binod Prasad

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions. Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL -1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface. Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl β-D-mannopyranoside (αMan-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1α)-D-Glc-1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A to mannoside and soybean agglutinin to galactose in these SAMs were found to be different on NPG than on flat polycrystalline gold, and was also sensitive to the chemical composition of the modified surfaces.

Detection of phase I IgG antibodies to Coxiella burnetii with EIA as a screening test for blood donations.

PubMed

van der Hoek, W; Wielders, C C H; Schimmer, B; Wegdam-Blans, M C A; Meekelenkamp, J; Zaaijer, H L; Schneeberger, P M

2012-11-01

The presence of a high phase I IgG antibody titre may indicate chronic infection and a risk for the transmission of Coxiella burnetii through blood transfusion. The outbreak of Q fever in the Netherlands allowed for the comparison of an enzyme immunoassay (EIA) with the reference immunofluorescence assay (IFA) in a large group of individuals one year after acute Q fever. EIA is 100 % sensitive in detecting high (≥1:1,024) phase I IgG antibody titres. The cost of screening with EIA and confirming all EIA-positive results with IFA is much lower than screening all donations with IFA. This should be taken into account in cost-effectiveness analyses of screening programmes.

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

PubMed Central

Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

2014-01-01

Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

Sensitive detection of respiratory syncytial virus based on a dual signal amplified plasmonic enzyme-linked immunosorbent assay.

PubMed

Zhan, Lei; Wu, Wen Bi; Yang, Lin; Huang, Cheng Zhi

2017-04-15

The timely detection of infectious pathogen is critical in clinical early diagnosis and treatment of infectious diseases. Plasmonic enzyme-linked immunosorbent assay (ELISA), by means of enzyme-mediated growth or aggregation of AuNPs, has received considerable attention because it allows a naked-eye detection of target in very low numbers. In this work, a dual-signal amplified plasmonic ELISA combined the high loading capacity of magnetic beads with the establishing stimulation effect of zinc ion has been developed to detect RSV as a model pathogen based on alkaline phosphatase-triggered dispersion of aggregated AuNPs. In ideal conditions, the proposed immunoassay can conveniently distinguish the concentration of RSV in a range of 0.1-30 pg/mL. In addition, the limit of detection of RSV of this immunoassay exceeds that of conventional ELISA by about 50 times. The high sensitivity makes this approach a good alternative to existing colorimetric immunoassays for pathogen detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo-immunoassays for Chemical Contaminants in Food and Environmental Samples.

PubMed

Chen, Chaochao; Luo, Jiaxun; Li, Chenglong; Ma, Mingfang; Yu, Wenbo; Shen, Jianzhong; Wang, Zhanhui

2018-03-21

The chemical contaminants in food and the environment are quite harmful to food safety and human health. Rapid, accurate, and cheap detection can effectively control the potential risks derived from these chemical contaminants. Among all detection methods, the immunoassay based on the specific interaction of antibody-analyte is one of the most widely used techniques in the field. However, biological antibodies employed in the immunoassay usually cannot tolerate extreme conditions, resulting in an unstable state in both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class of polymers with specific molecular recognition abilities, which are highly robust, showing excellent operational stability under a wide variety of conditions. Recently, MIPs have been used in biomimetic immunoassays for chemical contaminants as an antibody substitute in food and the environment. Here, we reviewed these applications of MIPs incorporated in different analytical platforms, such as enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, microfluidic paper-based immunoassay, and homogeneous immunoassay, and discussed current challenges and future trends in the use of MIPs in biomimetic immunoassays.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

PubMed

Ma, Youlong; Teng, Feiyue; Libera, Matthew

2018-06-05

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA - amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency; (c) inhibition of T7 RNA polymerase activity by AMV-RT.

Evaluation of third generation anti-HCV enzyme immunoassays.

PubMed

Panigrahi, A K; Nayak, B; Dixit, R; Acharya, S K; Panda, S K

1998-01-01

The Hepatitis C Virus (HCV) is a major cause of post transfusion hepatitis. The introduction of HCV antibody screening has reduced the risk of post transfusion hepatitis significantly. However, the test is yet to be used routinely in blood banks of several developing countries with limited resources. We have developed an Enzyme immunoassay using synthetic peptides. The test was compared to seven commercial tests available in the Indian market. The test was evaluated using a panel of 90 sera which were chosen from an earlier panel based on detection of HCV RNA by Reverse Transcription Polymerase Chain Reaction RT-PCR. In case of any discrepancy the sera were further analysed by Line immunoassay (LIA). The sensitivity of the in house EIA was 90%. The specificity of the commercial EIAs varied.

Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

PubMed

Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

2012-10-26

For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

IMMUNOASSAY METHODS FOR MEASURING ATRAZINE AND 3,5,6-TRICHLORO-2-PYRIDINOL IN FOODS

EPA Science Inventory

This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) methods for the analysis of two potential environmental contaminants in food sample media, atrazine and 3,5,6-trichloro-2-pyridinol (3,5,6-TCP). Two different immunoassay formats are employed: a magnetic...

Plasma cortisol and 11-ketotestosterone enzyme immunoassay (EIA) kit validation for three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus.

PubMed

Mills, S C; Mourier, J; Galzin, R

2010-08-01

Commercially available enzyme immunoassay (EIA) kits were validated for measuring steroid hormone concentrations in blood plasma from three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus. A minimum of 5 microl plasma was required to estimate hormone concentrations with both kits. These EIA kits are a simple method requiring minimal equipment, for measuring hormone profiles under field conditions.

A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA).

PubMed

Kai, Junhai; Puntambekar, Aniruddha; Santiago, Nelson; Lee, Se Hwan; Sehy, David W; Moore, Victor; Han, Jungyoup; Ahn, Chong H

2012-11-07

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

Automated homogeneous liposome immunoassay systems for anticonvulsant drugs.

PubMed

Kubotsu, K; Goto, S; Fujita, M; Tuchiya, H; Kida, M; Takano, S; Matsuura, S; Sakurabayashi, I

1992-06-01

We developed automated homogeneous immunoassays, based on immunolysis of liposomes, for measuring phenytoin, phenobarbital, and carbamazepine from serum. Liposome lysis was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. The procedure was fully automated on a routine automated clinical analyzer. Within-run, between-run, dilution, and recovery tests showed good accuracies and reproducibilities. Bilirubin, hemoglobin, triglycerides, and Intrafat did not affect assay results. The results obtained by liposome immunoassays for phenytoin, phenobarbital, and carbamazepine correlated well with those obtained by enzyme-multiplied immunoassay (Syva EMIT) kits (r = 0.995, 0.986, and 0.988, respectively) and fluorescence polarization immunoassay (Abbott TDx) kits (r = 0.990, 0.991, and 0.975, respectively). The proposed method should be useful for monitoring anticonvulsant drug concentrations in blood.

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.

PubMed

Nong, Rachel Yuan; Wu, Di; Yan, Junhong; Hammond, Maria; Gu, Gucci Jijuan; Kamali-Moghaddam, Masood; Landegren, Ulf; Darmanis, Spyros

2013-06-01

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

PubMed Central

Feyerabend, Martin; Weiler, Elmar W.

1987-01-01

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

PubMed

Hirakawa, Koji; Katayama, Masaaki; Soh, Nobuaki; Nakano, Koji; Imato, Toshihiko

2006-01-01

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.

Application of microstructural optical waveguides with hollow core for enzyme immunoassay

NASA Astrophysics Data System (ADS)

Pidenko, Pavel S.; Pidenko, Sergei A.; Burmistrova, Natalia A.; Shuvalov, Andrei A.; Chibrova, Anastasiya A.; Skibina, Yulia S.; Goryacheva, Irina Y.

2018-04-01

Microstructural optical waveguides with the hollow core are actively studied as a promising support for heterogeneous immunoassay in development of new optical biosensor elements for medicine and biology. Overcoming of the limitations associated with the low sorption capacity of glass used for the waveguides production is a crucial step for this assay format. In this work the possibility of silanization of microstructural optical waveguides with the hollow core using (3-glycidyloxypropyl) trimethoxysilane and their further application to enzymatic immunoassay was studied.

Monitoring of streptomycin and dihydrostreptomycin residual levels in porcine meat press juice and muscle via solid-phase fluorescence immunoassay and confirmatory analysis by liquid chromatography after post-column derivatization.

PubMed

Pyun, Chang-Won; Abd El-Aty, A M; Hashim, M M M; Shim, Jae-Han; Lee, Si-Kyung; Choi, Kang-Duk; Park, Kwan-Ha; Shin, Ho-Chul; Lee, Chiho

2008-03-01

A solid-phase fluorescence immunoassay (SPFIA) that was primarily developed for detection of antibiotic residues in milk was qualitatively applied for the pre-screening of the residues of aminoglycoside antibiotics, streptomycin and dihydrostreptomycin, in meat press juice. The confirmation of both analytes was performed using a validated method of highperformance liquid chromatography with post-column derivatization. The analytical performance was demonstrated by the analysis of pork meat samples spiked at three concentration levels, ranging from 0.25 to 2.5 ppm for each analyte. In general, the recoveries ranged from 80.4 to 81.5% and from 79.6 to 84.4% for streptomycin and dihydrostreptomycin, respectively, with relative standard deviations lower than 6%. The limits of detection were 0.1 and 0.15 ppm for streptomycin and dihydrostreptomycin, respectively, and the limits of quantification of 0.35 and 0.5 ppm are below the maximum residue limits of Codex, the European Union, and the Korean Food and Drug Administration (ranging from 0.5 to 0.6 ppm). Eight real samples collected from the Seoul area were first monitored using SPFIA, and none of them were found positive. These findings are in good accordance with those observed by HPLC analysis. To the best of our knowledge, this is the first report to monitor the aminoglycoside residues in pork meat press juice using SPFIA.

New antibody and immunoassay pretreatment strategy to screen polychlorinated biphenyls in Korean transformer oil.

PubMed

Terakado, Shingo; Ohmura, Naoya; Park, Seok-Un; Lee, Seung-Min; Glass, Thomas R

2013-01-01

Development and modifications are described that expand the application of an immunoassay from the detection of Kanechlors (Japanese technical PCBs mixtures) to the detection of Aroclors (U. S. technical PCB mixtures, used in Korea) in contaminated Korean transformer oil. The first necessary modification was the development of a new antibody with a reactivity profile favorable for Aroclors. The second modification was the addition of a second column to the solid-phase extraction method to reduce assay interference caused by the Korean oil matrix. The matrix interference is suspected to be caused by the presence of synthetic oils (or similar materials) present as contaminants. The modified assay was validated by comparison to high-resolution gas chromatography/high-resolution mass spectrometry analysis, and was shown to be tolerant of up to 10% of several common synthetic insulating oils. Finally the screening performance of the modified assay was evaluated using 500 used transformer oil samples of Korean origin, and was shown to have good performance in terms of false positive and false negative rates. This report provides evidence for the first establishment of immunoassay screening for Aroclor based PCB contamination in Korean transformer oil.

A comparison of five serological tests for bovine brucellosis.

PubMed Central

Dohoo, I R; Wright, P F; Ruckerbauer, G M; Samagh, B S; Robertson, F J; Forbes, L B

1986-01-01

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3539295

Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

NASA Astrophysics Data System (ADS)

Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

2014-06-01

In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6 days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4 °C with no additive up to 30 days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

Novel photoluminescence enzyme immunoassay based on supramolecular host-guest recognition using L-arginine/6-aza-2-thiothymine-stabilized gold nanocluster.

PubMed

Wang, Youmei; Lu, Minghua; Tang, Dianping

2018-06-30

A new photoluminescence (PL) enzyme immunoassay was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) via an innovative enzyme substrate, 6-aza-2-thiothymine-stabilized gold nanocluster (AAT-AuNC) with L-arginine. The enzyme substrate with strong PL intensity was formed through supramolecular host-guest assembly between guanidine group of L-arginine and AAT capped on the surface of AuNC. Upon arginase introduction, the captured L-arginine was hydrolyzed into ornithine and urea, thus resulting in the decreasing PL intensity. Based on this principle, a novel competitive-type immunoreaction was first carried out on AFB 1 -bovine serum albumin (AFB 1 -BSA) conjugate-coated microplate, using arginase-labeled anti-AFB 1 antibody as the competitor. Under the optimum conditions, the PL intensity increased with the increment of target AFB 1 , and allowed the detection of the analyte at concentrations as low as 3.2 pg mL -1 (ppt). Moreover, L-arginine-AAT-AuNC-based PL enzyme immunoassay afforded good reproducibility and acceptable specificity. In addition, the accuracy of this methodology, referring to commercial AFB 1 ELISA kit, was evaluated to analyze naturally contaminated or spiked peanut samples, giving well-matched results between two methods, thus representing a useful scheme for practical application in quantitative monitoring of mycotoxins in foodstuff. Copyright © 2018 Elsevier B.V. All rights reserved.

[The efficiency of the enzyme immunoassay test system opisthorchiasis-CIC-EIA-best to detect circulating immune complexes containing opisthorchis antigens in the serum of patients with opisthorchiasis].

PubMed

Starkova, T V; Poletaeva, O G; Kovrova, E A; Krasovskaia, N N; Tkachenko, T N; Masiago, A V; Ofitserov, V I; Tereshchenko, A Iu

2011-01-01

The efficacy of a kit of Opisthorchiasis-CIC-EIA-Best reagents was evaluated using 270 sera from patients in the study and control groups. The kit showed a sufficient sensitivity (not less than 87.2%) and a high specificity (not less than 97.9%). The use of the above kit of the reagents for enzyme immunoassay in practical healthcare enables one to increase detection rates among the infested subjects on comprehensive examination of those with suspected opisthorchiasis.

Comparison of the Vidas C. difficile GDH Automated Enzyme-Linked Fluorescence Immunoassay (ELFA) with Another Commercial Enzyme Immunoassay (EIA) (Quik Chek-60), Two Selective Media, and a PCR Assay for gluD for Detection of Clostridium difficile in Fecal Samples.

PubMed

Davies, K A; Berry, C E; Morris, K A; Smith, R; Young, S; Davis, T E; Fuller, D D; Buckner, R J; Wilcox, M H

2015-06-01

Prevention and management of Clostridium difficile infection (CDI) can be improved by rapid and reliable diagnostics. The Vidas C. difficile glutamate dehydrogenase assay had performance comparable to that of the Quik Chek-60 assay (overall agreement, 95%) and a sensitivity of >93%; thus, it is suitable as the first test in two-stage algorithms for a CDI diagnosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The significance for epidemiological studies anti-measles antibody detection examined by enzyme immunoassay (EIA) and plaque reduction neutralization test (PRNT).

PubMed

Siennicka, Joanna; Częścik, Agnieszka; Trzcińska, Agnieszka

2014-01-01

The paper discusses the role of anti-measles antibodies for protection and significance for epidemiological studies determination of antibodies by different serological methods. The comparison of anti-measles virus antibodies levels measured by enzyme immunoassay (EIA) and Plaque Reduction Neutralization Test (PRNT) was described. It was found that the 200 mIU/ml of anti-measles activity measured by PRNT (level protection against symp- tomatic disease) is equivalent of 636 mIU/ml measured by EIA (Enzygnost®Anti-Measles Virus/IgG, Simens).

Sources of variation in an enzyme-linked immunoassay of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae).

PubMed

Tabachnick, W J; Mecham, J O

1991-03-01

An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize.

PubMed

Roda, A; Mirasoli, M; Guardigli, M; Michelini, E; Simoni, P; Magliulo, M

2006-03-01

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.

A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

NASA Astrophysics Data System (ADS)

Yu, Fei; Wu, Yongjun; Yu, Songcheng; Zhang, Huili; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B.

With alkaline phosphatase (ALP)-adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR-McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350-1000 pg/mL and the detection limit of 0.24 ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%-96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

Chemiluminescence Resonance Energy Transfer Competitive Immunoassay Employing Hapten-Functionalized Quantum Dots for the Detection of Sulfamethazine.

PubMed

Ma, Mingfang; Wen, Kai; Beier, Ross C; Eremin, Sergei A; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

2016-07-20

We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL(-1), which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

1992-01-01

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

Method for immunodiagnostic detection of dioxins at low concentrations

DOEpatents

Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Petrovic, Peter; Gorbach, Siegbert

1995-01-01

A method is described for the use of monoclonal antibodies in a sensitive immunoassay for halogenated dioxins and dibenzofurans in industrial samples which contain impurities. Appropriate sample preparation and selective enzyme amplification of the immunoassay sensitivity permits detection of dioxin contaminants in industrial or environmental samples at concentrations in the range of a few parts per trillion.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

1992-04-28

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

EVALUATION OF IMMUNOASSAY METHODS FOR DETERMINATION OF 3,5,6-TRICHLORO-2-PYRIDINOL IN MULTIPLE SAMPLE MEDIA

EPA Science Inventory

Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by the RaPID (TM) commercial immunoassay testing ...

pH-dependent immobilization of urease on glutathione-capped gold nanoparticles.

PubMed

Garg, Seema; De, Arnab; Mozumdar, Subho

2015-05-01

Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Although the enzyme serves a significant role in several detoxification and analytical processes, its usability is restricted due to high cost, availability in small amounts, instability, and a limited possibility of economic recovery from a reaction mixture. Hence, there is a need to develop an efficient, simple, and reliable immobilization strategy for the enzyme. In this study, the carboxyl terminated surface of glutathione-capped gold nanoparticles have been utilized as a solid support for the covalent attachment of urease. The immobilization has been carried out at different pH conditions so as to elucidate its effect on the immobilization efficiency and enzyme bioactivity. The binding of the enzyme has been quantitatively and qualitatively analyzed through techniques like ultraviolet-visible spectroscopy, intrinsic steady state fluorescence, and circular dichorism. The bioactivity of the immobilized enzyme was investigated with respect to the native enzyme under different thermal conditions. Recyclability and shelf life studies of the immobilized enzyme have also been carried out. Results reveal that the immobilization is most effective at pH of 7.4 followed by that in an acidic medium and is least in alkaline environment. The immobilized enzyme also exhibits enhance activity in comparison to the native form at physiological temperature. The immobilized urease (on gold glutathione nanoconjugates surface) can be effectively employed for biosensor fabrication, immunoassays and as an in vivo diagnostic tool in the future. © 2014 Wiley Periodicals, Inc.

A highly efficient colorimetric immunoassay using a nanocomposite entrapping magnetic and platinum nanoparticles in ordered mesoporous carbon.

PubMed

Kim, Moon Il; Ye, Youngjin; Woo, Min-Ah; Lee, Jinwoo; Park, Hyun Gyu

2014-01-01

Nanocomposite to achieve ultrafast immunoassay: a new synergistically integrated nanocomposite consisting of magnetic and platinum nanoparticles, simultaneously entrapped in mesoporous carbon, is developed as a promising enzyme mimetic candidate to achieve ultrafast colorimetric immunoassays. Using new assay system, clinically important target molecules, such as human epidermal growth factor receptor 2 (HER2) and diarrhea-causing rotavirus, can be detected in only 3 min at room temperature with high specificity and sensitivity. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay.

PubMed

Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

2014-06-05

In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4°C with no additive up to 30days. These data were valuable for establishing CLEIA to quantify enrofloxacin. Copyright © 2014 Elsevier B.V. All rights reserved.

Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

NASA Astrophysics Data System (ADS)

Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

2013-10-01

The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies. Electronic supplementary information (ESI) available: Absorbance and fluorescence spectra of quantum dot nanoprobes, electrophoresis analysis, and experimental setup for fluorescence imaging with dual channels. See DOI: 10.1039/c3nr03291d

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

PubMed

Murphy, Brian M; Dandy, David S; Henry, Charles S

2009-04-27

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Dried venous blood samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay.

PubMed Central

Riddell, Michaela A.; Byrnes, Graham B.; Leydon, Jennie A.; Kelly, Heath A.

2003-01-01

OBJECTIVES: To determine whether samples of dried venous blood (DVB) were an acceptable alternative to serum for detecting measles-specific IgG in a commercial enzyme immunoassay. METHODS: Paired samples of serum and DVB were collected from 98 suspected cases of measles and 1153 schoolchildren in Victoria, Australia. All samples were tested using the Dade Behring Enzygnost Anti-Measles-Virus/IgG immunoassay. DVB samples were eluted using either the sample buffer provided with the kit or 5% dry milk powder in phosphate-buffered saline-Tween 20. FINDINGS: DVB samples eluted by sample buffer showed significantly better linear correlation to the serum samples than did DVB samples eluted in 5% dry milk in phosphate-buffered saline-Tween 20. To improve the comparability of serum and DVB samples an adjustment factor of 1.28 was applied to the optical density (OD) values of DVB. This adjustment also enabled quantification of the titre of measles IgG in mIU/ml directly from the OD value using the alpha calculation as specified by the kit protocol. For DVB samples stored for less than six months at 4 degrees C, the assay showed an overall sensitivity of 98.4% and a specificity of 97.2% compared with the results of serum testing. CONCLUSION: These results illustrate the potential for DVB samples to be widely used with the Dade Behring enzyme immunoassay system for determining the immunity of the individual and the population to the measles virus. PMID:14758429

COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY METHODS FOR MEASURING 3,5,6-TRICHLORO-2PYRIDINOL IN MULTIPLE SAMPLE MEDIA

EPA Science Inventory

Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by a commercial RaPID immunoassay testing kit. ...

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

PubMed

Hill, A S; Giersch, T M; Loh, C S; Skerritt, J H

1999-10-01

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

PubMed

Jadhao, Samadhan J; Anderson, Larry J

2016-01-01

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

Measurement of ring A-reduced progesterone metabolites by enzyme-linked immunoassay with colorimetric detection: baseline levels of six metabolites, including pregnanolone, in male rat plasma.

PubMed

Ocvirk, Rok; Franklin, Keith B J; Pearson Murphy, Beverley E

2009-02-01

The performance of an antiserum to progesterone and pregnane neurosteroids was assessed in two competitive assay setups: radioimmunoassay and enzyme-linked immunoassay with colorimetric detection, both with the same limit of detection of 2 pg. The enzyme-linked immunoassay was less labor-intensive and had better precision of measurement and was used to measure progesterone and six of its ring A-reduced metabolites in rat plasma. The measured levels of allopregnanolone and progesterone were in agreement with those reported previously when measured by gas chromatography/mass spectrometry and high-performance liquid chromatography coupled with radioimmunoassay and substantially lower than those previously measured by radioimmunoassay without chromatographic separation. Both isomers of dihydroprogesterone and all four isomers of pregnanolone were detected in rat plasma, indicating that progesterone is metabolized by reduction at the C5 and C3 position of the A ring, in both alpha and beta configurations. In addition to 5beta-dihydroprogesterone and isopregnanolone, which have not been previously detected in the rat, we found considerable amounts of pregnanolone, which is neuroactive, with similar potency to that of allopregnanolone but was previously thought not to be produced in rats.

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

PubMed Central

Crouch, C F

1995-01-01

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

Biomimetic Silica Nanoparticles Prepared by a Combination of Solid-Phase Imprinting and Ostwald Ripening.

PubMed

Piletska, Elena; Yawer, Heersh; Canfarotta, Francesco; Moczko, Ewa; Smolinska-Kempisty, Katarzyna; Piletsky, Stanislav S; Guerreiro, Antonio; Whitcombe, Michael J; Piletsky, Sergey A

2017-09-14

Herein we describe the preparation of molecularly imprinted silica nanoparticles by Ostwald ripening in the presence of molecular templates immobilised on glass beads (the solid-phase). To achieve this, a seed material (12 nm diameter silica nanoparticles) was incubated in phosphate buffer in the presence of the solid-phase. Phosphate ions act as a catalyst in the ripening process which is driven by differences in surface energy between particles of different size, leading to the preferential growth of larger particles. Material deposited in the vicinity of template molecules results in the formation of sol-gel molecular imprints after around 2 hours. Selective washing and elution allows the higher affinity nanoparticles to be isolated. Unlike other strategies commonly used to prepare imprinted silica nanoparticles this approach is extremely simple in nature and can be performed under physiological conditions, making it suitable for imprinting whole proteins and other biomacromolecules in their native conformations. We have demonstrated the generic nature of this method by preparing imprinted silica nanoparticles against targets of varying molecular mass (melamine, vancomycin and trypsin). Binding to the imprinted particles was demonstrated in an immunoassay (ELISA) format in buffer and complex media (milk or blood plasma) with sub-nM detection ability.

Microfabricated Renewable Beads-Trapping/Releasing Flow Cell for Rapid Antigen-Antibody Reaction in Chemiluminescent Immunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zhifeng; Shao, Guocheng; Wang, Jun

2011-04-01

A filter pillar-array microstructure was coupled with a pneumatic micro-valve to fabricate a reusable miniaturized beads-trapping/releasing flow cell, in which trapping and releasing beads can be conveniently realized by switching the micro-valve. This miniaturized device was suitable to construct automatic fluidic system for “renewable surface analysis”. The renewable surface strategy based on pneumatic micro-valve enabled capture of beads in beads chamber prior to each assay, and release of the used beads after the assay. Chemiluminescent competitive immunoassay of 3,5,6-trichloropyridinol (TCP) was performed as a model to demonstrate the application potential of this reusable miniaturized flow cell. The whole fluidic assaymore » process including beads trapping, immuno-binding, beads washing, beads releasing and signal collection could be completed in 10 min. Immunoassay of TCP using this miniaturized device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device had been successfully used for detection of TCP spiked in rat serum with average recovery of 97%. This investigation provides a rapid, sensitive, reusable, low-cost and automatic miniaturized device for solid-phase biochemical analysis for various purposes.« less

Enzyme-linked immunosorbent assay for determination of aflatoxin M1 based on magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Atanasova, M. K.; Ivanova, N. V.; Godjevargova, T. I.

2017-02-01

A sensitive enzyme immunoassay with magnetic nanoparticles (Method A) for the quantitative determination of aflatoxin M1 in milk was developed. This immunoassay was based on the immobilization of monoclonal antibody (mAb) on the modified magnetic nanoparticles (MNPs-NH2). It was observed that for each mg of the MNPs, 25 µg of antibody was immobilized. Both aflatoxin M1 in the sample and aflatoxin M1-BSA-peroxidase conjugate competed for the immobilized antibody. The proposed Method A was compared with other method (B). The Method B was based on the immobilization of aflatoxin M1-BSA conjugate on the MNPs-NH2, which competed with the aflatoxin M1 in the sample for binding to the added mAb. The binding of mAb to the aflatoxin M1-BSA-MNPs-NH2 was detected using a target secondary IgG-peroxidase antibody. The analytical characteristics of the two methods were compared. Real milk samples were investigated for present of aflatoxin M1. Two methods were based on the use of MNPs as a solid support for covalently immunoreagents immobilization. A comfortable separation of bound and free fraction of the tracer can be performed only through a simple collection of the MNPs by a permanent magnet. The application of MNPs helps to eliminate non-specific binding and to retain higher activity of bound biomolecules. The development of a MNPs-based ELISA for determination of aflatoxin M1 has a great potential to supersede the traditional ELISA for aflatoxin M1 diagnosis.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy.

PubMed

Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

2015-09-01

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detect Toxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Influence of surface-imprinted nanoparticles on trypsin activity.

PubMed

Guerreiro, António; Poma, Alessandro; Karim, Kal; Moczko, Ewa; Takarada, Jessica; de Vargas-Sansalvador, Isabel Perez; Turner, Nicholas; Piletska, Elena; de Magalhães, Cristiana Schmidt; Glazova, Natalia; Serkova, Anastasia; Omelianova, Aleksandra; Piletsky, Sergey

2014-09-01

Here, the modulation of enzyme activity is presented by protein-imprinted nanoparticles produced using a solid-phase approach. Using trypsin as target, binding of the nanoparticles to the enzyme results in its inhibition or in stabilization, depending on the orientation of the immobilized enzyme used during imprinting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The toxicology and immunology of detergent enzymes.

PubMed

Basketter, David; Berg, Ninna; Kruszewski, Francis H; Sarlo, Katherine; Concoby, Beth

2012-01-01

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

Steroid profiling reveals widespread local regulation of glucocorticoid levels during mouse development.

PubMed

Taves, Matthew D; Plumb, Adam W; Sandkam, Benjamin A; Ma, Chunqi; Van Der Gugten, Jessica Grace; Holmes, Daniel T; Close, David A; Abraham, Ninan; Soma, Kiran K

2015-02-01

Glucocorticoids (GCs) are produced by the adrenal glands and circulate in the blood to coordinate organismal physiology. In addition, different tissues may independently regulate their local GC levels via local GC synthesis. Here, we find that in the mouse, endogenous GCs show tissue-specific developmental patterns, rather than mirroring GCs in the blood. Using solid-phase extraction, HPLC, and specific immunoassays, we quantified endogenous steroids and found that in tissues of female and male mice, (1) local GC levels can be much higher than systemic GC levels, (2) local GCs follow age-related patterns different from those of systemic GCs, and (3) local GCs have identities different from those of systemic GCs. For example, whereas corticosterone is the predominant circulating adrenal GC in mice, high concentrations of cortisol were measured in neonatal thymus, bone marrow, and heart. The presence of cortisol was confirmed with liquid chromatography-tandem mass spectrometry. In addition, gene expression of steroidogenic enzymes was detected across multiple tissues, consistent with local GC production. Our results demonstrate that local GCs can differ from GCs in circulating blood. This finding suggests that steroids are widely used as local (paracrine or autocrine) signals, in addition to their classic role as systemic (endocrine) signals. Local GC regulation may even be the norm, rather than the exception, especially during development.

Analytical methods for characterization of explosives-contaminated sites on U.S. Army installations

NASA Astrophysics Data System (ADS)

Jenkins, Thomas F.; Walsh, Marianne E.; Thorne, Philip G.

1995-10-01

The U.S. Army manufactures munitions at facilities throughout the United States. Many of these facilities are contaminated with residues of explosives from production, disposal of off- specification, and out-of-data munitions. The first step in remediating these sites is careful characterization. Currently sites are being characterized using a combination of on-site field screening and off-site laboratory analysis. Most of the contamination is associated with TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-tri-nitro-1,3,5-triazine) and their manufacturing impurities and environmental transformation products. Both colorimetric and enzyme immunoassay-based field screening methods have been used successfully for on-site characterization. These methods have similar detection capabilities but differ in their selectivity. Although field screening is very cost-effective, laboratory analysis is still required to fully characterize a site. Laboratory analysis for explosives residues in the United States is generally conducted using high-performance liquid chromatography equipped with a UV detector. Air-dried soils are extracted with acetonitrile in an ultrasonic bath. Water is analyzed directly if detection limits in the range of 10 - 20 (mu) g/L are acceptable, or preconcentrated using either salting-out solvent extraction with acetonitrile or solid phase extraction.

Is there any relationship between proinflammatory mediator levels in disc material and myelopathy with cervical disc herniation and spondylosis? A non-randomized, prospective clinical study

PubMed Central

Asir, Alparslan; Cetinkal, Ahmet; Gedik, Nursal; Kutlay, Ahmet Murat; Çolak, Ahmet; Kurtar, Sedat; Simsek, Hakan

2007-01-01

The proinflammatory mediator (PIM) levels were assessed in surgically removed samples of herniated cervical intervertebral discs. The objective of this study was to investigate if there is a correlation between the levels of PIMs in disc material and myelopathy associated with cervical intervertebral disc herniation and spondylosis. The role of proinflammatory mediators in the degeneration of intervertebral disc and the inflammatory effects of disc herniations on radicular pain has been previously published. However, the possible relationship between PIMs and myelopathy related to cervical disc herniation and spondylosis has not been investigated before. Thirty-two patients undergoing surgery for cervical disc herniation and spondylosis were investigated. Surgically obtained disc materials, stored at 70°C, were classified into two groups: cervical disc herniation alone or with myelopathy. Biochemical preparation and solid phase enzyme amplified sensitivity immunoassay (ELISIA) analysis of the samples were performed to assess the concentration of mediators in the samples. Very similar values of interleukin-6 were found in both groups whereas the concentrations of mediators were significantly higher in myelopathy group. This study has demonstrated that PIMs are involved in cervical intervertebral disc degeneration with higher concentrations in the samples associated with myelopathy. PMID:17476536

Immunoassays for pesticide monitoring

NASA Astrophysics Data System (ADS)

Wengatz, Ingrid; Szurdoki, Ferenc; Swamy, Anand R.; Evans, Lawrence, III; Patonay, Gabor; Stimmann, Eric; Delwiche, Michael; Stoutamire, Donald; Gee, Shirley J.; Hammock, Bruce D.

1995-05-01

This study compares two formats of rapid assays for the detection of pesticides (bromacil and pyrethroid based metabolites): enzyme linked immunosorbent assay (ELISA) and immunoassay with near-infrared (NIR) fluorescence detection. NIR dye immunoassay (NIRDIA) measurements were carried out by using two different instruments, both having a silicon photodiode as the detector and a laser diode for excitation. ELISA and NIRDIA were performed in a tracer format, where the specific antibody is bound to the surface of a microtiter plate well and the tracer with enzyme or fluorescent dye label competes with the analyte for the antibody binding site. It was demonstrated that the NIRDIA is at least as sensitive as the ELISA. Both assays detect pesticides in the (mu) g/L (ppb) range. Hapten- macromolecule-NIR dye-conjugates have been synthesized with various biopolymers (e.g., proteins) as carriers. The use of carrier macromolecules enables convenient purification of the cyanine dye derivatives. The mild conjugation method of the dye is based on isothiocyanate chemistry.

Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics ▿

PubMed Central

Porwancher, Richard B.; Hagerty, C. Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J. B.; Kopnitsky, Mark; Steere, Allen C.; Kulas, Karen; Wong, Susan J.

2011-01-01

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted. PMID:21367982

Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

PubMed

Groome, N P

1981-10-01

The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

Determinants on an efficient cellulase recycling process for the production of bioethanol from recycled paper sludge under high solid loadings.

PubMed

Gomes, Daniel; Gama, Miguel; Domingues, Lucília

2018-01-01

In spite of the continuous efforts and investments in the last decades, lignocellulosic ethanol is still not economically competitive with fossil fuels. Optimization is still required in different parts of the process. Namely, the cost effective usage of enzymes has been pursued by different strategies, one of them being recycling. Cellulase recycling was analyzed on recycled paper sludge (RPS) conversion into bioethanol under intensified conditions. Different cocktails were studied regarding thermostability, hydrolysis efficiency, distribution in the multiphasic system and recovery from solid. Celluclast showed inferior stability at higher temperatures (45-55 °C), nevertheless its performance at moderate temperatures (40 °C) was slightly superior to other cocktails (ACCELLERASE ® 1500 and Cellic ® CTec2). Celluclast distribution in the solid-liquid medium was also more favorable, enabling to recover 88% of final activity at the end of the process. A central composite design studied the influence of solid concentration and enzyme dosage on RPS conversion by Celluclast. Solids concentration showed a significant positive effect on glucose production, no major limitations being found from utilizing high amounts of solids under the studied conditions. Increasing enzyme loading from 20 to 30 FPU/g cellulose had no significant effect on sugars production, suggesting that 22% solids and 20 FPU/g cellulose are the best operational conditions towards an intensified process. Applying these, a system of multiple rounds of hydrolysis with enzyme recycling was implemented, allowing to maintain the steady levels of enzyme activity with only 50% of enzyme on each recycling stage. Additionally, interesting levels of solid conversion (70-81%) were also achieved, leading to considerable improvements on glucose and ethanol production comparatively with the reports available so far (3.4- and 3.8-fold, respectively). Enzyme recycling viability depends on enzyme distribution between the solid and liquid phases at the end of hydrolysis, as well as enzymes thermostability. Both are critical features to be observed for a judicious choice of enzyme cocktail. This work demonstrates that enzyme recycling in intensified biomass degradation can be achieved through simple means. The process is possibly much more effective at larger scale, hence novel enzyme formulations favoring this possibility should be developed for industrial usage.

Establishment of a Simple and Convenient Method for Folic Acid Enzyme Chemiluminescence Immunoassay

NASA Astrophysics Data System (ADS)

Liu, Ting; Zeng, Ling; Yu, Zhengwei; Yang, Yanfei; Qu, Yunhuan

2018-01-01

The enzyme chemiluminescence new immunoassay for folic acid (FA) was established by competition model. Add FA samples to a microtiter plate precoated with the goat anti-mouse IgG firstly, then add enzyme abled FA and FA monoclonal antibody (McAb). The values of CLIA were measured to reflect the quantity of FA. The limit of detection(LOD) of assay is 0.37ng/mL. The assay shows good correlation during 1∼30 ng/mL with correlation coefficient 0.9976. The intra- and inter-assay coefficients of variation are 4.8 % ∼ 7.3 % and 6.1 % ∼ 12.2 %, respectively. The recovery of folic acid in serum is 90.4 %∼113.2 %. Compared with determine value clinically in chemiluminescence immunoassay kit from Roche company, the correlative equation is y = 0.9689x + 0.0228, and correlation coefficient is 0.9780. Various components and kit overall show good stabilities. This method is simple and convenient, and has low LOD value. The method has overcome the shortcomings of the present references. It is easy to apply and has broad clinical application prospect. It lays an experimental foundation for the preparation of Mc Ab against folic acid and the development of domestic kit.

Rapid quantitative analysis of 8-iso-prostaglandin-F(2alpha) using liquid chromatography-tandem mass spectrometry and comparison with an enzyme immunoassay method.

PubMed

Dahl, Jeffrey H; van Breemen, Richard B

2010-09-15

A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the measurement of urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), a biomarker of lipid peroxidation. Because urine contains numerous F(2) prostaglandin isomers, each with identical mass and similar mass spectrometric fragmentation patterns, chromatographic separation of 8-iso-PGF(2alpha) from its isomers is necessary for its quantitative analysis using MS/MS. We were able to achieve this separation using an isocratic LC method with a run time of less than 9min, which is at least threefold faster than previous methods, while maintaining sensitivity, accuracy, precision, and reliability. The limits of detection and quantitation were 53 and 178pg/ml urine, respectively. We compared our method with a commercially available affinity purification and enzyme immunoassay kit and found both assays to be in agreement. Despite the high sensitivity of the enzyme immunoassay method, it is more expensive and has a narrower dynamic range than LC-MS/MS. Our method was optimized for rapid measurement of 8-iso-PGF(2alpha) in urine, and it is ideally suited for clinical sample analysis. 2010 Elsevier Inc. All rights reserved.

Magnetic Affinity Enzyme-Linked Immunoassay for Diagnosis of Schistosomiasis Japonicum in Persons with Low-Intensity Infection

PubMed Central

Yu, Qin; Yang, Hai; Feng, Youmei; Zhu, Yanhong; Yang, Xiangliang

2012-01-01

Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections. PMID:22869635

An ultrasensitive chemiluminescence immunoassay for fumonisin B1 detection in cereals based on gold-coated magnetic nanoparticles.

PubMed

Jie, Mingsha; Yu, Songcheng; Yu, Fei; Liu, Lie; He, Leiliang; Li, Yanqiang; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B; Wu, Yongjun

2018-07-01

In the present study, a novel highly sensitive magnetic enzyme chemiluminescence immunoassay (MECLIA) was developed to detect fumonisin B 1 (FB 1 ) in cereal samples. The gold-coated magnetic nanoparticles (Fe 3 O 4 @Au, GoldMag) were used as solid phase carrier to develop a competitive CLIA for detecting FB 1 , in which FB 1 in samples would compete with FB 1 -ovalbumin coated on the surface of Fe 3 O 4 @Au nanoparticles for binding with FB 1 antibodies. Successively, horseradish peroxidase labeled goat anti-rabbit IgG (HRP-IgG) was conjugated with FB 1 antibodies on the microplate. In substrate solution containing luminol and H 2 O 2 , HRP-IgG catalyzed luminol oxidation by H 2 O 2 , generating a high chemiluminescence signal. The FB 1 immune GoldMag particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscope and zeta potential analysis, etc. RESULTS: The concentrations and the reaction times of these immunoreagents were optimized to improve the performances of this method. The established method could detect as low as 0.027 ng mL -1 FB 1 from 0.05 ng mL -1 to 25 ng mL -1 , demonstrating little cross-reaction (less than 2.4%) with other structurally related compounds. The average intrassay relative SD (RSD) (n = 6) was 3.4% and the average interassay RSD (n = 6) was 5.4%. This method was successfully applied for the determination of FB 1 in corn and wheat and gave recoveries of between 98-110% and 91-105%, respectively. The results of the present study suggest that the MECLIA approach has potential application for high-throughput fumonisin screening in cereals. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen

PubMed Central

Pandey, Binod; Demchenko, Alexei V.; Stine, Keith J.

2013-01-01

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL-1 for CEA and up to 30 ng mL-1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented. PMID:23935216

Clinical Characteristics and Outcomes of Hematologic Malignancy Patients With Positive Clostridium difficile Toxin Immunoassay Versus Polymerase Chain Reaction Test Results.

PubMed

Ziegler, Matthew; Landsburg, Daniel; Pegues, David; Alby, Kevin; Gilmar, Cheryl; Bink, Kristen; Gorman, Theresa; Moore, Amy; Bonhomme, Brittaney; Omorogbe, Jacqueline; Tango, Dana; Tolomeo, Pam; Han, Jennifer H

2018-04-25

In a cohort of inpatients with hematologic malignancy and positive enzyme immunoassay (EIA) or polymerase chain reaction (PCR) Clostridium difficile tests, we found that clinical characteristics and outcomes were similar between these groups. The method of testing is unlikely to predict infection in this population, and PCR-positive results should be treated with concern.Infect Control Hosp Epidemiol 2018;1-4.

Stool C difficile toxin

MedlinePlus

... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

[DIAGNOSTIC VALUE OF COMBINED USE OF COMBINED METHOD OF ENZYME IMMUNOASSAY AND POLYMERASE CHAIN REACTION TO DETECT OF INTRAUTERINE FETAL INFECTION BY PARVOVIRUS B19].

PubMed

Bondarenko, N P; Lakatosh, V P; Lakatosh, P V; Malanchuk, O B; Poladich, I V

2015-01-01

The combined method of diagnosis parvovirus infection during pregnancy by maternal serum enzyme immunoassay and deoxyribonucleic acid isolation parvovirus B19 polymerase chain reaction in amnniotic fluid and fetal cord blood newborns, can diagnose vertical transmission and anticipate a negative effect on the fetus parvovirus. Lack of maternal IgM antibodies in serum due to parvovirus seroconversion during pregnancy does not exclude the persistence of the virus in the fetus. To analyze the diagnostic value of the method for determining the LHP parvovirus B19 DNA in the amniotic fluid, umbilical cord blood of newborns to determine vertical transmission of parvovirus infection when infected mothers B19 during pregnancy.

Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities.

PubMed

Warkentin, Theodore E; Sheppard, Jo-Ann I; Chu, F Victor; Kapoor, Anil; Crowther, Mark A; Gangji, Azim

2015-01-01

Repeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera-when first testing SRA-negative-continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive. © 2015 by The American Society of Hematology.

[State of Fungal Lipases of Rhizopus microsporus, Penicillium sp. and Oospora lactis in Border Layers Water-Solid Phase and Factors Affecting Catalytic Properties of Enzymes].

PubMed

Khasanov, Kh T; Davranov, K; Rakhimov, M M

2015-01-01

We demonstrated that a change in the catalytic activity of fungal lipases synthesized by Rhizopus microsporus, Penicillium sp. and Oospora lactis and their ability to absorb on different sorbents depended on the nature of groups on the solid phase surface in the model systems water: lipid and water: solid phase. Thus, the stability of Penicillium sp. lipases increased 85% in the presence ofsorsilen or DEAE-cellulose, and 55% of their initial activity respectively was preserved. In the presence of silica gel and CM-cellulose, a decreased rate of lipid hydrolysis by Pseudomonas sp. enzymes was observed in water medium, and the hydrolysis rate increased by 2.4 and 1.5 times respectively in the presence of aminoaerosil and polykefamid. In an aqueous-alcohol medium, aminoaerosil and polykefamid decreased the rate of substrate hydrolysis by more than 30 times. The addition of aerosil to aqueous and aqueous-alcohol media resulted in an increase in the hydrolysis rate by 1.2-1.3 times. Sorsilen stabilized Penicillium sp. lipase activity at 40, 45, 50 and 55 degrees C. Either stabilization or inactivation of lipases was observed depending on the pH of the medium and the nature of chemical groups localized on the surface of solid phase. The synthetizing activity of lipases also changed depending on the conditions.

Monitoring methotrexate in clinical samples from cancer patients during chemotherapy with a LSPR-based competitive sensor.

PubMed

Zhao, Sandy Shuo; Bichelberger, Mathilde A; Colin, Damien Y; Robitaille, Robert; Pelletier, Joelle N; Masson, Jean-François

2012-10-21

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.

Effects of freezer storage time on levels of complement biomarkers.

PubMed

Morgan, Angharad R; O'Hagan, Caroline; Touchard, Samuel; Lovestone, Simon; Morgan, B Paul

2017-11-06

There is uncertainty regarding how stable complement analytes are during long-term storage at - 80 °C. As part of our work program we have measured 17 complement biomarkers (C1q, C1 inhibitor, C3, C3a, iC3b, C4, C5, C9, FB, FD, FH, FI, TCC, Bb, sCR1, sCR2, Clusterin) and the benchmark inflammatory marker C-reactive protein (CRP) in a large set of plasma samples (n = 720) that had been collected, processed and subsequently stored at - 80 °C over a period of 6.6-10.6 years, prior to laboratory analysis. The biomarkers were measured using solid-phase enzyme immunoassays with a combination of multiplex assays using the MesoScale Discovery Platform and single-plex enzyme-linked immunosorbent assays (ELISAs). As part of a post hoc analysis of extrinsic factors (co-variables) affecting the analyses we investigated the impact of freezer storage time on the values obtained for each complement analyte. With the exception of five analytes (C4, C9, sCR2, clusterin and CRP), storage time was significantly correlated with measured plasma concentrations. For ten analytes: C3, FI, FB, FD, C5, sCR1, C3a, iC3b, Bb and TCC, storage time was positively correlated with concentration and for three analytes: FH, C1q, and C1 inhibitor, storage time was negatively correlated with concentration. The results suggest that information on storage time should be regarded as an important co-variable and taken into consideration when analysing data to look for associations of complement biomarker levels and disease or other outcomes.

Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever.

PubMed

Boisen, Matthew L; Hartnett, Jessica N; Shaffer, Jeffrey G; Goba, Augustine; Momoh, Mambu; Sandi, John Demby; Fullah, Mohamed; Nelson, Diana K S; Bush, Duane J; Rowland, Megan M; Heinrich, Megan L; Koval, Anatoliy P; Cross, Robert W; Barnes, Kayla G; Lachenauer, Anna E; Lin, Aaron E; Nekoui, Mahan; Kotliar, Dylan; Winnicki, Sarah M; Siddle, Katherine J; Gbakie, Michael; Fonnie, Mbalu; Koroma, Veronica J; Kanneh, Lansana; Kulakosky, Peter C; Hastie, Kathryn M; Wilson, Russell B; Andersen, Kristian G; Folarin, Onikepe O; Happi, Christian T; Sabeti, Pardis C; Geisbert, Thomas W; Saphire, Erica Ollmann; Khan, S Humarr; Grant, Donald S; Schieffelin, John S; Branco, Luis M; Garry, Robert F

2018-04-12

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient's clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

Preparation of milk samples for immunoassay and liquid chromatographic screening using matrix solid-phase dispersion.

PubMed

Barker, S A; Long, A R

1994-01-01

The use of drugs to maintain the health and maximize the output of dairy cattle has made the monitoring of milk for such agents essential. Screening tests based on immunological, microbial inhibition, and bacterial receptor assays have been developed for the detection of violative levels of therapeutic substances. However, such assays are not infallible, and false positive or negative results can occur when contaminants bind receptors or compete for the binding of the target residues. Such effects may arise from dietary sources, diseases, or other variables. Thus, a violation by such a test is not definitive until further confirmation is obtained. Our laboratory has developed extraction procedures for several drugs used in dairy production. Our method uses matrix solid-phase dispersion (MSPD) to isolate drugs away from contaminants and to eliminate many possible interferences. MSPD can also be used to enhance the specificity of such assays by fractionating various classes of drugs that may cross-react. Similarly, such methods may be used for liquid chromatographic screening and confirmation of a suspect sample.

Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples

USDA-ARS?s Scientific Manuscript database

A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, de...

Enzyme immunoassays for detection of gypsy moth nuclear polyhedrosis virus

Treesearch

Michael Ma

1985-01-01

Enzyme-linked immunosorbent assays (ELISA) were developed for detecting gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (NPV). They were used to detect the presence of NPV in hemoplymph samples collected from infected larvae. The incorporation of hybridoma antibodies with these procedures would make them even more specific for gypsy moth...

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration.

PubMed

Moreno-Paz, Mercedes; Gómez-Cifuentes, Ana; Ruiz-Bermejo, Marta; Hofstetter, Oliver; Maquieira, Ángel; Manchado, Juan M; Morais, Sergi; Sephton, Mark A; Niessner, Reinhard; Knopp, Dietmar; Parro, Victor

2018-04-11

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth. Key Words: Planetary exploration-Molecular biomarkers-D- and L- aromatic amino acids-Life detection-Multiplex inhibitory/competitive immunoassay-Kerogen type IV. Astrobiology 18, xxx-xxx.

Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw.

PubMed

Rodrigues, Ana Cristina; Felby, Claus; Gama, Miguel

2014-03-01

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50°C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

PubMed

Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-06-07

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

PubMed

Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

2016-01-15

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

[Effects of low-intensity infrared impulse laser therapy on inflammation activity markers in patients with rheumatoid arthritis].

PubMed

Ilich-Stoianovich, O; Nasonov, E L; Balabanova, R M

2000-01-01

To evaluate effects of low-intensity infrared impulse laser therapy (IRILT) on concentration of immunity activation [not readable: see text] (soluble receptors of TNF-alpha and neopterin) and indicator of the inflammation activity (concentration of C-reactive protein) in patients with rheumatoid arthritis (RA). Enzyme immunoassay, radioimmunoassay, enzyme immunoassay and radial immunodiffusion were used to measure soluble receptors of TNF-alpha, neopterin and C-reactive protein in 38 females with verified RA receiving IRILT or sham procedures. IRILT induced lowering of neopterin, TNF-alpha soluble receptors (p < 0.01) and C-reactive protein (p < 0.01). The findings give pathogenetical grounds for IRILT use in RA as this treatment suppresses functional activity of macrophages which serve the main source of neopterin and the receptors synthesis.

Determination of blood sirolimus concentrations in liver and kidney transplant recipients using the Innofluor® fluorescence polarization immunoassay: Comparison with the microparticle enzyme immunoassay and high-performance liquid chromatography-ultraviolet method

PubMed Central

Bouzas, Lorena; Hermida, Jesús

2009-01-01

Background Although high-performance liquid chromatography (HPLC) is the method of choice for blood sirolimus determination, the microparticle enzyme immunoassay (MEIA) run on the IMx® analyser is widely used in therapeutic monitoring of this immunosuppressant agent. The aim of our study was to evaluate the possible determination of sirolimus using the fluorescence polarization immunoassay (FPIA) commercialized for everolimus quantification. Methods Sirolimus concentrations were determined in whole-blood samples from liver and kidney transplant recipients using the Innofluor® Certican® FPIA (Seradyn Inc.) run on a TDx® analyser (Abbott Laboratories), Sirolimus MEIA run on an IMx® analyser (Abbott Laboratories), and HPLC (UV detection) methods. Results The Innofluor® FPIA has a similar cross-reactivity with everolimus and sirolimus, and the within- and between-run coefficients of variation obtained for sirolimus determination were 2.7%–13.3%. In analysing different blood samples from liver and kidney transplant patients the linear regressions obtained were: FPIA = 1.12 HPLC + 0.43 (n=104, r=0.874), MEIA = 1.14 HPLC (n=146, r=0.892), and FPIA = 1.00 MEIA + 0.29 (n=106, r=0.941). Better correlation coefficients were obtained between the methods in the liver transplant samples (r≥0.900) than in the kidney transplant samples (r≥0.849). No significant effect was found for sirolimus clearance or the blood hematocrit on the relationship between the results produced by both immunoassays and HPLC. Conclusion The Innofluor® FPIA is a valid alternative with an analogous performance to the MEIA for the therapeutic monitoring of sirolimus. PMID:19242874

Enzyme immunoassay of two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL).

PubMed

Chen, P; Tian, Z; Digenis, G A; Tai, H H

1996-06-01

Specific and sensitive enzyme immunoassays for two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL) have been developed. The hydroxyl group of hydroxymethyl at position 8 of either MDL or MMDL was carboxymethylated to introduce a carboxyl group for protein conjugation. Antibodies generated from O-carboxymethyl MDL or MMDL recognized the spacer arm between the hapten and the carrier protein and the molecular domain near the conjugation site as well. A heterologous bridge strategy was used to improve the affinity of the hapten-enzyme conjugate to the antibodies. The sensitivity of both assays was greatly increased by using such an approach. Both antibodies are specific for their own haptens. Little cross reactivity was observed with nicergoline and other metabolites. Determination of MDL and MMDL from both spiked plasma and urine showed nearly quantitative recovery. Detection of MDL and MMDL can be as sensitive as 10 pg/ml.

Development of immunoassays for human urokinase

NASA Technical Reports Server (NTRS)

Atassi, M. Zouhair

1988-01-01

Radioimmune assays (RIA) and enzyme linked immune assays for measurement of pro-urokinase and the two active forms of the enzyme were developed. Polyclonal and monoclonal antibodies, with desired specificities against preselected synthetic regions of urokinase (UK), were obtained by immunization with the respective synthetic peptides and used to develop RIA for zymogen and the two activated forms of UK.

Amplification of the antigen-antibody interaction from quartz crystal microbalance immunosensors via back-filling immobilization of nanogold on biorecognition surface.

PubMed

Tang, Dian-Quan; Zhang, Da-Jun; Tang, Dian-Yong; Ai, Hua

2006-10-20

A new quartz crystal microbalance immunoassay method based on a novel transparent immunoaffinity reactor was developed for clinical immunoassay. To construct such an affinity reactor, resonators with a frequency of 10 MHz were fabricated by affinity binding of functionalized gold nanoparticles (nanogold) to quartz crystal with immobilized specific ligand for the label-free analysis of the affinity reaction between a ligand and its receptor. [Recombinant human tumor markers, carcinoembryonic antigen (CEA) was chosen as a model ligand.] The binding of target molecules onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was proportional to the CEA concentration in the range of 3.0-50 ng/ml with a detection limit of 1.5 ng/ml at a signal/noise ration of 3. A glycine-HCl solution (pH 2.3) was used to release antigen-antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of CEA into normal human sera was diagnosed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable, implying a promising alternative approach for detecting CEA in clinical immunoassay. Compared with the conventional enzyme-linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.

Enhanced production of bioethanol from waste of beer fermentation broth at high temperature through consecutive batch strategy by simultaneous saccharification and fermentation.

PubMed

Khattak, Waleed Ahmad; Khan, Taous; Ha, Jung Hwan; Ul-Islam, Mazhar; Kang, Min-Kyung; Park, Joong Kon

2013-10-10

Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new 'one-pot consecutive batch strategy' was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25-67°C and 50rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67°C. The ethanol production increased from 50 to 102.5g/L when bare supernatant was used in the first phase, and then to 219g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33°C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production. Copyright © 2013 Elsevier Inc. All rights reserved.

Laboratory diagnosis of viral infections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lennette, E.H.

1985-01-01

This book contains 30 chapters. Some of the chapter titles are: Radioimmunoassay Systems; Enzyme Immunoassay Systems; Concepts of Clinical Diagnostic Virology; Role of Tissue Culture Systems, Adenoviruses; Influenza Viruses; Rabies Virus; and Rubella.

Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate.

PubMed

Lai, Wenqiang; Tang, Dianping; Zhuang, Junyang; Chen, Guonan; Yang, Huanghao

2014-05-20

This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (<1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody.

Reproducibility of risk figures in 2nd-trimester maternal serum screening for down syndrome: comparison of 2 laboratories.

PubMed

Benn, Peter A; Makowski, Gregory S; Egan, James F X; Wright, Dave

2006-11-01

Analytical error affects 2nd-trimester maternal serum screening for Down syndrome risk estimation. We analyzed the between-laboratory reproducibility of risk estimates from 2 laboratories. Laboratory 1 used Bayer ACS180 immunoassays for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), Diagnostic Systems Laboratories (DSL) RIA for unconjugated estriol (uE3), and DSL enzyme immunoassay for inhibin-A (INH-A). Laboratory 2 used Beckman immunoassays for AFP, hCG, and uE3, and DSL enzyme immunoassay for INH-A. Analyte medians were separately established for each laboratory. We used the same computational algorithm for all risk calculations, and we used Monte Carlo methods for computer modeling. For 462 samples tested, risk figures from the 2 laboratories differed >2-fold for 44.7%, >5-fold for 7.1%, and >10-fold for 1.7%. Between-laboratory differences in analytes were greatest for uE3 and INH-A. The screen-positive rates were 9.3% for laboratory 1 and 11.5% for laboratory 2, with a significant difference in the patients identified as screen-positive vs screen-negative (McNemar test, P<0.001). Computer modeling confirmed the large between-laboratory risk differences. Differences in performance of assays and laboratory procedures can have a large effect on patient-specific risks. Screening laboratories should minimize test imprecision and ensure that each assay performs in a manner similar to that assumed in the risk computational algorithm.

Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron-specific enolase.

PubMed

Yang, Tingzhen; Vdovenko, Marina; Jin, Xue; Sakharov, Ivan Yu; Zhao, Shulin

2014-07-01

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron-specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate-labeled antibody binds with HRP-labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP-NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two-order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanoparticle-based biologic mimetics

PubMed Central

Cliffel, David E.; Turner, Brian N.; Huffman, Brian J.

2009-01-01

Centered on solid chemistry foundations, biology and materials science have reached a crossroad where bottom-up designs of new biologically important nanomaterials are a reality. The topics discussed here present the interdisciplinary field of creating biological mimics. Specifically, this discussion focuses on mimics that are developed using various types of metal nanoparticles (particularly gold) through facile synthetic methods. These methods conjugate biologically relevant molecules, e.g., small molecules, peptides, proteins, and carbohydrates, in conformationally favorable orientations on the particle surface. These new products provide stable, safe, and effective substitutes for working with potentially hazardous biologicals for applications such as drug targeting, immunological studies, biosensor development, and biocatalysis. Many standard bioanalytical techniques can be used to characterize and validate the efficacy of these new materials, including quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA). Metal nanoparticle–based biomimetics continue to be developed as potential replacements for the native biomolecule in applications of immunoassays and catalysis. PMID:20049778

Preparation of a Magnetically Switchable Bioelectrocatalytic System Employing Cross-Linked Enzyme Aggregates in Magnetic Mesocellular Carbon Foam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jinwoo; Lee, Dohun; Oh, Eunkeu

2005-11-18

Nanostructured magnetic materials (NMMs)[1] have attracted much attention recently because of their broad biotechnological applications including support matrices for enzyme immobilization,[2] immunoassays,[3] drug delivery,[4] and biosensors.[ 5] Specifically, the easy separation and controlled placement of NMMs by means of an external magnetic field enables their application in the development of immobilized enzyme processes[2] and the construction of magnetically controllable bio-electrocatalytic systems.[5, 6] Herein, we demonstrate the use of immobilized enzymes in NMMs for magnetically switchable bio-electrocatalysis.

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate.

PubMed

Tao, Xiaoqi; Wang, Wenjun; Wang, Zhanhui; Cao, Xingyuan; Zhu, Jinghui; Niu, Lanlan; Wu, Xiaoping; Jiang, Haiyang; Shen, Jianzhong

2014-06-01

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.

Evaluation of three commercial rapid tests for detecting antibodies to human immunodeficiency virus.

PubMed

Ng, K P; Saw, T L; Baki, A; Kamarudin, R

2003-08-01

Determine HIV-1/2, Chembio HIV-1/2 STAT-PAK and PenTest are simple/rapid tests for the detection of antibodies to HIV-1 and HIV-2 in human whole blood, serum and plasma samples. The assay is one step and the result is read visually within 15 minutes. Using 92 known HIV-1 reactive sera and 108 known HIV-1 negative sera, the 3 HIV tests correctly identified all the known HIV-1 reactive and negative samples. The results indicated that Determine HIV-1/2, Chembio HIV-1/2 STAT-PAK and PenTest HIV are as sensitive and specific (100% concordance) as Microparticle Enzyme Immunoassay. The data indicated that these 3 HIV tests are effective testing systems for diagnosis of HIV infection in a situation when the conventional Enzyme Immunoassay is not suitable.

Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine

NASA Astrophysics Data System (ADS)

Wu, Yongjun; Yu, Songcheng; Yu, Fei; Yan, Nali; Qu, Lingbo; Zhang, Hongquan

2011-10-01

Sulfamethoxydiazine (SMD), which is often used for animal disease treatment, is harmful to human health. No SMD residue should be detected in food in some countries, such as USA and Japan. Therefore, it is significant to develop a high-throughput, high-sensitivity and accurate method for the determination of the content of SMD in food. In this paper, chemiluminescence enzyme immunoassay (CLEIA) was developed for quantification of SMD. For this method, the limit of detection was 3.2 pg/ml, the linear range was from 10 to 2000 pg/ml, the within-day and inter-day precision were below 13% and below 18%, respectively, and the recovery was from 85% to 105%. Milk and egg were selected as samples to be examined with this method, and the result indicated that this CLEIA method was suitable for screening and quality control of food.

A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenic Aspergillus species.

PubMed

Wegner, S; Bauer, J I; Dietrich, R; Märtlbauer, E; Usleber, E; Gottschalk, C; Gross, M

2017-02-01

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml -1 . The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0·87%) and negligible reactivity with aflatoxins (<0·02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (<0·1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains. The carcinogenic mycotoxin sterigmatocystin (STC) is produced by several Aspergillus species, either alone or together with aflatoxins. Here, we report a very simple and straightforward procedure to obtain highly sensitive and specific anti-STC antibodies, and their use in the first ever real STC-specific competitive direct enzyme immunoassay (EIA). In combination with a previous EIA for aflatoxins, this study for the first time demonstrates the potential of a STC/aflatoxin EIA pair for what is branded as 'immunochemotaxonomic' identification of mycotoxigenic Aspergillus species. This new analytical tool enhances analytical possibilities for differential analysis of STC and aflatoxins. © 2016 The Society for Applied Microbiology.

Impact of HIV Subtype on Performance of the Limiting Antigen-Avidity Enzyme Immunoassay, the Bio-Rad Avidity Assay, and the BED Capture Immunoassay in Rakai, Uganda

PubMed Central

Longosz, Andrew F.; Serwadda, David; Nalugoda, Fred; Kigozi, Godfrey; Franco, Veronica; Gray, Ronald H.; Quinn, Thomas C.; Eshleman, Susan H.

2014-01-01

Abstract Previous studies demonstrated that individuals with subtype D HIV infection who had been infected for 2 or more years were frequently misclassified as assay positive using cross-sectional incidence assays. Samples from 510 subjects (212 subtype A, 298 subtype D) who were infected for 2.2 to 14.5 years (median 5.4 years) and were not virally suppressed were tested using an LAg-Avidity enzyme immunoassay (LAg-Avidity EIA), Bio-Rad Avidity assay, and BED capture enzyme immunoassay (BED-CEIA). The performance of these three assays was evaluated using various assay cutoff values [LAg-Avidity EIA: <1.0 OD-n and <2.0 OD-n; Bio-Rad Avidity assay: <40% avidity index (AI) and <80% AI; BED-CEIA: <0.8 OD-n]. The mean LAg-Avidity EIA result was higher for subtype A than D (4.54±0.95 vs. 3.86±1.26, p<0.001); the mean Bio-Rad Avidity assay result was higher for subtype A than D (88.9%±12.5% vs. 75.1±30.5, p<0.001); and the mean BED-CEIA result was similar for the two subtypes (2.2±1.2 OD-n for subtype A, 2.2±1.3 OD-n for subtype D, p<0.9). The frequency of misclassification was higher for individuals with subtype D infection compared to those with subtype A infection, using either the LAg-Avidity EIA with a cutoff of <2.0 OD-n or the Bio-Rad Avidity assay with cutoffs of <40% or <80% AI. No subtype-specific differences in assay performance were observed using the BED-CEIA. Sex and age were not significantly associated with misclassification by any assay. The LAg-Avidity EIA with a cutoff <1.0 OD-n had the lowest frequency of misclassification in this Ugandan population. PMID:24083837

Surface plasmon resonance biosensing: Approaches for screening and characterising antibodies for food diagnostics.

PubMed

Yakes, B J; Buijs, J; Elliott, C T; Campbell, K

2016-08-15

Research in biosensing approaches as alternative techniques for food diagnostics for the detection of chemical contaminants and foodborne pathogens has increased over the last twenty years. The key component of such tests is the biorecognition element whereby polyclonal or monoclonal antibodies still dominate the market. Traditionally the screening of sera or cell culture media for the selection of polyclonal or monoclonal candidate antibodies respectively has been performed by enzyme immunoassays. For niche toxin compounds, enzyme immunoassays can be expensive and/or prohibitive methodologies for antibody production due to limitations in toxin supply for conjugate production. Automated, self-regenerating, chip-based biosensors proven in food diagnostics may be utilised as rapid screening tools for antibody candidate selection. This work describes the use of both single channel and multi-channel surface plasmon resonance (SPR) biosensors for the selection and characterisation of antibodies, and their evaluation in shellfish tissue as standard techniques for the detection of domoic acid, as a model toxin compound. The key advantages in the use of these biosensor techniques for screening hybridomas in monoclonal antibody production were the real time observation of molecular interaction and rapid turnaround time in analysis compared to enzyme immunoassays. The multichannel prototype instrument was superior with 96 analyses completed in 2h compared to 12h for the single channel and over 24h for the ELISA immunoassay. Antibodies of high sensitivity, IC50's ranging from 4.8 to 6.9ng/mL for monoclonal and 2.3-6.0ng/mL for polyclonal, for the detection of domoic acid in a 1min analysis time were selected. Although there is a progression for biosensor technology towards low cost, multiplexed portable diagnostics for the food industry, there remains a place for laboratory-based SPR instrumentation for antibody development for food diagnostics as shown herein. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of drugs in human tissues by supercritical fluid extraction/immunoassay

NASA Astrophysics Data System (ADS)

Furton, Kenneth G.; Sabucedo, Alberta; Rein, Joseph; Hearn, W. L.

1997-02-01

A rapid, readily automated method has been developed for the quantitative analysis of phenobarbital from human liver tissues based on supercritical carbon dioxide extraction followed by fluorescence enzyme immunoassay. The method developed significantly reduces sample handling and utilizes the entire liver homogenate. The current method yields comparable recoveries and precision and does not require the use of an internal standard, although traditional GC/MS confirmation can still be performed on sample extracts. Additionally, the proposed method uses non-toxic, inexpensive carbon dioxide, thus eliminating the use of halogenated organic solvents.

Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

PubMed

Bong, Ji-Hong; Song, Hyun-Woo; Kim, Tae-Hun; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2018-04-15

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity. Copyright © 2017 Elsevier B.V. All rights reserved.

IMMUNOCHEMICAL DETERMINATION OF DIOXINS IN SEDIMENT SAMPLES

EPA Science Inventory

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are considered highly toxic contaminants with the environmental monitoring of these compounds being of great importance. Immunoassay procedures such as the enzyme-linked immunosorbent assay (ELIS...

ELEGANT ENVIRONMENTAL IMMUNOASSAYS

EPA Science Inventory

Immunochemical methods are based on selective antibodies directed to a particular target analyte. The specific binding between antibody and analyte can be used for detection and quantitation. Methods such as the enzyme-linked immunosorbent assay (ELISA) can provide a sensitiv...

SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration.

PubMed

Parro, Víctor; de Diego-Castilla, Graciela; Rodríguez-Manfredi, José A; Rivas, Luis A; Blanco-López, Yolanda; Sebastián, Eduardo; Romeral, Julio; Compostizo, Carlos; Herrero, Pedro L; García-Marín, Adolfo; Moreno-Paz, Mercedes; García-Villadangos, Miriam; Cruz-Gil, Patricia; Peinado, Verónica; Martín-Soler, Javier; Pérez-Mercader, Juan; Gómez-Elvira, Javier

2011-01-01

The search for unequivocal signs of life on other planetary bodies is one of the major challenges for astrobiology. The failure to detect organic molecules on the surface of Mars by measuring volatile compounds after sample heating, together with the new knowledge of martian soil chemistry, has prompted the astrobiological community to develop new methods and technologies. Based on protein microarray technology, we have designed and built a series of instruments called SOLID (for "Signs Of LIfe Detector") for automatic in situ detection and identification of substances or analytes from liquid and solid samples (soil, sediments, or powder). Here, we present the SOLID3 instrument, which is able to perform both sandwich and competitive immunoassays and consists of two separate functional units: a Sample Preparation Unit (SPU) for 10 different extractions by ultrasonication and a Sample Analysis Unit (SAU) for fluorescent immunoassays. The SAU consists of five different flow cells, with an antibody microarray in each one (2000 spots). It is also equipped with an exclusive optical package and a charge-coupled device (CCD) for fluorescent detection. We demonstrated the performance of SOLID3 in the detection of a broad range of molecular-sized compounds, which range from peptides and proteins to whole cells and spores, with sensitivities at 1-2 ppb (ng mL⁻¹) for biomolecules and 10⁴ to 10³ spores per milliliter. We report its application in the detection of acidophilic microorganisms in the Río Tinto Mars analogue and report the absence of substantial negative effects on the immunoassay in the presence of 50 mM perchlorate (20 times higher than that found at the Phoenix landing site). Our SOLID instrument concept is an excellent option with which to detect biomolecules because it avoids the high-temperature treatments that may destroy organic matter in the presence of martian oxidants.

Hemostatic Function of Apheresis Platelets Stored at 4 deg C and 22 deg C

DTIC Science & Technology

2014-05-01

utilized. Thromboxane B2 (TxB2) enzyme immunoassay kits were purchased from Cayman Chemicals (Ann Arbor, MI), and human soluble CD40L (sCD40L) extra...sensitive platinum enzyme linked immunosorbent assay kits were pur chased from eBioscience (Vienna, Austria). CG4+ and CHEM8+ cartridges were purchased from...TruCount tubes (BD Biosciences). Enzyme linked immunosorbent assay Commercially available kits were used to assess sCD40L and TxB2 levels released into

Highly sensitive determination of diclofenac based on resin beads and a novel polyclonal antibody by using flow injection chemiluminescence competitive immunoassay

NASA Astrophysics Data System (ADS)

Shi, Jing; Xu, Mingxia; Tang, Qinghui; Zhao, Kang; Deng, Anping; Li, Jianguo

2018-02-01

A novel flow injection chemiluminescence immunoassay for simple, sensitive and low-cost detection of diclofenac was established based on specific binding of antigen and antibody. Carboxylic resin beads used as solid phase carrier materials provided good biocompatibility and large surface-to-volume ratio for modifying more coating antigen. There was a competitive process between the diclofenac in solution and the immobilized coating antigen to react with the limited binding sites of the polyclonal antibody to form the immunocomplex. The second antibody labelled with horseradish peroxidase was introduced into the immunosensor and trapped by captured polyclonal antibody against diclofenac, which could effectively amplify chemiluminescence signals of luminol-PIP-H2O2. Under optimal conditions, the diclofenac could be detected quantitatively. The chemiluminescence intensity decreased linearly with the logarithm of the diclofenac concentration in the range of 0.1-100 ng mL- 1 with a detection limit of 0.05 ng mL- 1 at a signal-to-noise ratio of 3. The immunosensor exhibited high sensitivity, specificity and acceptable stability. This easy-operated and cost-effective analytical method could be valuable for the diclofenac determination in real water samples.

Antibody class capture assays for varicella-zoster virus.

PubMed Central

Forghani, B; Myoraku, C K; Dupuis, K W; Schmidt, N J

1984-01-01

Pooled monoclonal antibodies to varicella-zoster virus (VZV) were used as "detector" antibodies in a four-phase enzyme immunofluorescence assay for determination of immunoglobulin M (IgM), IgA, and IgG antibodies to VZV. Polyclonal antisera specific for heavy chains of human IgM, IgA, and IgG were employed as "capture" antibodies on the solid phase. The antibody class capture assay (ACCA) for VZV IgM antibody detected high titers of virus-specific IgM in all patients with varicella and in 5 of 10 zoster patients. VZV IgM antibody was not detected in patients with primary herpes simplex virus infections or in other individuals without active VZV infection, with one exception, a patient with encephalitis who had other serological findings compatible with a reactivated VZV infection. VZV-specific IgA and IgG antibody titers demonstrable by ACCA were compared with those measured by solid-phase indirect enzyme immunofluorescence assay (EIFA). VZV IgA antibody titers detected in patients with varicella and zoster were variable and could not be considered to be reliable markers of active VZV infection. IgA antibody titers detected by ACCA tended to be higher than those demonstrated by solid-phase indirect EIFA in varicella and zoster patients. VZV IgG antibody titers detected by ACCA in patients with varicella, and to a lesser extent in zoster patients, were as high as or higher than those demonstrated by solid-phase indirect EIFA. However, ACCA was totally insensitive in detecting VZV IgG antibody in individuals with past infections with VZV and would not be a suitable approach for determination of immunity status to VZV. PMID:6330163

Immunoassay as an analytical tool in agricultural biotechnology.

PubMed

Grothaus, G David; Bandla, Murali; Currier, Thomas; Giroux, Randal; Jenkins, G Ronald; Lipp, Markus; Shan, Guomin; Stave, James W; Pantella, Virginia

2006-01-01

Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

Production and partial purification of tannase from Aspergillus ficuum Gim 3.6.

PubMed

Ma, Wan-liang; Zhao, Fen-fen; Ye, Qin; Hu, Zhen-xing; Yan, Dong; Hou, Jie; Yang, Yang

2015-01-01

A novel fungal strain, Aspergillus ficuum Gim 3.6, was evaluated for its tannase-producing capability in a wheat bran-based solid-state fermentation. Thin-layer chromatography (TLC) analysis revealed that the strain was able to degrade tannic acid to gallic acid and pyrogallol during the fermentation process. Quantitation of enzyme activity demonstrated that this strain was capable of producing a relatively high yield of extracellular tannase. Single-factor optimization of process parameters resulted in high yield of tannase after 60 hr of incubation at a pH of 5.0 at 30°C, 1 mL of inoculum size, and 1:1 solid-liquid ratio in the presence of 2.0% (w/v) tannic acid as inducer. The potential of aqueous two-phase extraction (ATPE) for the purification of tannase was investigated. Influence of various parameters such as phase-forming salt, molecular weight of polyethylene glycol (PEG), pH, and stability ratio on tannase partition and purification was studied. In all the systems, the target enzyme was observed to preferentially partition to the PEG-rich top phase, and the best result of purification (2.74-fold) with an enzyme activity recovery of 77.17% was obtained in the system containing 17% (w/w) sodium citrate and 18.18% (w/w) PEG1000, at pH 7.0.

PCP IMMUMOASSAY TECHNOLOGIES - INNOVATIVE TECHNOLOGY EVALUATION REPORT

EPA Science Inventory

Three enzyme-linked immunosorbent assay technologies for pentachlorophenol (PCP) testing in soil and water were evaluated. Penta RISc Test Systems (formerly ENSYS, Inc.), EnviroGard™ PCP Immunoassay Test Kit (Millipore Corp.), and Pentachlorophenol RaPID Assay (formerly Ohmicron ...

A prospective field evaluation of an enzyme immunoassay: Detection of eastern equine encephalomyelitis virus antigen in pools of Culiseta melanura

USGS Publications Warehouse

Scott, T.W.; Olson, J.G.; Lewis, T.E.; Carpenter, J.W.; Lorenz, L.H.; Lembeck, L.A.; Joseph, S.R.; Pagac, B.B.

1987-01-01

A prospective field study was conducted to determine the sensitivity and specificity of an enzyme immunoassay (EIA) compared to virus isolation in cell culture for the detection of eastern equine encephalomyelitis (EEE) virus in naturally infected mosquitoes. A total of 10,811 adult female Culiseta melanura were collected in light traps during 1985 from four locations in Maryland. Eastern equine encephalomyelitis virus was isolated from 5 of 495 mosquito pools in African green monkey kidney and baby hamster kidney cell cultures. All five virus-infected pools were detected by the EIA, and all 490 uninfected pools were correctly scored as not containing virus. The EIA did not produce false positive or false negative results. Results support the assertion of previous researchers that the antigen detection EIA is a rapid, sensitive, specific, and simple alternative to traditional bioassays for the detection of EEE virus in mosquitoes.

Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

PubMed

Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

1993-08-01

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

The Geochemical Behavior and Transport Characteristics of Xenoestrogens

NASA Astrophysics Data System (ADS)

Wallace, T. C.; Bennett, P. C.

2003-12-01

Xenoestrogens are estrogenic active synthetic chemicals that mimic the actions of female sex hormones. Xenoestrogens can be produced synthetically and naturally, and exposure can occur from a variety of sources- food additives, plastics, pesticides, or pharmaceuticals. These environmental chemicals are also known as endocrine disruptors because exposure to low doses (ng/L) have been linked to adverse effects in the reproductive and developmental stages in aquatic species (i.e. reproductive anomalies, feminization, infertility, alterations in growth during life cycles, and changes in community structures). Determining the exposure risks of these toxicological compounds, however, requires a better understanding of the geochemical behavior and transport of synthetic estrogens it is discharged to. Estrogen and its metabolites are also useful tracers because of their specific medical usage (sources from birth control pills, estrogen replacement therapy, and livestock farming), slow degradation before excretion, and unique physiochemical properties (low volatility, hydrophobicity, and high Kow). Estradiol concentrations analyzed by an enzyme-linked immunoassay (ELISA) show that <2-55 ng/L are discharged to Walnut Creek, a stream that also connects to the Colorado River(TX). The bioavailability of these compounds is affected by sorption processes, where xenoestrogens become associated with solid phases. A series of batch sorption experiments using sediment collected from Walnut Creek downstream of an Austin waste water treatment plant and synthetic estrogen standards (Simga@ Estrone, 17B-Estradiol, and Estriol), examined the distribution of estrogen between solid and aqueous phases. Analysis of the concentrations sorbed to sediment result in Freundlich sorption isotherms using HPLC/UV techniques (High Performance Liquid Chromatography with UV detectors- 220 and 280nm). Sorption occurs rapidly with 98% of 17B-Estradiol sorbed within 30 minutes (Estriol=80%, Estrone=95%), which is compared to photolysis degradation rates under UV and a broader spectrum sun lamp. Ultraviolet/Visible (UV/VIS) spectroscopy of the estrogen standards with dilute fulvic acid may indicate complexing with organic material. The hydrophobic nature of estrogen molecules due to a phenolic group seem to play a large role in the sorption rate. This sorption may alter direct photolysis decay rates, thereby acting as both a `sunscreen' and a carrier by increasing the exposure distance and bioavailability of xenoestrogens in the aquatic environment.

False Elevation of the Blood Tacrolimus Concentration, as Assessed by an Affinity Column-mediated Immunoassay (ACMIA), Led to Acute T Cell-mediated Rejection after Kidney Transplantation.

PubMed

Kono, Momoko; Hasegawa, Jumpei; Ogawa, Hina; Yoshikawa, Kanae; Ishiwatari, Ayumi; Wakai, Sachiko; Tanabe, Kazunari; Shirakawa, Hiroki

2018-05-01

Tacrolimus is the most commonly used immunosuppressant. Because of its narrow therapeutic range, it is necessary to frequently monitor its concentration. We report the case of a 25-year-old man who underwent kidney transplantation whose tacrolimus concentrations, as measured by an affinity column-mediated immunoassay, were falsely elevated. As we reduced the dose of tacrolimus, the recipient developed T cell-mediated rejection. Using the same blood samples, an enzyme-multiplied immunoassay technique showed that the patient's levels of tacrolimus were extremely low. A further examination indicated that the false increase in the tacrolimus concentration was likely due to an unknown interfering substance. We administered methylprednisolone and antithymocyte-globulin. The patient's serum creatinine level decreased and remained stable after these treatments.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

PubMed

Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

2016-06-07

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Field demonstration of on-site analytical methods for TNT and RDX in ground water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Craig, H.; Ferguson, G.; Markos, A.

1996-12-31

A field demonstration was conducted to assess the performance of eight commercially-available and emerging colorimetric, immunoassay, and biosensor on-site analytical methods for explosives 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ground water and leachate at the Umatilla Army Depot Activity, Hermiston, Oregon and US Naval Submarine Base, Bangor, Washington, Superfund sites. Ground water samples were analyzed by each of the on-site methods and results compared to laboratory analysis using high performance liquid chromatography (HPLC) with EPA SW-846 Method 8330. The commercial methods evaluated include the EnSys, Inc., TNT and RDX colorimetric test kits (EPA SW-846 Methods 8515 and 8510) with amore » solid phase extraction (SPE) step, the DTECH/EM Science TNT and RDX immunoassay test kits (EPA SW-846 Methods 4050 and 4051), and the Ohmicron TNT immunoassay test kit. The emerging methods tested include the antibody-based Naval Research Laboratory (NRL) Continuous Flow Immunosensor (CFI) for TNT and RDX, and the Fiber Optic Biosensor (FOB) for TNT. Accuracy of the on-site methods were evaluated using linear regression analysis and relative percent difference (RPD) comparison criteria. Over the range of conditions tested, the colorimetric methods for TNT and RDX showed the highest accuracy of the emerging methods for TNT and RDX. The colorimetric method was selected for routine ground water monitoring at the Umatilla site, and further field testing on the NRL CFI and FOB biosensors will continue at both Superfund sites.« less

Serum tumour necrosis factor alpha in osteopenic and osteoporotic postmenopausal females: A cross-sectional study in Pakistan.

PubMed

Murad, Rafat; Shezad, Zahra; Ahmed, Saara; Ashraf, Mussarat; Qadir, Murad; Rehman, Rehana

2018-03-01

To compare biochemical parameters serum tumour necrosis factor alpha, calcium, magnesium, bone-specific alkaline phosphatase and vitamin D in postmenopausal women. This cross-sectional study was carried out from June 2015 to July 2016 at Jinnah Medical and Dental College, Karachi, and comprised postmenopausal women. Bone mineral density done by dual energy X-ray absorptiometryscan categorised subjects by World Health Organisation classification into normal (T score > -1) osteopenic (T score between -1 and -2.5) and osteoporotic (T score < -2.5). Biochemical parameters like tumour necrosis alpha, calcium, magnesium, bone-specific alkaline phosphatase and vitamin D were measured by solid phase enzyme amplified sensitivity immunoassay method. SPSS 16 was used to analyse the data. Of the 146 women, 34(23%) were normal, 93(67%) were osteopenic and 19(13%) were osteoporotic. There was significant difference in mean body mass index, serum tumour necrosis factor alpha and calcium in all the three groups (p<0.01). Significant mean difference was observed in serum calcium levels between normal and osteopenic, and between normal and osteoporotic group (p<0.05 each) without any significant mean difference between osteopenic and osteoporotic groups (p>0.05). A significant difference was observed for mean tumour necrosis factor alpha values between normal and osteoporotic groups (p<0.05). Tumour necrosis factor alpha showed negative correlation with bone mineral density in osteopenic and osteoporotic groups (p>0.05). Increased bone turnover in postmenopausal osteopenic women can be predicted by increased serum cytokine.

Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps.

PubMed Central

Harmsen, T; Jongerius, M C; van der Zwan, C W; Plantinga, A D; Kraaijeveld, C A; Berbers, G A

1992-01-01

A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA. PMID:1500523

Pore-scale dynamics of enzyme adsorption, swelling and reactive dissolution determine sugar yield in hemicellulose hydrolysis for biofuel production

PubMed Central

Dutta, Sajal Kanti; Chakraborty, Saikat

2016-01-01

Hemicelluloses are the earth’s second most abundant structural polymers, found in lignocellulosic biomass. Efficient enzymatic depolymerization of xylans by cleaving their β-(1 → 4)-glycosidic bonds to produce soluble sugars is instrumental to the cost-effective production of liquid biofuels. Here we show that the multi-scale two-phase process of enzymatic hydrolysis of amorphous hemicelluloses is dominated by its smallest scale–the pores. In the crucial first five hours, two to fourfold swelling of the xylan particles allow the enzymes to enter the pores and undergo rapid non-equilibrium adsorption on the pore surface before they hydrolyze the solid polymers, albeit non-competitively inhibited by the products xylose and xylobiose. Rapid pore-scale reactive dissolution increases the solid carbohydrate’s porosity to 80–90%. This tightly coupled experimental and theoretical study quantifies the complex temporal dynamics of the transport and reaction processes coupled across scales and phases to show that this unique pore-scale phenomenon can be exploited to accelerate the depolymerization of hemicelluloses to monomeric sugars in the first 5–6 h. We find that an ‘optimal substrate loading’ of 5 mg/ml (above which substrate inhibition sets in) accelerates non-equilibrium enzyme adsorption and solid hemicellulose depolymerization at the pore-scale, which contributes three-quarters of the soluble sugars produced for bio-alcohol fermentation. PMID:27905534

Pore-scale dynamics of enzyme adsorption, swelling and reactive dissolution determine sugar yield in hemicellulose hydrolysis for biofuel production

NASA Astrophysics Data System (ADS)

Dutta, Sajal Kanti; Chakraborty, Saikat

2016-12-01

Hemicelluloses are the earth’s second most abundant structural polymers, found in lignocellulosic biomass. Efficient enzymatic depolymerization of xylans by cleaving their β-(1 → 4)-glycosidic bonds to produce soluble sugars is instrumental to the cost-effective production of liquid biofuels. Here we show that the multi-scale two-phase process of enzymatic hydrolysis of amorphous hemicelluloses is dominated by its smallest scale-the pores. In the crucial first five hours, two to fourfold swelling of the xylan particles allow the enzymes to enter the pores and undergo rapid non-equilibrium adsorption on the pore surface before they hydrolyze the solid polymers, albeit non-competitively inhibited by the products xylose and xylobiose. Rapid pore-scale reactive dissolution increases the solid carbohydrate’s porosity to 80-90%. This tightly coupled experimental and theoretical study quantifies the complex temporal dynamics of the transport and reaction processes coupled across scales and phases to show that this unique pore-scale phenomenon can be exploited to accelerate the depolymerization of hemicelluloses to monomeric sugars in the first 5-6 h. We find that an ‘optimal substrate loading’ of 5 mg/ml (above which substrate inhibition sets in) accelerates non-equilibrium enzyme adsorption and solid hemicellulose depolymerization at the pore-scale, which contributes three-quarters of the soluble sugars produced for bio-alcohol fermentation.

Bupropion interference with immunoassays for amphetamines and LSD.

PubMed

Vidal, Christian; Skripuletz, Thomas

2007-06-01

A 50-year-old male patient suddenly had lost consciousness, although he had previously been healthy. On arrival at hospital seizures arose. The authors investigated a urine sample of the patient, and performed toxicological drug screening with immunochemical Cloned Enzyme Donor Immunoassay (CEDIA) assays. Positive findings for amphetamines and LSD could not be confirmed. Using gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS), the authors identified bupropion, a drug used to aid in smoking cessation, as the interfering compound, which may cause false-positive results for amphetamines and LSD using the CEDIA assays.

Phase 1 first-in-human studies of the reactogenicity and immunogenicity of a recombinant meningococcal NspA vaccine in healthy adults.

PubMed

Halperin, Scott A; Langley, Joanne M; Smith, Bruce; Wunderli, Peter; Kaufman, Lisa; Kimura, Alan; Martin, Denis

2007-01-05

Neisserial surface protein A (NspA) is a highly conserved, surface-exposed outer membrane protein of Neisseria meningitidis that has been shown to induce a bactericidal immune response in animals against all pathogenic Neisserial serogroups. Healthy 18-50-year-old adults were assigned to receive, in a dose escalating manner, 3 doses of 1 of 5 formulations of an experimental, unfolded, recombinant NspA (rNspA) vaccine or placebo, or 1 dose of commercially available quadravalent (A, C, Y, W-135) meningococcal polysaccharide vaccine (Menomune((R))). Adverse events were collected during the first week post-immunization, prior to the next dose and 1 month after the last dose. Serum for measurement of hematological and biochemical parameters and antibodies by enzyme immunoassay and bactericidal assay were measured before the first dose, prior to the second dose and 1 month after the last dose of vaccine. The rNspA vaccine was well tolerated by recipients. Injection-site pain was reported more frequently by recipients of the three highest doses of rNspA compared to placebo but at similar rates to the licensed meningococcal polysaccharide vaccine. Adverse events were reported less frequently after subsequent doses in the three-dose series. An antibody rise measured by enzyme immunoassay was elicited with a dose-related increase that reached a maximum with the 125mug dose. Prolongation of the dosing interval between the second and third dose appeared to be associated with increased antibody levels. No bactericidal antibodies were detected after any of the rNspA formulations. The unfolded rNspA meningococcal vaccine was well tolerated and immunogenic in healthy adult volunteers but did not elicit bactericidal antibodies.

Anti-colitis and -adhesion effects of daikenchuto via endogenous adrenomedullin enhancement in Crohn's disease mouse model.

PubMed

Kono, Toru; Kaneko, Atsushi; Hira, Yoshiki; Suzuki, Tatsuya; Chisato, Naoyuki; Ohtake, Nobuhiro; Miura, Naoko; Watanabe, Tsuyoshi

2010-06-01

Adrenomedullin (ADM) is a member of the calcitonin family of regulatory peptides, and is reported to have anti-inflammatory effects in animal models of Crohn's disease (CD). We investigated the therapeutic effects of daikenchuto (DKT), an extracted Japanese herbal medicine, on the regulation of endogenous ADM in the gastrointestinal tract in a CD mouse model. Colitis was induced in mice by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS); afterwards, DKT was given orally. Colonic damage was assessed on day 3 by macroscopic and microscopic observation, enzyme immunoassays of proinflammatory cytokines in the colonic mucosa, and serum amyloid A (SAA), a hepatic acute-phase protein. To determine the involvement of ADM, an ADM antagonist was instilled intrarectally before DKT administration. The effect of DKT on ADM production by intestinal epithelial cells was evaluated by enzyme immunoassay and real-time PCR. DKT significantly attenuated mucosal damage and colonic inflammatory adhesions, and inhibited elevations of SAA in plasma and the proinflammatory cytokines TNFα and IFNγ in the colon. Small and large intestinal epithelial cells produced higher levels of ADM after DKT stimulation. A DKT-treated IEC-6 cell line also showed enhanced ADM production at protein and mRNA levels. Abolition of this effect by pretreatment with an ADM antagonist shows that DKT appears to exert its anti-colitis effect via up-regulation of endogenous ADM in the intestinal tract. DKT exerts beneficial effects in a CD mouse model through endogenous release and production of ADM. Endogenous ADM may be a therapeutic target for CD. Copyright © 2009 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Surface immobilized antibody orientation determined using ToF-SIMS and multivariate analysis.

PubMed

Welch, Nicholas G; Madiona, Robert M T; Payten, Thomas B; Easton, Christopher D; Pontes-Braz, Luisa; Brack, Narelle; Scoble, Judith A; Muir, Benjamin W; Pigram, Paul J

2017-06-01

Antibody orientation at solid phase interfaces plays a critical role in the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture as compared to their randomly oriented counterparts. Direct characterization of oriented proteins with surface analysis methods still remains a challenge however surface sensitive techniques such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody orientation. Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. Herein, ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr are used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) test against each of the models indicated preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab') 2 region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. Mass fragments were then compared to amino acid fragments and amino acid composition in F(ab') 2 and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab') 2 fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp. The systematic data analysis methodology employed herein offers a new approach for the investigation of antibody orientation applicable to a range of substrates. Controlled orientation of antibodies at solid phases is critical for maximizing antigen detection in biosensors and immunoassays. Surface-sensitive techniques (such as ToF-SIMS), capable of direct characterization of surface immobilized and oriented antibodies, are under-utilized in current practice. Selection of a small number of mass fragments for analysis, typically pertaining to amino acids, is commonplace in literature, leaving the majority of the information-rich spectra unanalyzed. The novelty of this work is the utilization of a comprehensive, unbiased mass fragment list and the employment of principal component analysis (PCA) and artificial neural network (ANN) models in a unique methodology to prove antibody orientation. This methodology is of significant and broad interest to the scientific community as it is applicable to a range of substrates and allows for direct, label-free characterization of surface bound proteins. Copyright © 2017 Acta Materialia Inc. All rights reserved.

Regulatory effect of Epithalon on production of melatonin and cortisol in old monkeys.

PubMed

Goncharova, N D; Khavinson, B K; Lapin, B A

2001-04-01

The effect of Epithalon on melatonin and cortisol secretion in female rhesus monkeys of various ages was evaluated by enzyme immunoassay. Epithalon stimulated evening melatonin production and normalized circadian rhythms of cortisol production in old monkeys.

Stalking The Silent Killers.

ERIC Educational Resources Information Center

DeYoung, H. Garrett

1983-01-01

New technologies have been developed to diagnose such disorders as cancer, heart disease, and defects of the newborn in presymptomatic stages. Several technologies, including amniocentesis, radioimmunoassay, enzyme immunoassay, multichannel analysis and others are discussed. Development of these technologies and issues related to their use are…

First 25-hydroxyvitamin D assay for general chemistry analyzers.

PubMed

Saida, Fakhri B; Chen, Xiaoru; Tran, Kiet; Dou, Chao; Yuan, Chong

2015-03-01

25-Hydroxyvitamin D [25(OH)D], the predominant circulating form of vitamin D, is an accurate indicator of the general vitamin D status of an individual. Because vitamin D deficiencies have been linked to several pathologies (including osteoporosis and rickets), accurate monitoring of 25(OH)D levels is becoming increasingly important in clinical settings. Current 25(OH)D assays are either chromatographic or immunoassay-based assays. These assays include HPLC, liquid chromatography-tandem mass spectrometry (LC-MS/MS), enzyme-immunosorbent, immunochemiluminescence, immunofluorescence and radioimmunoassay. All these assays use heterogeneous formats that require phase separation and special instrumentations. In this article, we present an overview of these assays and introduce the first homogeneous assay of 25(OH)D for use on general chemistry analyzers. A special emphasis is put on the unique challenges posed by the 25(OH)D analyte. These challenges include a low detection limit, the dissociation of the analyte from its serum transporter and the inactivation of various binding proteins without phase separation steps.

Effect of monensin on the levels of tachykinins and their processing enzyme activity in rat dorsal root ganglia.

PubMed

Chikuma, Toshiyuki; Inomata, Yuji; Tsuchida, Ken; Hojo, Hiroshi; Kato, Takeshi

2002-06-28

Th effect of monensin, which inhibits trans-Golgi function, on the levels of tachykinins and their processing enzyme activity was examined in organ-cultured rat dorsal root ganglia (DRG). Using an enzyme immunoassay method, we measured neurokinin A and substance P immunoreactivity in the DRG cultured for 72 h with and without 0.1 microM monensin. Both tachykinins were reduced in the DRG treated with monensin. Treatment with monensin also reduced the activity of carboxypeptidase E, which is one of the proteolytic processing enzymes of neuropeptides. These data suggest that proteolytic processing enzymes may in part modulate the biological activity of neuropeptides within a trans-Golgi apparatus.

Recovery Act: Innovative CO 2 Sequestration from Flue Gas Using Industrial Sources and Innovative Concept for Beneficial CO 2 Use

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dando, Neal; Gershenzon, Mike; Ghosh, Rajat

2012-07-31

The overall goal of this DOE Phase 2 project was to further develop and conduct pilot-scale and field testing of a biomimetic in-duct scrubbing system for the capture of gaseous CO 2 coupled with sequestration of captured carbon by carbonation of alkaline industrial wastes. The Phase 2 project, reported on here, combined efforts in enzyme development, scrubber optimization, and sequestrant evaluations to perform an economic feasibility study of technology deployment. The optimization of carbonic anhydrase (CA) enzyme reactivity and stability are critical steps in deployment of this technology. A variety of CA enzyme variants were evaluated for reactivity and stabilitymore » in both bench scale and in laboratory pilot scale testing to determine current limits in enzyme performance. Optimization of scrubber design allowed for improved process economics while maintaining desired capture efficiencies. A range of configurations, materials, and operating conditions were examined at the Alcoa Technical Center on a pilot scale scrubber. This work indicated that a cross current flow utilizing a specialized gas-liquid contactor offered the lowest system operating energy. Various industrial waste materials were evaluated as sources of alkalinity for the scrubber feed solution and as sources of calcium for precipitation of carbonate. Solids were mixed with a simulated sodium bicarbonate scrubber blowdown to comparatively examine reactivity. Supernatant solutions and post-test solids were analyzed to quantify and model the sequestration reactions. The best performing solids were found to sequester between 2.3 and 2.9 moles of CO 2 per kg of dry solid in 1-4 hours of reaction time. These best performing solids were cement kiln dust, circulating dry scrubber ash, and spray dryer absorber ash. A techno-economic analysis was performed to evaluate the commercial viability of the proposed carbon capture and sequestration process in full-scale at an aluminum smelter and a refinery location. For both cases the in-duct scrubber technology was compared to traditional amine- based capture. Incorporation of the laboratory results showed that for the application at the aluminum smelter, the in-duct scrubber system is more economical than traditional methods. However, the reverse is true for the refinery case, where the bauxite residue is not effective enough as a sequestrant, combined with challenges related to contaminants in the bauxite residue accumulating in and fouling the scrubber absorbent. Sensitivity analyses showed that the critical variables by which process economics could be improved are enzyme concentration, efficiency, and half-life. At the end of the first part of the Phase 2 project, a gate review (DOE Decision Zero Gate Point) was conducted to decide on the next stages of the project. The original plan was to follow the pre-testing phase with a detailed design for the field testing. Unfavorable process economics, however, resulted in a decision to conclude the project before moving to field testing. It is noted that CO 2 Solutions proposed an initial solution to reduce process costs through more advanced enzyme management, however, DOE program requirements restricting any technology development extending beyond 2014 as commercial deployment timeline did not allow this solution to be undertaken.« less

Immunoanalysis Methods for the Detection of Dioxins and Related Chemicals

PubMed Central

Tian, Wenjing; Xie, Heidi Qunhui; Fu, Hualing; Pei, Xinhui; Zhao, Bin

2012-01-01

With the development of biotechnology, approaches based on antibodies, such as enzyme-linked immunosorbent assay (ELISA), active aryl hydrocarbon immunoassay (Ah-I) and other multi-analyte immunoassays, have been utilized as alternatives to the conventional techniques based on gas chromatography and mass spectroscopy for the analysis of dioxin and dioxin-like compounds in environmental and biological samples. These screening methods have been verified as rapid, simple and cost-effective. This paper provides an overview on the development and application of antibody-based approaches, such as ELISA, Ah-I, and multi-analyte immunoassays, covering the sample extraction and cleanup, antigen design, antibody preparation and immunoanalysis. However, in order to meet the requirements for on-site fast detection and relative quantification of dioxins in the environment, further optimization is needed to make these immuno-analytical methods more sensitive and easy to use. PMID:23443395

The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer.

PubMed

Fu, Xiaoling; Liu, Yangyang; Qiu, Ruiyun; Foda, Mohamed F; Zhang, Yong; Wang, Tao; Li, Jinshan

2018-03-01

The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0-1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.

An evaluation of the DRI-ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post-mortem urine.

PubMed

Turfus, Sophie C; Vo, Tu; Niehaus, Nadia; Gerostamoulos, Dimitri; Beyer, Jochen

2013-06-01

A commercial enzyme immunoassay for the qualitative and semi-quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post-mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EtG and ethyl sulfate. Using a cut-off of 0.5 µg/ml and LC-MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post-mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut-off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post-mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade-offs between sensitivity and specificity for LC-MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut-offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC-MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi-quantitative presumptive detection of ethyl glucuronide in urine. Copyright © 2012 John Wiley & Sons, Ltd.

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells.

PubMed

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla; Hansson, Stefan; Casslén, Bertil

2009-02-01

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

Identification and optimization of parameters for the semi-continuous production of garbage enzyme from pre-consumer organic waste by green RP-HPLC method.

PubMed

Arun, C; Sivashanmugam, P

2015-10-01

Reuse and management of organic solid waste, reduce the environmental impact on human health and increase the economic status by generating valuable products for current and novel applications. Garbage enzyme is one such product produced from fermentation of organic solid waste and it can be used as liquid fertilizer, antimicrobial agents, treatment of domestic wastewater, municipal and industrial sludge treatment, etc. The semi-continuous production of garbage enzyme in large quantity at minimal time period and at lesser cost is needed to cater for treatment of increasing quantities of industrial waste activated sludge. This necessitates a parameter for monitoring and control for the scaling up of current process on semi-continuous basis. In the present study a RP-HPLC (Reversed Phase-High Performance Liquid Chromatography) method is used for quantification of standard organic acid at optimized condition 30°C column oven temperature, pH 2.7, and 0.7 ml/min flow rate of the mobile phase (potassium dihydrogen phosphate in water) at 50mM concentration. The garbage enzyme solution collected in 15, 30, 45, 60, 75 and 90 days were used as sample to determine the concentration of organic acid. Among these, 90th day sample showed the maximum concentration of 78.14 g/l of acetic acid in garbage enzyme, whereas other organic acids concentration got decreased when compare to the 15th day sample. This result confirms that the matured garbage enzyme contains a higher concentration of acetic acid and thus it can be used as a monitoring parameter for semi-continuous production of garbage enzyme in large scale. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detection of anti-nuclear antibody by immunofluorescence assay and enzyme immunoassay in childhood systemic lupus erythematosus: experience from Bangladesh.

PubMed

Dipti, Tanjeem Rabika; Azam, Mohammad Shaiful; Sattar, Mohammad Humayun; Rahman, Shahana Akhter

2012-02-01

Systemic lupus erythematosus (SLE) is a multisystem, chronic but often episodic, autoimmune disease that is characterized by the presence of antinuclear antibodies (ANA). The criteria set by American College of Rheumatology are widely used for diagnosis of SLE. Elevation of ANA titer is the most sensitive of the ACR criteria. There are different methods for detection of ANA. Indirect immunofluorescence (ANA-IFA) and enzyme immunoassay (ANA-EIA) are commonly used methods. The sensitivity of ANA-IFA using HEp-2 cell substrate is 90-100% in systemic rheumatic diseases. In Bangladesh most of the laboratories use ANA-EIA for detection of ANA. As the sensitivity of ANA-EIA is lower than ANA-IFA it might be that we are missing many cases of ANA positivity in childhood SLE cases. To detect ANA by immunofluorescence assay using HEp-2 cell substrate and enzyme immunoassay in childhood SLE and to compare the diagnostic performance of these methods. This is a cross-sectional analytical study. A total of 40 patients were enrolled. Among them 20 were childhood SLE cases. Another 20 patients of childhood rheumatic diseases other than SLE were taken as the disease control group. In childhood SLE cases, 100% were ANA-positive by IFA and 55% were ANA positive by EIA. The sensitivity of ANA-IFA was 100%. In contrast, sensitivity of ANA-EIA was 55%.   ANA-IFA is superior to ANA-EIA for detection of ANA in childhood SLE patients. ANA-IFA should be the primary screening test for children with clinical features suggestive of SLE. © 2011 The Authors. International Journal of Rheumatic Diseases © 2011 Asia Pacific League of Associations for Rheumatology and Blackwell Publishing Asia Pty Ltd.

Determination of chondroitin-6-sulphate by a competitive enzyme immunoassay using a biotinylated antigen.

PubMed

Kähnert, H; Brinkmann, T; Gässler, N; Kleesiek, K

1994-04-01

A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitin-6-sulphate in the samples and the biotinylated antigen. This assay enables the quantification of chondroitin-6-sulphate in the low concentration range of 16-120 micrograms/l. The intra-assay and inter-assay coefficients of variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/l phosphate-buffered saline, an albumin solution (40 g/l in phosphate-buffered saline) and cell culture medium (containing 100 ml/l foetal calf serum). Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal range was 55-169 micrograms/l. In men the mean value was estimated at 102.2 +/- 37.1 micrograms/l and in women at 98.7 +/- 26.4 micrograms/l. No age or sex dependence was observed. The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 +/- 21.1 mg/kg creatinine (mean +/- standard deviation) and in females (n = 10) 53.5 +/- 21.3 mg/kg creatinine. The clearance rate in men was 0.41 +/- 0.22 ml x min-1 and in women 0.38 +/- 0.15 ml x min-1. No sex dependence was found. Furthermore, the enzyme immunoassay was modified to measure the specific incorporation of a radioactively labelled precursor ([14C]galactosamine) into chondroitin-6-sulphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.

PubMed

Milnerowicz, Halina; Bizoń, Anna

2010-01-01

Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate).

Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains.

PubMed

Bong, Ji-Hong; Yoo, Gu; Park, Min; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2014-11-01

The outer membrane of Escherichia coli was previously isolated as a liposome-like outer membrane particle using an enzymatic treatment for lysozymes; for immunoassays, the particles were subsequently layered on solid supports via hydrophobic interactions. This work presents an enzyme-free isolation method for the E. coli outer membrane with autodisplayed Z-domains using ultrasonication. First, the properties of the outer membrane particle, such as the particle size, zeta potential, and total protein, were compared with the properties of particles obtained using the previous preparation methods. Compared with the conventional isolation method using an enzyme treatment, the ultrasonic method exhibited a higher efficiency at isolating the outer membrane and less contamination by cytosolic proteins. The isolated outer membrane particles were layered on a gold surface, and the roughness and thickness of the layered outer membrane layers were subsequently analyzed using AFM analysis. Finally, the antibody-binding activity of two outer membrane layers with autodisplayed Z-domains created from particles that were isolated using the enzymatic and ultrasonic isolation methods was measured using fluorescein-labeled antibody as a model analyte, and the activity of the outer membrane layer that was isolated from the ultrasonic method was estimated to be more than 20% higher than that from the conventional enzymatic method. Copyright © 2014 Elsevier Inc. All rights reserved.

Optical Sensors for Biomolecules Using Nanoporous Sol-Gel Materials

NASA Technical Reports Server (NTRS)

Fang, Jonathan; Zhou, Jing C.; Lan, Esther H.; Dunn, Bruce; Gillman, Patricia L.; Smith, Scott M.

2004-01-01

An important consideration for space missions to Mars is the ability to detect biosignatures. Solid-state sensing elements for optical detection of biological entities are possible using sol-gel based biologically active materials. We have used these materials as optical sensing elements in a variety of bioassays, including immunoassays and enzyme assays. By immobilizing an appropriate biomolecule in the sol-gel sensing element, we have successfully detected analytes such as amino acids and hormones. In the case of the amino acid glutamate, the enzyme glutamate dehydrogenase was the immobilized molecule, whereas in the case of the hormone cortisol, an anti-cortisol antibody was immobilized in the sensing element. In this previous work with immobilized enzymes and antibodies, excellent sensitivity and specificity were demonstrated in a variety of formats including bulk materials, thin films and fibers. We believe that the sol-gel approach is an attractive platform for bioastronautics sensing applications because of the ability to detect a wide range of entities such as amino acids, fatty acids, hopanes, porphyrins, etc. The sol-gel approach produces an optically transparent 3D silica matrix that forms around the biomolecule of interest, thus stabilizing its structure and functionality while allowing for optical detection. This encapsulation process protects the biomolecule and leads to a more "rugged" sensor. The nanoporous structure of the sol-gel matrix allows diffusion of small target molecules but keeps larger, biomolecules immobilized in the pores. We are currently developing these biologically active sol-gel materials into small portable devices for on-orbit cortisol detection

Association of homocysteine with global DNA methylation in vegetarian Indian pregnant women and neonatal birth anthropometrics.

PubMed

Gadgil, Maithili S; Joshi, Kalpana S; Naik, Sadanand S; Pandit, Anand N; Otiv, Suhas R; Patwardhan, Bhushan K

2014-11-01

The present study was designed to evaluate if plasma maternal folate, vitamin B-12 and homocysteine levels had an effect on maternal global DNA methylation and neonatal anthropometrics in Indian pregnant women. A total of 49 participants having completed ≥36 weeks of pregnancy were enrolled in the study. Estimation of folate was by Ion capture assay, vitamin B-12 by microparticle enzyme immunoassay, total homocysteine by fluorescence polarization immunoassay and global DNA methylation using Cayman's DNA methylation enzyme immunoassay (EIA) kit. Folate and vitamin B-12 were inversely correlated to homocysteine in pregnant women consuming vegetarian and non-vegetarian diet. No difference in global DNA methylation was found between the vegetarian and non-vegetarian pregnant women. Folate and vitamin B-12 did not show association with global DNA methylation, however plasma total homocysteine of the vegetarian group showed significant correlation to global DNA methylation (r(2 )= 0.49, p = 0.011). Plasma total homocysteine was inversely related to tricep skinfold (r(2 )= -0.484, p = 0.01) and chest circumference (r(2 )= -0.104, p = 0.04) of neonates in vegetarian group. Moderate vitamin B-12 deficiency in vegetarian pregnant women might be the cause of hyperhomocystinemia, hypermethylation when compared to vitamin B-12 sufficient non-vegetarian group.

Detection of antibodies to hepatitis B core antigen using the Abbott ARCHITECT anti-HBc assay: analysis of borderline reactive sera.

PubMed

Ollier, Laurence; Laffont, Catherine; Kechkekian, Aurore; Doglio, Alain; Giordanengo, Valérie

2008-12-01

Routine use of the automated chemiluminescent microparticle immunoassay Abbott ARCHITECT anti-HBc for diagnosis of hepatitis B is limited in case of borderline reactive sera with low signal close to the cut-off index. In order to determine the significance of anti-HBc detection when borderline reactivity occurs using the ARCHITECT anti-HBc assay, a comparative study was designed. 3540 serum samples collected over a 2-month period in the hospital of Nice were examined for markers of HBV infection (HBsAg, anti-HBs and anti-HBc). One hundred seven samples with sufficient volume and with borderline reactivity by the ARCHITECT assay were tested by two other anti-HBc assays, a microparticle enzyme immunoassay (MEIA, AxSYM Core, Abbott Laboratories, IL, USA) and an enzyme linked fluorescent assay (ELFA, VIDAS Anti-HBc Total II, bioMérieux, Lyon, France). Only 46 samples were confirmed by the AxSYM and the VIDAS assays. Additional serological information linked to patient history showed that the remaining samples (61) were false positives (11), had low titer of anti-HBc antibodies (13), or were inconclusive (37). This comparative study highlighted the existence of a grey zone around the cut-off index. Confirmative results through a different immunoassay are needed to confirm the diagnosis of HBV on borderline reactive sera using the ARCHITECT anti-HBc assay.

Detection of anticentromere antibodies using cloned autoantigen CENP-B.

PubMed

Rothfield, N; Whitaker, D; Bordwell, B; Weiner, E; Senecal, J L; Earnshaw, W

1987-12-01

A solid-phase enzyme-linked immunosorbent assay has been established using a cloned fusion protein, CtermCENP-B [beta-gal], as antigen. The fusion protein carries the major epitope of CENP-B, the major centromeric autoantigen. The enzyme-linked immunosorbent assay was more sensitive than immunofluorescence techniques in detecting anticentromere antibodies in patients with scleroderma or Raynaud's disease, and was weakly positive in 3% of normal controls and in 3% of 70 patients with other connective tissue diseases.

Evaluation of ColorPAC Giardia/Cryptosporidium Rapid Assay and ProSpecT Giardia/Cryptosporidium Microplate Assay for Detection of Giardia and Cryptosporidium in Fecal Specimens

PubMed Central

Katanik, M. T.; Schneider, S. K.; Rosenblatt, J. E.; Hall, G. S.; Procop, G. W.

2001-01-01

Detection of Giardia and Cryptosporidium in clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement for Giardia detection and 98.1% agreement for Cryptosporidium detection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay for Giardia and Cryptosporidium detection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody. PMID:11724874

ENZYME IMMUNOASSAY WITH MONOCLONAL ANTIBODIES FOR THE DETECTION OF ROTAVIRUS IN STOOL SPECIMENS

EPA Science Inventory

Rotavirus infections are generally recognized as a major problem in young children; however, they have also been associated with severe gastroenteritis in adults and neonates. Infections in neonates are usually asymptomatic, although the incidence of infection may be high. Adult ...

Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

PubMed

Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

2014-02-01

The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

NASA Astrophysics Data System (ADS)

Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

1994-03-01

Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

USGS Publications Warehouse

Thurman, E.M.; Aga, D.S.

2001-01-01

Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

PubMed

Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia

2016-11-01

A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Combining supercritical fluid extraction of soil herbicides with enzyme immunoassay analysis.

PubMed

Stearman, G K

2001-10-01

Supercritical fluid extraction (SFE) of soil herbicides followed by enzyme immunoassay analysis (EIA) is explained in a step-by-step process. Extracted herbicides, include 2,4-D, simazine, atrazine, and alachlor. The herbicide, trifluralin was not successfully analyzed by EIA because of crossreacting metabolites. Problems with SFE, including uneven packing of cells, leaks, uneven flow and clogging, can largely be eliminated as the method parameters are optimized. It was necessary to add modifiers including methanol or acetone to the SF CO2 to increase the solubility of the analytes. Detection limits of 2.5 ng/g soil for atrazine and alachlor and 15 ng/g soil for simazine and 2,4-D without concentration of the sample were achieved. Recoveries above 80% and relative standard deviations (RSDs) less than 15% for 2,4-D simazine, atrazine and alachlor were achieved. Atrazine and alachlor recoveries were above 90% with RSDs below 10%. Forty soil samples could be extracted and analyzed in an 8-h day.

SNAP Assay Technology.

PubMed

O'Connor, Thomas P

2015-12-01

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A Review of the Extraction and Determination Methods of Thirteen Essential Vitamins to the Human Body: An Update from 2010.

PubMed

Zhang, Yuan; Zhou, Wei-E; Yan, Jia-Qing; Liu, Min; Zhou, Yu; Shen, Xin; Ma, Ying-Lin; Feng, Xue-Song; Yang, Jun; Li, Guo-Hui

2018-06-19

Vitamins are a class of essential nutrients in the body; thus, they play important roles in human health. The chemicals are involved in many physiological functions and both their lack and excess can put health at risk. Therefore, the establishment of methods for monitoring vitamin concentrations in different matrices is necessary. In this review, an updated overview of the main pretreatments and determination methods that have been used since 2010 is given. Ultrasonic assisted extraction, liquid⁻liquid extraction, solid phase extraction and dispersive liquid⁻liquid microextraction are the most common pretreatment methods, while the determination methods involve chromatography methods, electrophoretic methods, microbiological assays, immunoassays, biosensors and several other methods. Different pretreatments and determination methods are discussed.

Simplified urinary immunoassay for 2,4-D: validation and exposure assessment.

PubMed

Lyubimov, A V; Garry, V F; Carlson, R E; Barr, D B; Baker, S E

2000-08-01

Urinary monitoring of exposed workers by either analytic chemical methods or radioimmunoassay suggests that urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) exceeding 30 ppb are indicative of occupational exposure. However, the current methods do not lend themselves to clinical laboratory use in the rural medical setting. The major goal of this project was to provide medical practitioners who care for members of the agricultural community with a cost-efficient way to conduct exposure assessment. This project used a direct 2,4-D enzyme immunoassay (EIA) and measurement of the ratio between 2,4-D-spiked and non-spiked samples of the same urine to quantify 2,4-D levels. This simplified approach minimizes the effects of non-specific interfering substances in urine and eliminates the need for sample extraction and clean-up. Possible urine co-contaminants (2,4-dichlorophenol and 2,5-dichlorophenol) do not significantly interfere with this immunoassay. Twenty-two forest pesticide applicators who apply and use chlorophenoxy herbicides in their work and 14 comparable control subjects were studied to validate the assay in the occupational setting. Coded urine specimens were examined for levels of 2,4-D by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and compared with immunoassay results from the same specimens. A correlation coefficient of r = 0.982 with a P value of .0001 for a plot of HPLC-MS/MS versus immunoassay demonstrated that the results from these methods were comparable over urinary dose levels ranging from not detectable (<19 ppb) to 1700 ppb 2,4-D, as determined by immunoassay.

Recombinant antibodies and their use in biosensors.

PubMed

Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

2012-04-01

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

PubMed

Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth

2016-11-10

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags 1 . Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Homogeneous Immunoassays: Historical Perspective and Future Promise

NASA Astrophysics Data System (ADS)

Ullman, Edwin F.

1999-06-01

The founding and growth of Syva Company is examined in the context of its leadership role in the development of homogeneous immunoassays. The simple mix and read protocols of these methods offer advantages in routine analytical and clinical applications. Early homogeneous methods were based on insensitive detection of immunoprecipitation during antigen/antibody binding. The advent of reporter groups in biology provided a means of quantitating immunochemical binding by labeling antibody or antigen and physically separating label incorporated into immune complexes from free label. Although high sensitivity was achieved, quantitative separations were experimentally demanding. Only when it became apparent that reporter groups could provide information, not only about the location of a molecule but also about its microscopic environment, was it possible to design practical non-separation methods. The evolution of early homogenous immunoassays was driven largely by the development of improved detection strategies. The first commercial spin immunoassays, developed by Syva for drug abuse testing during the Vietnam war, were followed by increasingly powerful methods such as immunochemical modulation of enzyme activity, fluorescence, and photo-induced chemiluminescence. Homogeneous methods that quantify analytes at femtomolar concentrations within a few minutes now offer important new opportunities in clinical diagnostics, nucleic acid detection and drug discovery.

COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY FOR MEASUREMENT OF POLYCYCLIC AROMATIC HYDROCARBONS IN CONTAMINATED SOIL

EPA Science Inventory

Polycyclic aromatic hydrocarbons (PAHs) are frequently encountered in the environment and may pose health concerns due to their carcinogenicity. A commercial enzyme-linked immunosorbent assay (ELISA), was evaluated as a screening method for monitoring PAHs at contaminated site...

DEVELOPMENT OF A CLASS-SPECIFIC IMMUNOASSAY FOR THE TYPE I PYRETHROID INSECTICIDES. (R825433)

EPA Science Inventory

A general enzyme-linked immunosorbent assay was developed for the type I pyrethroid insecticides, such as permethrin, phenothrin, resmethrin and bioresmethrin. Polyclonal antibodies were generated by immunizing with a permethrin derivative, 2,2-dimethyl-3-(5′-carboxy-pe...

Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

PubMed

Jacobson, R H; Downing, D R; Lynch, T J

1982-11-15

A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

PubMed

van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

2015-05-01

A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost-effective alternative to the current mass spectrometry based techniques for the quantitation of cocaine at forensically significant concentrations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Development Of Antibody-Based Fiber-Optic Sensors

NASA Astrophysics Data System (ADS)

Tromberg, Bruce J.; Sepaniak, Michael J.; Vo-Dinh, Tuan

1988-06-01

The speed and specificity characteristic of immunochemical complex formation has encouraged the development of numerous antibody-based analytical techniques. The scope and versatility of these established methods can be enhanced by combining the principles of conventional immunoassay with laser-based fiber-optic fluorimetry. This merger of spectroscopy and immunochemistry provides the framework for the construction of highly sensitive and selective fiber-optic devices (fluoroimmuno-sensors) capable of in-situ detection of drugs, toxins, and naturally occurring biochemicals. Fluoroimmuno-sensors (FIS) employ an immobilized reagent phase at the sampling terminus of a single quartz optical fiber. Laser excitation of antibody-bound analyte produces a fluorescence signal which is either directly proportional (as in the case of natural fluorophor and "antibody sandwich" assays) or inversely proportional (as in the case of competitive-binding assays) to analyte concentration. Factors which influence analysis time, precision, linearity, and detection limits include the nature (solid or liquid) and amount of the reagent phase, the method of analyte delivery (passive diffusion, convection, etc.), and whether equilibrium or non-equilibrium assays are performed. Data will be presented for optical fibers whose sensing termini utilize: (1) covalently-bound solid antibody reagent phases, and (2) membrane-entrapped liquid antibody reagents. Assays for large-molecular weight proteins (antigens) and small-molecular weight, carcinogenic, polynuclear aromatics (haptens) will be considered. In this manner, the influence of a system's chemical characteristics and measurement requirements on sensor design, and the consequence of various sensor designs on analytical performance will be illustrated.

Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.

PubMed

Clem, T R; Yolken, R H

1978-01-01

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories.

Binding Assays for the Quantitative Detection of P. brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

DTIC Science & Technology

1987-12-01

editions are obsolete. -I Block 19 continued structure. Preliminary experiments involving conversion of the radio- immunoassay to a urease enzyme linked...the radioimmunoassay to a urease I enzyme linked form have been successful. DTIC GTAB Di tributioul AV~i~b~±~YCoded Avsi abi11i ntY___ tat Special...necessary prior to thin- layer chromatography. A preparative thin- layer chromatography step using silica gel plates (1000 u thickness) utilizes acetone

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

NASA Technical Reports Server (NTRS)

Roux, S. J.

1990-01-01

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

PubMed Central

Neuwelt, E A; Johnson, W G; Blank, N K; Pagel, M A; Maslen-McClure, C; McClure, M J; Wu, P M

1985-01-01

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model. Images PMID:4040927

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

PubMed

Neuwelt, E A; Johnson, W G; Blank, N K; Pagel, M A; Maslen-McClure, C; McClure, M J; Wu, P M

1985-08-01

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

Characterization of the binding specificity of Anguilla anguilla agglutinin (AAA) in comparison to Ulex europaeus agglutinin I (UEA-I).

PubMed

Baldus, S E; Thiele, J; Park, Y O; Hanisch, F G; Bara, J; Fischer, R

1996-08-01

Using immunochemical and immunohistochemical methods, the binding site of Anguilla anguilla agglutinin (AAA) was characterized and compared with the related fucose-specific lectin from Ulex europaeus (UEA-I). In solid-phase enzyme-linked immunoassays, the two lectins recognized Fuc alpha 1-2Gal beta-HSA. AAA additionally cross-reacted with neoglycolipids bearing lacto-N-fucopentaose (LNFP) I [H type 1] and II [Le(a)] and lactodifucotetraose (LDFT) as glycan moieties. UEA-I, on the other hand, bound to a LDFT-derived neoglycolipid but not to the other neoglycolipids tested. Binding of AAA to gastric mucin was competitively neutralized by Le(a)-specific monoclonal antibodies. UEA-I binding, on the other hand, was reduced after co-incubation with H type 2- and Le(y)-specific monoclonal antibodies. According to our results, AAA reacts with fucosylated type 1 chain antigens, whereas UEA-I binds only to the alpha 1-2-fucosylated LDFT-derived neoglycolipid. In immunohistochemical studies, the reactivity of AAA and UEA-I in normal pyloric mucosa from individuals with known Lewis and secretor status was analysed. AAA showed a broad reaction in the superficial pyloric mucosa from secretors and non-secretors, but AAA reactivity was more pronounced in Le(a+b-) individuals. On the other hand, UEA-I stained the superficial pyloric mucosa only from secretor individuals. A staining of deep mucous glands by the lectins was found in all specimens. Both reacted with most human carcinomas of different origin. Slight differences in their binding pattern were observed and may be explained by the different fine-specificities of the lectins.

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

PubMed

Brinãs, Raymond P; Sundgren, Andreas; Sahoo, Padmini; Morey, Susan; Rittenhouse-Olson, Kate; Wilding, Greg E; Deng, Wei; Barchi, Joseph J

2012-08-15

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii.

PubMed Central

Herwaldt, B L; Lew, J F; Moe, C L; Lewis, D C; Humphrey, C D; Monroe, S S; Pon, E W; Glass, R I

1994-01-01

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents. Images PMID:8027335

Seroprevalence of Chlamydia trachomatis in Enugu, Nigeria.

PubMed

Ikeme, A C; Ezegwui, H U; Ikeako, L C; Agbata, I; Agbata, E

2011-01-01

Chlamydia infections in women cause pelvic inflammatory disease, which often results in devastating consequences of infertility, ectopic pregnancy, or chronic pelvic pain. The infection is largely asymptomatic. To determine the seroprevalence of Chlamydia trachomatis in Enugu, South Eastern Nigeria. A population-based prospective study comprising female residents of Enugu, South Eastern Nigeria. Indirect solid phase enzyme immunoassay of Chlamydia antibodies was done using ImmunoComb C0. Trachomatis IgG Kit (Orgenics). The population comprised 136 female undergraduate students and 150 non-student women. The overall prevalence of C. trachomatis in the population studied was 29.4%. The percentage of subjects who admitted to be having multiple sexual partners was higher among the student population (71.2%) compared to those from the non-student population (28.8%). The highest percentage of seroprevalence was 28 (33.3%) in the age group of 20-24 years for the student population and 18 (21.4%) in the age group of 25-29 years for the non-student population. The highest seroprevalence of C. trachomatis antibodies (69.0%) in both populations was observed in females without any history of infection. Females that had pelvic inflammatory disease, sexually transmitted infection, and secondary infertility assayed for C. trachomatis had seroprevalence levels of 19%, 9.5%, and 2.4%, respectively. There was a positive correlation between positive Chlamydia assay and the type of subject population (student or non-student) with r2 value of 1.55 at P < 0.01. C. trachomatis infection is largely underdiagnosed and remains a silent disease in the apparently healthy population of Enugu, South eastern Nigeria.

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis.

PubMed

Mokhtarian, Kobra; Meamar, Ahmad Reza; Khoshmirsafa, Majid; Razmjou, Elham; Masoori, Leila; Khanmohammadi, Majid; Akhlaghi, Lame; Falak, Reza

2018-01-01

Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions.

Epidemic of Febrile Disease in Berbera, Somalia

DTIC Science & Technology

1993-10-01

situated on the Gulf all negative by Widal tests. In July 1989, of Aden, adjacent to the Indian Ocean , is a a U.S. Navy team administered a coastal town...A. (1976): Microplate enzyme linked immunosorbent assay antibody immunoassays for the immunodiagnosis response to C. burnetii in experimentally of

Method 546: Determination of Total Microcystins and Nodularins in Drinking Water and Ambient Water by Adda Enzyme-Linked Immunosorbent Assay

EPA Pesticide Factsheets

This method is listed as Tier I for presumptive analysis of microcystins in water samples and Tier II all other environmental sample types. The method determines total microcystins and nodularins using an indirect competitive immunoassay (ELISA).

Tobago Prostate Survey: Prostate Cancer Risk in a Large Population-Based Study of Men of African Descent

DTIC Science & Technology

2001-06-01

Pittsburgh Central Pathology Laboratory using the automated Microparticle Enzyme Immunoassay, Abbot AxSYM PSA assay (Abbott Laboratories, Abbott Park, IL, USA...1997) pesticide applicators in Sweden (Dich & Wik/und, 1998) "* No evidence of an association (Giovannuccietal., 1997; * Increased risk for soap

Immuno-PCR assay for sensitive detection of proteins in real time

USDA-ARS?s Scientific Manuscript database

The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10–1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). Thi...

Comparison of three luminescent immunoassays for hepatitis B virus surface antigen quantification during the natural history of chronic hepatitis B virus infection.

PubMed

Cheng, Xiao-Dong; Song, Liu-Wei; Fang, Lin-Lin; Yang, Lin; Wu, Yong; Ge, Sheng-Xiang; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao; Hao, Xiao-Ke

2014-11-01

Hepatitis B surface antigen (HBsAg) quantification has garnered attention because of its high predictive value in determining treatment responses. The HBsAg quantification assays, such as Architect and Elecsys, are commercially available, and more assays are in development. We aimed to compare the results of the Architect and Elecsys assays with those of a new assay, WTultra. The WTultra HBsAg assay is a sandwich chemiluminescent microplate enzyme immunoassay and provides an alternative choice which is more cost-effective and potentially applicable in developing or resource-constrained countries and areas. A total of 411 serum samples were collected from patients during various phases of chronic hepatitis B (CHB) infection. The samples were assessed using the three assays, and the results were compared and analyzed. The results for the Architect, Elecsys, and WTultra assays were well correlated according to the overall results for the samples (correlation coefficients, rArchitect versus WTultra = 0.936, rArchitect versus Elecsys = 0.952, and rWTultra versus Elecsys = 0.981) and the various infection phases (rArchitect versus WTultra ranging from 0.67 to 0.975, rArchitect versus Elecsys ranging from 0.695 to 0.982, and rWTultra versus Elecsys ranging from 0.877 to 0.99). Additionally, consistent results were observed according to genotype (genotype B: rArchitect versus WTultra = 0.976, rArchitect versus Elecsys = 0.978, and rWTultra versus Elecsys = 0.979; genotype C: rArchitect versus WTultra = 0.950, rArchitect versus Elecsys = 0.963, and rWTultra versus Elecsys = 0.981) and hepatitis B virus (HBV) DNA levels (rArchitect = 0.540, rWTultra = 0.553, and rElecsys = 0.580). In conclusion, the Elecsys and WTultra assays were well correlated with the Architect assay, irrespective of the CHB infection phase or genotype. All of these assays are reliable for HBsAg quantification. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Regulation of coal polymer degradation by fungi. Eighth quarterly report, [January--March 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Irvine, R.L.; Bumpus, J.A.

1996-07-28

Progress is reported on solubilization of low-rank coal by enzyme activity derived from Trametes versicolor or P. chrysosporium. Specifically during the reporting period efforts were directed towards the determining the effect of pH on solubilization of leonardite, the role of laccase in low coal solubilization and metabolism, the decolorization of soluble coal macromolecule by P. chrysosprium and T. versicolor in solid agar gel, and the solubilization of low rank coal in slurry cultures and solid phase reactors.

Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay.

PubMed

Abdelshafi, Nahla A; Panne, Ulrich; Schneider, Rudolf J

2017-04-01

This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm 2 of one banknote by swabbing 1cm 2 : 0, 14, and 21pg/cm 2 . Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note. Copyright © 2017 Elsevier B.V. All rights reserved.

The Occurrence of Abscisic Acid and Abscisyl-β-d-Glucopyranoside in Developing and Mature Citrus Fruit as Determined by Enzyme Immunoassay 1

PubMed Central

Harris, Michael J.; Dugger, William M.

1986-01-01

The contents of (+)-cis-abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate(s) were analyzed by means of enzyme immunoassay in partially purified extracts of developing and mature sweet orange fruit (Citrus sinensis [L.] Osbeck cv Washington navel). A relatively small increase in ABA was observed in the fruit exocarp during the natural color transition from green to orange. At the same time, the ABA-conjugate level increased approximately 12-fold in this tissue. The contents of ABA and ABA-conjugate equaled 15.0 ± 0.7 and 107.8 ± 2.1 nanomoles per gram fresh weight, respectively, in the exocarp at harvest. Other tissues also contained considerable quantities of these compounds. Whereas the highest ABA content was observed in the exocarp, the highest ABA-conjugate content was observed in the central vascular axis of the fruit and equaled 187.0 ± 10.3 nanomoles per gram fresh weight. The only immunoreactive conjugate found in significant quantity in mature fruit was identified as abscisyl-β-d-glucopyranoside (ABA-GE) based on (a) immunological cross-reactivity, (b) thin layer chromatography co-chromatography with authentic standards in two solvent systems, (c) susceptibility to both chemical and enzymic degradation, and (d) mass spectroscopy. PMID:16665032

Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids.

PubMed

Vidal, Juan C; Bertolín, Juan R; Bonel, Laura; Asturias, Laura; Arcos-Martínez, M Julia; Castillo, Juan R

2016-06-05

Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h). Copyright © 2016 Elsevier B.V. All rights reserved.

The influences of inoculants from municipal sludge and solid waste on compost stability, maturity and enzyme activities during chicken manure composting.

PubMed

Li, Shuyan; Li, Jijin; Yuan, Jing; Li, Guoxue; Zang, Bing; Li, Yangyang

2017-07-01

The aim of this study was to investigate the influence of inoculants on compost stability, maturity and enzyme activities during composting of chicken manure and cornstalk. Two microbial inoculants (originated from aerobic municipal sludge and municipal solid waste, respectively) were used in composting at the rate of 0.3% of initial raw materials (wet weight). No microbial inoculums were added to the control. The experiment was conducted under aerobic conditions for 53 days. The results show that enzyme activity is an important index to comprehensively evaluate the composting stability and maturity. Microbes originated from sludge works best in terms of composting stability and maturity (C:N ratio decreased from 15.5 to 10, and germination index increased to 109%). Microbial inoculums originated from sludge and municipal solid waste extended the time of thermophilic phase for 11 and 7 days, respectively. Microbial inoculums originated from sludge and MSW significantly increased the average of catalase activity (by 15.0% and 12.1%, respectively), urease activity (by 21.5% and 12.2%, respectively) and cellulase activity (by 32.1% and 26.1%, respectively) during composting.

Industrial Fungal Enzymes: An Occupational Allergen Perspective

PubMed Central

Green, Brett J.; Beezhold, Donald H.

2011-01-01

Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis.

PubMed

Jisha, Veloorvalappil Narayanan; Smitha, Robinson Babysarojam; Priji, Prakasan; Sajith, Sreedharan; Benjamin, Sailas

2015-02-01

This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Synthesis and biological properties of enzyme-resistant analogues of substance P.

PubMed

Sandberg, B E; Lee, C M; Hanley, M R; Iversen, L L

1981-02-01

Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-P-degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogues described, an heptapeptide analogue of residues 5-11, less than Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide. Fluorenylmethyloxycarbonyl was used as an alpha-amino protecting group in conjunction with t-butyloxycarbonyl as an epsilon-amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ion-exchanger. The peptides were finally purified on a semi-preparative reverse-phase column.

Multilayers enzyme-coated carbon nanotubes as biolabel for ultrasensitive chemiluminescence immunoassay of cancer biomarker.

PubMed

Bi, Sai; Zhou, Hong; Zhang, Shusheng

2009-06-15

A novel and ultrasensitive chemiluminescence immunoassay (CLIA) method based on multiple enzyme layers assembled multiwall carbon nanotubes (MWCNTs) as signal amplification labels was developed by employing luminol-H(2)O(2)-HRP-bromophenol blue (BPB) enhanced chemiluminescence (CL) system for the detection of a cancer biomarker in human serum samples, as exemplified by the measurement of alpha-fetoprotein (AFP) as a model protein. In this study, horseradish peroxidase (HRP) was assembled onto MWCNTs templates layer-by-layer (LBL) through electrostatic interactions with polyion PDDA, and further conjugated with AFP secondary antibodies (Ab(2)) as the enzyme label. The resulting LBL assembly could maximize the ratio of HRP/Ab(2) which could amplify the sensitivity greatly. To the best of our knowledge, it was the first time for this strategy applied in CLIA to date. Under the optimum conditions of luminol-H(2)O(2)-HRP-BPB CL system and the sandwich immunoreactions, a linear range from 0.02 to 2.0 ng/mL (R=0.9980) was obtained with the detection limit of 8.0 pg/mL (3sigma) which was two orders of magnitude lower than standard ELISA method. Furthermore, accurate detection of AFP in human serum samples was also demonstrated by comparison to ELISA assays. From the above results, such signal amplification strategy proposed by the novel CNT-LBL enzyme label showed an excellent promise for ultrasensitive detection of cancer biomarkers in clinical laboratory.

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

PubMed

Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

2016-12-01

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of a new amplified enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in male urine, female endocervical swab, and patient obtained vaginal swab specimens

PubMed Central

Tanaka, M.; Nakayama, H.; Sagiyama, K.; Haraoka, M.; Yoshida, H.; Hagiwara, T.; Akazawa, K.; Naito, S.

2000-01-01

Aims—To compare the performance of a new generation dual amplified enzyme immunoassay (EIA) with a molecular method for the diagnosis of Chlamydia trachomatis, using a range of urogenital samples, and to assess the reliability of testing self collected vaginal specimens compared with clinician collected vaginal specimens. Methods—Two population groups were tested. For the first population group, first void urine samples were collected from 193 male patients with urethritis, and endocervical swabs were collected from 187 high risk commercial sex workers. All urine and endocervical specimens were tested by a conventional assay (IDEIA chlamydia), a new generation amplified immunoassay (IDEIA PCE chlamydia), and the Amplicor polymerase chain reaction (PCR). Discrepant results obtained among the three sample types were confirmed using a nested PCR test with a different plasmid target region. For the second population group, four swab specimens, including one patient obtained vaginal swab, two clinician obtained endocervical swabs, and one clinician obtained vaginal swab, were collected from 91 high risk sex workers. Self collected and clinician collected vaginal swabs were tested by IDEIA PCE chlamydia. Clinician obtained endocervical swabs were assayed by IDEIA PCE chlamydia and Amplicor PCR. Results—The performance of the IDEIA PCE chlamydia test was comparable to that of the Amplicor PCR test when male urine and female endocervical swab specimens were analysed. The relative sensitivities of IDEIA, IDEIA PCE, and Amplicor PCR on male first void urine specimens were 79.3%, 91.4%, and 100%, respectively. The relative sensitivities of the three tests on female endocervical specimens were 85.0%, 95.0%, and 100%, respectively. The positivity rates for patient collected vaginal specimens and clinician collected vaginal specimens by IDEIA PCE were 25.2% and 23.1%, respectively, whereas those for clinician collected endocervical swabs by PCR and IDEIA PCE were both 27.5%. Conclusions—IDEIA PCE chlamydia is a lower cost but sensitive alternative test to PCR for testing male urine samples and female endocervical swabs. In addition, self collected or clinician collected vaginal specimens tested by IDEIA PCE chlamydia are a reliable alternative to analysing endocervical specimens. Key Words: Chlamydia trachomatis • enzyme immunoassay • clinical specimens PMID:10889816

Molecularly Imprinted Polymers for Ochratoxin A Extraction and Analysis

PubMed Central

Yu, Jorn C. C.; Lai, Edward P. C.

2010-01-01

Molecularly imprinted polymers (MIPs) are considered as polymeric materials that mimic the functionality of antibodies. MIPs have been utilized for a wide variety of applications in chromatography, solid phase extraction, immunoassays, and sensor recognition. In this article, recent advances of MIPs for the extraction and analysis of ochratoxins are discussed. Selection of functional monomers to bind ochratoxin A (OTA) with high affinities, optimization of extraction procedures, and limitations of MIPs are compared from different reports. The most relevant examples in the literature are described to clearly show how useful these materials are. Strategies on MIP preparation and schemes of analytical methods are also reviewed in order to suggest the next step that would make better use of MIPs in the field of ochratoxin research. The review ends by outlining the remaining issues and impediments. PMID:22069649

A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability

NASA Technical Reports Server (NTRS)

Hejtmancik, Kelly E.

1987-01-01

It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.

Measurements in international units of antibody to hepatitis B surface antigen(anti-HBs) after immunization with a yeast-derived, subtype adr hepatitis B vaccine are considerably different between chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA).

PubMed

Ogata, Norio

2006-04-01

The worldwide consensus of the minimum protective anti-HBs level against HBV infection is 10 mIU/mL on assays standardized by the World Health Organization (WHO) reference preparations. To investigate whether this value could be applied to recipients of yeast-derived recombinant HB vaccine containing the major surface protein of subtype adr (Bimmugen, Astellas Pharmaceutical, Tokyo), we compared anti-HBs measurements between chemiluminescent immunoassay (CLIA) (Architect Ausab, Abbott Japan, Tokyo) and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse Forte, Fujirebio, Tokyo) in given serum samples obtained from the vaccinees. The vaccine and the two assay methods are currently in a wide use in Japan. The study included 300 medical students who completed a standard vaccination course (0, 1 and 6 months). Serum samples obtained 1 month or 13 months after completing the vaccination were simultaneously tested for anti-HBs by CLIA and CLEIA. In 147 samples with quantifiable values on both CLIA and CLEIA (10 to 1000 mIU/mL) the geometric mean titer on CLEIA (225.0 mIU/mL) was significantly higher than that on CLIA (94.5 mIU/mL) (p < 0.0001). Of 26 subjects with CLIA measurements below 10 mIU/mL, 15 samples (57.7%) showed CLEIA measurements more than 10 mIU/mL. Thus, in the subtype adr-vaccinees CLEIA demonstrated considerably high serum anti-HBs measurements compared to CLIA and discordance in determining critical anti-HBs level of 10 mIU/mL was observed in more than half the samples. This suggests that the minimum HBV-protective anti HBs titer of 10 mIU/mL is difficult to be introduced to Japan where subtype adr-HB vaccines or -HBV infection are prevalent, unless characteristics of assay methods are carefully evaluated.

Reliability of the cloned-enzyme donor immunoassay (CEDIA) for cocaine in human serum in the range between the detection limit and the cut-off.

PubMed

Schütz, Harald; Auch, Jürgen; Erdmann, Freidoon; Weiler, Günter; Verhoff, Marcel A

2006-01-01

Immunoassays are used worldwide for the rapid screening of drugs. Despite the fact that they are highly valuable tools for the testing of legal and illicit drugs, there is a non-negligable risk of false-positive and false-negative findings and many pitfalls must be taken into account when using these tests in an uncritical manner and without valid confirmation procedures. In order to check the correlation between cloned-enzyme donor immunoassay (CEDIA) readings and exact determined gas chromatography/mass spectrometry (GC/MS) values for benzoylecgonine, a total of 472 serum samples was measured with an immunoassay (CEDIA) as well as GC/MS. As a result, it was shown that in the lower area of concentration, up to approx. 300 ng equ. benzoylecgonine/ml, there is a semiquantitative useful correlation. With higher concentrations, the values shift more and more apart and many runaways are visible. Generally the use of tests for materials other than those declared is excluded from the manufacturer's warranty and is at the user's own risk, but the authors found that many immunochemical tests with a certification only for the use in urine are well suited to the screening of serum samples, and even low readings could be confirmed by GC/MS. With blood or serum as sample, it could be regularly observed that generally very small readings could not only be confirmed with mass spectrometry but could in most cases also be quantified. This was the key factor in determining the rate of confirmation in 495 cases. In total, an incredibly high reliability for the CEDIA cocaine test (via benzoylecgonine) was shown up to the area of detection (detection limit), far below the cut-off. These results confirm the extraordinary sensitivity and specificity of this test also for blood or serum as sample material to be tested.

Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens.

PubMed Central

Garcia, L S; Shimizu, R Y

1997-01-01

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of interpretation of results (color, intensity of fluorescence), equipment, available personnel, and number of tests ordered are also important considerations prior to kit selection. PMID:9163474

Detection of alpha-fetoprotein in magnetic immunoassay of thin channels using biofunctional nanoparticles

NASA Astrophysics Data System (ADS)

Tsai, H. Y.; Gao, B. Z.; Yang, S. F.; Li, C. S.; Fuh, C. Bor

2014-01-01

This paper presents the use of fluorescent biofunctional nanoparticles (10-30 nm) to detect alpha-fetoprotein (AFP) in a thin-channel magnetic immunoassay. We used an AFP model biomarker and s-shaped deposition zones to test the proposed detection method. The results show that the detection using fluorescent biofunctional nanoparticle has a higher throughput than that of functional microparticle used in previous experiments on affinity reactions. The proposed method takes about 3 min (versus 150 min of previous method) to detect 100 samples. The proposed method is useful for screening biomarkers in clinical applications, and can reduce the run time for sandwich immunoassays to less than 20 min. The detection limits (0.06 pg/ml) and linear ranges (0.068 pg/ml-0.68 ng/ml) of AFP using fluorescent biofunctional nanoparticles are the same as those of using functional microparticles within experimental errors. This detection limit is substantially lower and the linear range is considerably wider than those of enzyme-linked immunosorbent assay (ELISA) and other methods in sandwich immunoassay methods. The differences between this method and an ELISA in AFP measurements of serum samples were less than 12 %. The proposed method provides simple, fast, and sensitive detection with a high throughput for biomarkers.

Development and comparison of three diagnostic immunoassay formats for the detection of azoxystrobin.

PubMed

Furzer, Gordon S; Veldhuis, Linda; Hall, J Christopher

2006-02-08

The currently accepted method of detection for azoxystrobin, a strobilurin fungicide, involves a labor-intensive organic solvent extraction and gas chromatography analysis. Three diagnostic assay formats, i.e., enzyme-linked immunosorbent assay (ELISA), fluorescence polarization (FP), and time-resolved fluorescence (TR-FIA), were developed and compared with regard to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples. These three assay formats require no initial sample extraction and were not affected by any of the environmental matrices tested, and each had a linear working range of 0-400 pg/mL. The polyclonal antibodies used for each of the immunoassays were specific to azoxystrobin; that is, the highest cross-reactivity to other pesticides observed was 5.7%. The limits of detection of the immunoassays were similar at 3 (ELISA), 46 (FP), and 28 (TR-FIA) pg/mL, as were the respective IC50 values of 306, 252, and 244 pg/mL. Each of the three immunoassays developed was less labor-intensive and approximately 100-fold more sensitive than the gas chromatographic method. While the three formats were comparable in terms of performance, the fluorescence polarization assay was the least labor-intensive and required the least time to perform.

Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

NASA Astrophysics Data System (ADS)

Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

2001-09-01

To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

Amphetamine concentrations in human urine following single-dose administration of the calcium antagonist prenylamine-studies using fluorescence polarization immunoassay (FPIA) and GC-MS.

PubMed

Kraemer, Thomas; Roditis, Susanne K; Peters, Frank T; Maurer, Hans H

2003-03-01

Prenylamine (R,S-N-(3,3-diphenylpropyl-methyl-2-phenethylamine), a World Health Organization class V calcium antagonist, is known to be metabolized to amphetamine. In this study, amphetamine concentrations after a single-dose administration of prenylamine were determined to check if they reached values that could be of analytical and/or pharmacological importance in clinical and forensic toxicology. Enantiomeric composition of amphetamine was also studied. Five volunteers received a single 120-mg oral dose of prenylamine. Urine samples were analyzed using the Abbott TDx immunoassay Amphetamine/Methamphetamine II and using our routine systematic toxicological analysis (STA) gas chromatography-mass spectrometry (GC-MS) procedure. For quantitation purposes, GC-MS was used in the selected-ion monitoring (SIM) mode (ions m/z 118, 122, 240, 244) after solid-phase extraction (Isolute Confirm HCX) and derivatization (heptafluorobutyric anhydride). Amphetamine-d5 was used as internal standard (IS). Chiral separation of the heptafluorobutyrated amphetamine enantiomers was achieved using an Astec Chiraldex G-PN column. The TDx results showed a great variability for the different volunteers. A urine sample of one volunteer showed results as high as 3200 ng/mL, whereas the urine samples of another volunteer never gave results greater than the TDx detection limit (100 ng/mL). Using the STA procedure, the presence of amphetamine could be confirmed in all urine samples with TDx results greater than the cutoff value (300 ng/mL). Using the GC-MS SIM method, amphetamine concentrations up to 1280 ng/mL were determined. Chiral analysis revealed that both enantiomers of amphetamine were present in the samples with a surplus of the S(+)-enantiomer in the early phase of excretion. Forensic implications are discussed.

A highly sensitive and selective immunoassay for the detection of tetrabromobisphenol A in soil and sediment

USDA-ARS?s Scientific Manuscript database

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) for the detection of TBBPA was developed. Six haptens (T1-T6) mimicking different structural elements of TBBPA were synthesized and coupled to keyhole...

Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

USDA-ARS?s Scientific Manuscript database

A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

DETECTION OF NORWALK VIRUS AND OTHER GENOGROUP I HUMAN CALICIVIRUSES BY A MONOCLONAL ANTIBODY, RECOMBINANT ANTIGEN-BASED IMMUNOGLOBULIN M CAPTURE ENZYME IMMUNOASSAY. (R826139)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Simultaneous detection of multiple chemical residues in milk using broad-specifity antibodies in a hybrid immunosorbent assay

USDA-ARS?s Scientific Manuscript database

The wide array of applications using quantum dots (QDs) for detection of multiple analytes reflects the versatility of the technology. In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different low-molecular...

Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a transmissible spongiform encephalopathy that affects sheep and goats. As detected by enzyme-linked immunoassay, previous studies suggested scrapie prions in the blood of sheep might be associated with B lymphocytes but not with monocytes or T lymphocytes. The association of sc...

Toxicity comparison of chlorinated and brominated dibenzo-p-dioxins and dibenzofurans in industrial source samples by HRGC/HRMS and enzyme immunoassay

EPA Science Inventory

Limited information is available on the applicability of polychlorinated dibenzo-p-dioxin/furan (PCDD/F) toxicity assays to their brominated counterparts: polybrominated dibenzo-p-dixoins/furans (PBDDs/Fs). We estimated the toxicity of mixtures of chlorinated, brominated, and mi...

A competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic mice.

PubMed

Yang, Xi; Zhou, Tao; Yu, Lei; Tan, Wenwen; Zhou, Rui; Hu, Yonggang

2015-03-01

A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05-80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2. Copyright © 2014 John Wiley & Sons, Ltd.

An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.

PubMed

Ordronneau, P; Abdullah, L H; Petrusz, P

1991-09-13

In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.

Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics.

PubMed

Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

2012-04-11

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.

Specific Immunoassays for Placental Alkaline Phosphatase As a Tumor Marker

PubMed Central

Stinghen, Sérvio T.; Moura, Juliana F.; Zancanella, Patrícia; Rodrigues, Giovanna A.; Pianovski, Mara A.; Lalli, Enzo; Arnold, Dodie L.; Minozzo, João C.; Callefe, Luis G.; Ribeiro, Raul C.; Figueiredo, Bonald C.

2006-01-01

Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71) of hPLAP (ICA-PEP assay); the working range was 0.1–11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%–6.5% (ICA-PLAP assay) and 9.0%–9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes. PMID:17489017

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Dan; Wang, Jun; Wang, Limin

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic beads (MBs) immunosensing platform. The principle of this approach is based on the combination of MBs immuno-capture based enzyme activity assay and competitive immunoassay of total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target total BChE in a sample (mixture of OP-inhibited BChE and active BChE) competes with the BChE modified on themore » MBs to bind to the limited anti-BChE antibody labeled with quantum dots (QDs-anti-BChE), and followed by electrochemical stripping analysis of the bound QDs conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1~20 nM. Simultaneously, real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with microflow injection system after immuno-capture by MBs-anti-BChE conjugate. Therefore, the formed phosphorylated adduct (OP-BChE) can be estimated by the difference values of the total amount BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values, but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective and inexpensive quantification of phosphorylated adducts and enzyme inhibition for biomonitoring of OP and nerve agent exposures.« less

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. Electronic supplementary information (ESI) available: Additional circular dichroism data and nanoparticle tracking analysis trace. See DOI: 10.1039/c6nr02009g

Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.

PubMed Central

Clem, T R; Yolken, R H

1978-01-01

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories. Images PMID:342540

Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical Microbiology. 2007. 45(10): 3377–3380)

EPA Pesticide Factsheets

This procedure describes a rapid optical immunoassay for the detection of Stx-1 and Stx-2 using a commercially available kit for presumptive analysis of aerosol, solid, particulate, liquid and water samples contaminated with Shiga and Shiga-like toxin.

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

NASA Astrophysics Data System (ADS)

Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

2016-04-01

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Regional water-quality analysis of 2,4-D and dicamba in river water using gas chromatography-isotope dilution mass spectrometry

USGS Publications Warehouse

Thurman, E.M.; Zimmerman, L.R.; Aga, D.S.; Gilliom, R.J.

2001-01-01

Gas chromatography with isotope dilution mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA) were used in regional National Water Quality Assessment studies of the herbicides, 2,4-D and dicamba, in river water across the United States. The GC-MS method involved solid-phase extraction, derivatized with deutemted 2,4-D, and analysis by selected ion monitoring. The ELISA method was applied after preconcentration with solid-phase extraction. The ELISA method was unreliable because of interference from humic substances that were also isolated by solid-phase extraction. Therefore, GC-MS was used to analyzed 80 samples from river water from 14 basins. The frequency of detection of dicamba (28%) was higher than that for 2,4-D (16%). Concentrations were higher for dicamba than for 2,4-D, ranging from less than the detection limit (<0.05 ??g/L) to 3.77 ??g/L, in spite of 5 times more annual use of 2,4-D as compared to dicamba. These results suggest that 2,4-D degrades more rapidly in the environment than dicamba.

Long-term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer-independent peroxidase purified from Jatropha curcas leaves.

PubMed

Duan, Peipei; Cai, Feng; Luo, Yongting; Chen, Yangxi; Zou, Shujuan

2015-09-01

Isoenzyme c of horseradish peroxidase (HRP-C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP-C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H2O2). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H2O2. Compared with HRP-C, the JcGP1-induced reaction was enhancer independent, which made the enzyme-linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long-term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H2 O2, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long-term stable CL signal combined with enhancer-independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed Central

Ackerman, S B; Kelley, E A

1983-01-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units. Images PMID:6341399

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed

Ackerman, S B; Kelley, E A

1983-03-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

EQCM Immunoassay for Phosphorylated Acetylcholinesterase as a Biomarker for Organophosphate Exposures Based on Selective Zirconia Adsorption and Enzyme-Catalytic Precipitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hua; Wang, Jun; Choi, Daiwon

2009-03-01

A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulationmore » of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was comparably studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of phosphorylated AChE in human plasma. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.« less

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

PubMed

Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

2015-02-15

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

PubMed

Féraudet-Tarisse, Cécile; Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R; Dorner, Brigitte G; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

PubMed Central

Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R.; Dorner, Brigitte G.; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test. PMID:28700661

Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

PubMed

Li, Xiangmei; Wang, Wenjun; Wang, Limiao; Wang, Qi; Pei, Xingyao; Jiang, Haiyang

2015-10-01

Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

Validation of two new immunoassays for sensative detection of a broad range of shiga toxins

USDA-ARS?s Scientific Manuscript database

The objective of this study was to evaluate two newly developed commercial assays, Abraxis Stx1 and Stx2 enzyme-linked immunosorbent assays (ELISAs), for their ability to detect Shiga toxin (Stx) produced by Escherichia coli (E. coli). The performance of these two assays were compared to a widely us...

Eastern Equine Encephalomyelitis Virus and Culiseta Melanura Activity at the Patuxent Wildlife Research Center, 1985-90

DTIC Science & Technology

1992-09-01

melanura ( Coq .), and the virus can cause for virus in 1985, 1989 and 1990. Mosquitoes for Snf severe disease or death in horses, game birds viral...prospective field evaluation of an enzyme immunoassay: detection of eastern equine Carpenter. J. W.. (G. G. Clark and D. M. Watts. 199,9

Seroepidemiology of Toxoplasma gondii infection in general population in a Northern Mexican city

USDA-ARS?s Scientific Manuscript database

There is a lack of information about the seroepidemiology of T. gondii infection in the general population of Durango City, Mexico. Anti-T. gondii IgG and IgM antibodies were sought in 974 inhabitants in Durango City, Mexico using enzyme-linked immunoassays. In total, 59 (6.1%) of 974 participants (...

Antibodies to Nipah-Like Virus in Bats (Pteropus lylei), Cambodia

PubMed Central

Olson, James G.; Rupprecht, Charles; Rollin, Pierre E.; An, Ung Sam; Niezgoda, Michael; Clemins, Travis; Walston, Joe

2002-01-01

Serum specimens from fruit bats were obtained at restaurants in Cambodia. We detected antibodies cross-reactive to Nipah virus by enzyme immunoassay in 11 (11.5%) of 96 Lyle’s flying foxes (Pteropus lylei). Our study suggests that viruses closely related to Nipah or Hendra viruses are more widespread in Southeast Asia than previously documented. PMID:12194780

Distinguishing between respiratory syncytial virus subgroups by protein profile analysis.

PubMed Central

Walpita, P; Mufson, M A; Stanek, R J; Pfeifer, D; Connor, J D

1992-01-01

We subgrouped 75 strains of respiratory syncytial virus by a protein profile method (PPM) which relies on different mobilities of the phosphoprotein in one-dimensional polyacrylamide gel electrophoresis and does not require monoclonal antibodies. When compared with enzyme immunoassay, PPM correctly subgrouped 54 of 56 subgroup A and all 19 subgroup B strains. Images PMID:1572961

Evaluation of the Alexon-Trend ProSpecT Campylobacter Microplate Assay

PubMed Central

Tolcin, Rita; LaSalvia, Margaret M.; Kirkley, Barbara A.; Vetter, Emily A.; Cockerill, Franklin R.; Procop, Gary W.

2000-01-01

We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection. PMID:11015419

Mathematical modeling of ethanol production in solid-state fermentation based on solid medium' dry weight variation.

PubMed

Mazaheri, Davood; Shojaosadati, Seyed Abbas; Zamir, Seyed Morteza; Mousavi, Seyyed Mohammad

2018-04-21

In this work, mathematical modeling of ethanol production in solid-state fermentation (SSF) has been done based on the variation in the dry weight of solid medium. This method was previously used for mathematical modeling of enzyme production; however, the model should be modified to predict the production of a volatile compound like ethanol. The experimental results of bioethanol production from the mixture of carob pods and wheat bran by Zymomonas mobilis in SSF were used for the model validation. Exponential and logistic kinetic models were used for modeling the growth of microorganism. In both cases, the model predictions matched well with the experimental results during the exponential growth phase, indicating the good ability of solid medium weight variation method for modeling a volatile product formation in solid-state fermentation. In addition, using logistic model, better predictions were obtained.

Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

PubMed

Wei, Hui; Wang, Erkang

2013-07-21

Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage.

PubMed

Bechman, Allison; Phillips, Robert D; Chen, Jinru

2012-06-01

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 ¹¹ Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry. © 2012 Institute of Food Technologists®

Sandwich-type enzyme immunoassay for big endothelin-I in plasma: concentrations in healthy human subjects unaffected by sex or posture.

PubMed

Aubin, P; Le Brun, G; Moldovan, F; Villette, J M; Créminon, C; Dumas, J; Homyrda, L; Soliman, H; Azizi, M; Fiet, J

1997-01-01

A sandwich-type enzyme immunoassay has been developed for measuring human big endothelin-1 (big ET-1) in human plasma and supernatant fluids from human cell cultures. Big ET-1 is the precursor of endothelin 1 (ET-1), the most potent vasoconstrictor known. A rabbit antibody raised against the big ET-1 COOH-terminus fragment was used as an immobilized antibody (anti-P16). The Fab' fragment of a monoclonal antibody (1B3) raised against the ET-1 loop fragment was used as the enzyme-labeled antibody, after being coupled to acetylcholinesterase. The lowest detectable value in the assay was 1.2 pg/mL (0.12 pg/well). The assay was highly specific for big ET-1, demonstrating no cross-reactivity with ET-1, <0.4% cross-reactivity with big endothelin-2 (big ET-2), and <0.1% with big endothelin-3 (big ET-3). We used this assay to evaluate the effect of two different postural positions (supine and standing) on plasma big ET-1 concentrations in 11 male and 11 female healthy subjects. Data analysis revealed that neither sex nor body position influenced plasma big ET-1 concentrations. This assay should thus permit the detection of possible variations in plasma concentrations of big ET-1 in certain pathologies and, in association with ET-1 assay, make possible in vitro study of endothelin-converting enzyme activity in cell models. Such studies could clarify the physiological and clinical roles of this family of peptides.

Production of monoclonal antibody against clonazepam for immunoassay of benzodiazepine drugs in swine tissues.

PubMed

Shan, Wen C; Cui, Ya L; He, Xin; Zhang, Lei; Liu, Jing; Wang, Jian P

2015-01-01

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods.

Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

NASA Astrophysics Data System (ADS)

Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

2018-04-01

In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

Biomarkers in the Diagnosis and Prognosis of Alzheimer's Disease.

PubMed

Schaffer, Cole; Sarad, Nakia; DeCrumpe, Ashton; Goswami, Disha; Herrmann, Sara; Morales, Jose; Patel, Parth; Osborne, Jim

2015-10-01

Alzheimer's disease (AD) is a neurodegenerative disease that inhibits cognitive functions and has no cure. This report reviews the current diagnostic standards for AD with an emphasis on early diagnosis using the cerebrospinal fluid (CSF) biomarkers amyloid-beta, t-tau, and p-tau and fluorodeoxyglucose positron emission tomography imaging. Abnormal levels of these CSF biomarkers and decreased cerebral uptake of glucose have recently been used in the early diagnosis of AD in experimental studies. These promising biomarkers can be measured using immunoassays performed in singleplex or multiplex formats. Although presently, there are no Food and Drug Administration-approved in vitro diagnostics (IVDs) for early detection of AD, a multiplex immunoassay measuring a panel of promising AD biomarkers in CSF may be a likely IVD candidate for the clinical AD diagnostic market. Specifically, the INNO-BIA AlzBio3 immunoassay kit, performed using bead arrays on the xMAP Luminex analyzer, allows simultaneous quantification of amyloid-beta, t-tau, and p-tau biomarkers. AD biomarkers can also be screened using enzyme-linked immunosorbent assays that are offered as laboratory-developed tests. © 2014 Society for Laboratory Automation and Screening.

Development of a field-friendly technique for fecal steroid extraction and storage using the African wild dog (Lycaon pictus).

PubMed

Santymire, R M; Armstrong, D M

2010-01-01

Hormonal analysis provides information about wildlife populations, but is difficult to conduct in the field. Our goal was to develop a rapid and effective field method for fecal steroid analysis by comparing: (1) three extraction methods (laboratory (LAB), homogenize (HO) and handshake (HS)) and (2) two storage methods (solid-phase extraction (SPE) tubes vs. plastic tubes (PT)). Samples (n=23) from captive African wild dogs (Lycaon pictus) were thoroughly mixed, three aliquots of each were weighed ( approximately 0.5 g) and 5 ml of 90% ethanol was added. For LAB, samples were agitated (mixer setting 60; 30 min), centrifuged (1,500 rpm; 20 min) and poured into glass tubes. Or aliquots were HO (1 min) or HS (1 min) and poured through filter paper into glass tubes. Samples were split, analyzed for corticosterone (C) and testosterone (T) metabolites using enzyme immunoassays or stored in SPE or PT. Samples were stored (room temperature) for 30, 60 or 180 days, reconstituted in buffer and analyzed. Mean C and T recoveries of HO were greater (P=0.03) than HS compared with LAB, which was similar to HO (P>0.05). After 30 days <21% of C and T was recovered from SPE, but approximately 100% of each was recovered from HO-PT and HS-PT. Similarly, after 60 and 180 days, approximately 100% of C and T was recovered from HO-PT and HS-PT. Results demonstrated that, for C and T, HO was more comparable (P<0.001) to LAB than HS and PT storage was more efficient than SPE (P<0.001). (c) 2009 Wiley-Liss, Inc.

Correlation between oestrogen receptor protein expression in infiltrating ductal carcinoma of the breast by immunohistochemistry and cytosol measurements.

PubMed

Looi, L M; Yap, S F; Cheah, P L

1997-11-01

Fresh frozen neoplastic tissues from 70 infiltrating ductal breast carcinomas were analysed for cytosolic oestrogen receptor (ER) protein content using a solid phase enzyme immunoassay (EIA) method based on a "sandwich" principle (Abbott ER-EIA monoclonal). Formalin-fixed, paraffin-embedded sections from the same carcinomas were examined for nuclear immunoreactivity against a monoclonal antibody for ER protein (Dako) using the standard avidin-biotin complex immunoperoxidase (IP) method after microwave antigen retrieval. The degree of ER positivity by IP was also scored according to a visual estimation of the percentage of cells expressing immunopositivity and the intensity of staining. Twenty-eight (40%) of the carcinomas were ER-positive by EIA and 34 (48.6%) were positive by IP. Twenty-five (35.7%) were ER-positive and 33 (47.1%) were ER-negative by both methods. Nine (12.9%) were ER-negative by EIA but were positive by IP, this discrepancy being ascribed to sampling inadequacy for EIA. However, 3 (4.3%) tumours were ER-positive by EIA and negative by IP. This discrepancy may be variously due to inadequate antigen retrieval, faulty technique and the possibility that the two methods do not measure identical ER proteins. IP appears to have an advantage over EIA in that it has a higher pick-up rate, does not require fresh tissue and can be applied to archival material. However, to reduce false negative estimations, it may be necessary to run IP staining using more than one ER antibody. Standardisation of the IP method for ER is desirable before this method is to be widely adopted in Malaysian laboratories. Quantitation of ER positivity by IP scoring correlated poorly with actual cytosolic levels. Caution should be exercised in attaching patient management value to visual IP scoring.

Cow's milk challenge through human milk evokes immune responses in infants with cow's milk allergy.

PubMed

Järvinen, K M; Mäkinen-Kiljunen, S; Suomalainen, H

1999-10-01

In order to measure the immune response evoked in breast-fed infants with cow's milk allergy (CMA) by cow's milk challenge through human milk, mothers were given increasing doses of cow's milk after they had been on a cow's milk elimination diet. Another objective was to study the secretion of beta-lactoglobulin (BLG) into human milk before and during milk challenge in relation to the appearance of symptoms in infants. Seventeen asymptomatic mothers who had infants with challenge-proven CMA and 10 asymptomatic mothers who had healthy infants were recruited. Infants ranged in age from 1.8 to 9.4 months. A solid-phase enzyme-linked immunoassay (ELISPOT) was used to assess the total number of immunoglobulin-secreting and specific antibody-secreting cells. Flow cytometry was used to enumerate different lymphocyte subpopulations among peripheral blood lymphocytes primed during provocation by cow's milk antigens. BLG levels were assessed in human milk before the challenge and 1, 2, 3, and 4 hours after the commencement of the challenge. All but one of the infants with CMA showed symptoms of CMA during cow's milk challenge through human milk. There was a significant rise in the total number of immunoglobulin-secreting cells in the IgA and IgG classes associated with a positive cow's milk challenge response, but the proportions of peripheral blood B cells bearing CD19, CD23, CD19 and 23, CD5, or CD19 and CD5 were comparable. BLG levels were comparable in both study groups. Most of the infants with CMA reacted to cow's milk challenge through human milk. Hypersensitivity reactions to food antigens through human milk may be more common than previously thought.

The usage of phage mini-antibodies as a means of detecting ferritin concentration in animal blood serum.

PubMed

Staroverov, Sergey A; Volkov, Alexei A; Fomin, Alexander S; Laskavuy, Vladislav N; Mezhennyy, Pavel V; Kozlov, Sergey V; Larionov, Sergey V; Fedorov, Michael V; Dykman, Lev A; Guliy, Olga I

2015-01-01

Mini-antibodies that have specific ferritin response have been produced for the first time using sheep's phage libraries (Griffin.1, Medical Research Council, Cambridge, UK). Produced phage antibodies were used for the first time for the development of diagnostic test kits for ferritin detection in the blood of cattle. The immunodot assay with secondary biospecific labeling is suggested as means of ferritin detection in cow blood serum (antiferritin phage antibodies and rabbit antiphage antibodies conjugated with different labels). Сolloidal gold, gold nanoshells, and horse reddish peroxidase used as labels have shown a similar response while detecting concentration of ferritin (0.2 mg/mL). It is shown that the method of solid-phase immunoassay with a visual view of the results allows determination of the minimum concentration of ferritin in the blood of cows at 0.225 g/mL.

Development of an incurred cornbread model for gluten detection by immunoassays.

PubMed

Sharma, Girdhari M; Khuda, Sefat E; Pereira, Marion; Slate, Andrew; Jackson, Lauren S; Pardo, Christopher; Williams, Kristina M; Whitaker, Thomas B

2013-12-11

Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision.

Pepsin as a Marker for Pulmonary Aspiration

PubMed Central

Metheny, Norma A.; Chang, Yie-Hwa; Ye, Jing Song; Edwards, Sharon J.; Defer, Julie; Dahms, Thomas E.; Stewart, Barbara J.; Stone, Kathleen S.; Clouse, Ray E.

2008-01-01

BACKGROUND Although assessment for aspiration of small volumes of gastric contents in tube-fed patients receiving mechanical ventilation is important, available methods for this purpose are not wholly satisfactory. A potential method is immunoassay of tracheal secretions for the gastric enzyme pepsin. OBJECTIVES To determine the frequency with which pepsin in suctioned tracheal secretions from acutely ill, tube-fed patients receiving mechanical ventilation could be detected via an immunoassay. METHODS A convenience sample of 136 specimens of suctioned tracheal secretions was collected from 30 acutely ill, tube-fed adults receiving mechanical ventilation. Multiple samples were obtained from 26 of the 30 patients (range, 2−11 per subject). An immunoassay with rooster polyclonal antibodies to purified human pepsin was used to detect pepsin in the secretions. RESULTS Fourteen specimens tested positive for pepsin. Secretions from 5 patients accounted for the 14 pepsin-positive results. A significant relationship was found between the position of the head of the bed and the presence of pepsin in tracheal secretions (P< .001). Of the 14 pepsin-positive specimens, 13 (92.9%) were obtained from subjects in a flat position. CONCLUSIONS A pepsin immunoassay can be used to detect pepsin in human tracheal secretions. If pepsin in tracheal secretions is considered an indicator of aspiration of gastric contents, aspiration occurred in 5 of the 30 subjects. A flat position is strongly associated with the presence of pepsin in tracheal secretions. PMID:11888127

A new automated turbidimetric immunoassay for the measurement of canine C-reactive protein.

PubMed

Piñeiro, Matilde; Pato, Raquel; Soler, Lourdes; Peña, Raquel; García, Natalia; Torrente, Carlos; Saco, Yolanda; Lampreave, Fermín; Bassols, Anna; Canalias, Francesca

2018-03-01

In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use. © 2018 American Society for Veterinary Clinical Pathology.

Design of experiment (DOE) based screening of factors affecting municipal solid waste (MSW) composting.

PubMed

Kazemi, Khoshrooz; Zhang, Baiyu; Lye, Leonard M; Cai, Qinghong; Cao, Tong

2016-12-01

A design of experiment (DOE) based methodology was adopted in this study to investigate the effects of multiple factors and their interactions on the performance of a municipal solid waste (MSW) composting process. The impact of four factors, carbon/nitrogen ratio (C/N), moisture content (MC), type of bulking agent (BA) and aeration rate (AR) on the maturity, stability and toxicity of compost product was investigated. The statistically significant factors were identified using final C/N, germination index (GI) and especially the enzyme activities as responses. Experimental results validated the use of enzyme activities as proper indices during the course of composting. Maximum enzyme activities occurred during the active phase of decomposition. MC has a significant effect on dehydrogenase activity (DGH), β-glucosidase activity (BGH), phosphodiesterase activity (PDE) and the final moisture content of the compost. C/N is statistically significant for final C/N, DGH, BGH, and GI. The results provided guidance to optimize a MSW composting system that will lead to increased decomposition rate and the production of more stable and mature compost. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microwave-assisted synthesis of triple-helical, collagen-mimetic lipopeptides

PubMed Central

Banerjee, Jayati; Hanson, Andrea J; Muhonen, Wallace W; Shabb, John B; Mallik, Sanku

2018-01-01

Collagen-mimetic peptides and lipopeptides are widely used as substrates for matrix degrading enzymes, as new biomaterials for tissue engineering, as drug delivery systems and so on. However, the preparation and subsequent purification of these peptides and their fatty-acid conjugates are really challenging. Herein, we report a rapid microwave-assisted, solid-phase synthetic protocol to prepare the fatty-acid conjugated, triple-helical peptides containing the cleavage site for the enzyme matrix metalloproteinase-9 (MMP-9). We employed a PEG-based resin as the solid support and the amino acids were protected with Fmoc- and tert-butyl groups. The amino acids were coupled at 50 °C (25 W of microwave power) for 5 min. The deprotection reactions were carried out at 75 °C (35 W of microwave power) for 3 min. Using this protocol, a peptide containing 23 amino acids was synthesized and then conjugated to stearic acid in 14 h. PMID:20057380

A redox-mediated chromogenic reaction and application in immunoassay.

PubMed

Yu, Ru-Jia; Ma, Wei; Peng, Mao-Pan; Bai, Zhi-Shan; Long, Yi-Tao

2016-08-31

A novel redox-mediated chromogenic reaction was demonstrated based on the reaction between HAuCl4 and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which generate various color responses from red to green in the resulting solutions. Various redox substance could be used to mediate the reaction and trigger a distinct color response. We established a sensitive hydrogen peroxide colorimetric sensor based on the redox-mediated chromogenic reaction and depicted the application both in detection of enzyme and in an immunoassay. Combining the traditional chromogenic reagent with gold nanoparticles, our assay has the advantage in short response time (within three minutes), high sensitivity (10(-12) g mL(-1) for HBsAg) and stability. Copyright © 2016. Published by Elsevier B.V.

Enzyme immunoassay of Chlamydia in birds.

PubMed

Ruppanner, R; Behymer, D E; DeLong, W J; Franti, C E; Schulz, T

1984-01-01

Serum samples from 192 free-living birds (27 species) were tested for antibodies against Chlamydia using the enzyme-linked immunosorbent assay (ELISA); 97 (51%) were seropositive. The highest antibody prevalence was among pheasants (96%), ducks (88%), and blackbirds (86%). None of 41 starlings tested were seropositive. The serotesting of 42 confined pheasants indicated 100% exposure to the organism. The ELISA is a sensitive, rapid serologic method that can be of epidemiologic and diagnostic value for detecting exposure to Chlamydia. The ELISA could also be used for mass-screening of pet birds where chlamydiosis may be considered a potential public health hazard.

Development of a bioreactor system for the decolorization of dyes by Coriolus versicolor f. antarcticus.

PubMed

Diorio, L A; Mercuri, A A; Nahabedian, D E; Forchiassin, F

2008-05-01

Decolorization of 100 microM malachite green (MG) by Coriolus versicolor f. antarcticus using a two-phase bioreactor, was investigated. In the first phase the decolorization ability of this fungus, growing under conditions of solid-state fermentation (SSF), was proved; in the second phase the capacity of the enzymes present in extracts from the solid residues was exploited. During the first phase using the same culture in the bioreactor, five consecutive charges were made, each with 75 ml of 100 microM MG solution, at 28 degrees C. Each cycle ended when MG solution reached a decolorization of 50%, at this time the bioreactor was discharged to a stainless steel coil at 50 degrees C, initiating the second phase of decolorization. Time required in order to reach 50% decolorization during the first phase varied between 25 and 65 min, with an average retention time of 48 min. The second stage had a retention time of 120 min. Residual MG after this phase varied from 0% to 6.3%. The role of laccase and Mn-peroxidase in MG decolorization is discussed. Toxicity of MG solutions before and after decolorization treatments was assayed using Lumbriculus variegatus as test organism.

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr; Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri; Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com

2016-04-18

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC{sub 50} values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (Thismore » research was supported by Ataturk University. Project Number: BAP-2015/98).« less

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2010 CFR

2010-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2011 CFR

2011-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2012 CFR

2012-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

Development of an Immunoassay and a Sol-Gel-Based Immunoaffinity Cleanup Method for Coplanar PCBs from Soil and Sediment Samples

EPA Science Inventory

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL⁻¹) with sensitivities in the range of 6–11 ng m...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2014 CFR

2014-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

Code of Federal Regulations, 2013 CFR

2013-01-01

... enzyme-linked immunosorbent assay (ELISA) or a gpI Particle Concentration Fluorescence Immunoassay (PCFIA... negative; (iv) The date of the gpI ELISA or the gpI PCFIA approved differential pseudorabies test; and (v) The name of the laboratory that conducted the gpI ELISA or the gpI PCFIA approved differential...

A Semester-Long Student-Directed Research Project Involving Enzyme Immunoassay: Appropriate for Immunology, Endocrinology, or Neuroscience Courses

ERIC Educational Resources Information Center

Goyette, Sharon Ramos; DeLuca, Jane

2007-01-01

The following project aimed at promoting integrated and long-lasting learning is described for an Immunology course, but it may be adapted to other disciplines. Students were asked to develop and carry out a research project to examine the relationship between immune function and stress. The experiments were required to include the assessment of…

Detection of Circulating Paracoccidioides brasiliensis Antigen in Urine of Paracoccidioidomycosis Patients before and during Treatment

PubMed Central

Salina, Margarete Aparecida; Shikanai-Yasuda, Maria Aparecida; Mendes, Rinaldo Poncio; Barraviera, Benedito; Mendes Giannini, Maria José Soares

1998-01-01

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with “apparent cure.” The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse. PMID:9620407

[Fundamental evaluation of apolipoprotein B-48 by chemiluminescence enzyme immunoassay--identification of apolipoprotein B-48 with immunoblotting].

PubMed

Sato, Itsuko; Fujioka, Yoshio; Hayashi, Fujio; Mukai, Masahiko; Kawano, Seiji; Ishikawa, Yuichi; Yamashita, Shizuya; Kumagai, Shunichi

2007-06-01

Apolipoprotein B-48 (apo B-48) is a constituent of chylomicrons and chylomicron remnants, and its fasting concentration has been reported to be a marker of postprandial hyperlipidemia, which is thought to be a risk factor of atherosclerosis. We evaluated the serum apo B-48 concentrations by chemiluminescence enzyme immunoassay (CLEIA), which was recently introduced as Lumipulse f fully automated immunosaasy analyzer by Fujirebio Inc (Tokyo, Japan), and performed immunoblotting on agarose gel electrophoresis with anti-apo B-48 antibody. Apo B-48 assay was intra-assay reproducible (CVs: 1.9-3.1%) and inter-assay reproducible (CVs: 2.2-4.4%). The assay range for apo B-48 was from 0.2 to 40.0 microg/ml. The effects of interfering substances such as free/conjugated birirubin, hemoglobin, Intrafat, ascorbic acid and rheumatoid factor were negligible. For storage, it was preferable to freeze, and to avoid frozen-thaw process as much as possible. Anti-apo B-48 antibody was reactive over a wide range from origin to the position of very-low-density lipoproteins in immunoblotting after agarose gel electrophoresis. Apo B-48 measurement by CLEIA was feasible to clinical use for the assessment of lipoprotein metabolism.

Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, J.W.; Grindon, A.J.; Feorino, P.M.

1986-07-18

The enzyme immunoassays (EIAs) for antibody to human T-cell lymphotropic virus type III (HTLV-III) were rapidly adopted for screening donated blood and plasma. To evaluate the significance of a positive EIA reaction, test performance was examined in a blood bank screening program. Specimens were tested by EIA, Western blot assay, and HTLV-III/lymphadenopathy-associated virus (LAV) culture. The EIA was positive in 0.25% of 67 190 blood donations. Specimens were categorized and 57.3% had low (weak) reactivity, 12.7% had moderate reactivity, and 30.0% had high reactivity. Highly reactive specimens were strongly associated with a positive Western blot or culture (86.7%) in contrastmore » to moderately and weekly reactive specimens (1.9%). Twenty-five of 29 donors interviewed with a highly reactive EIA had risk factors for HTLV-III/LAV infection. Risk factors were not identified for 74 of 75 interviewed donors with specimens of lower reactivity. The minimum calculated specificity was 99.82%. The use of the HTLV-III EIA has virtually eliminated the use of blood and plasma for HTLV-III/LAV infected donors.« less

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

PubMed Central

Turunen, H J

1983-01-01

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis. PMID:6345574

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

PubMed

Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Liu, J Q; Zhang, Y; Wang, N N; Hu, P; Lu, S Y; Ren, H L; Liu, Z S

2014-11-15

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample. Copyright © 2014 Elsevier B.V. All rights reserved.

Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

PubMed

Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

2015-01-01

The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli.

PubMed

Birnbaum, S; Bülow, L; Hardy, K; Danielsson, B; Mosbach, K

1986-10-01

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

NASA Astrophysics Data System (ADS)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

Determination of catechins and catechin gallates in tissues by liquid chromatography with coulometric array detection and selective solid phase extraction.

PubMed

Chu, Kai On; Wang, Chi Chiu; Chu, Ching Yan; Rogers, Michael Scott; Choy, Kwong Wai; Pang, Chi Pui

2004-10-25

Catechins levels in organ tissues, particularly liver, determined by published methods are unexpectedly low, probably due to the release of oxidative enzymes, metal ions and reactive metabolites from tissue cells during homogenization and to the pro-oxidant effects of ascorbic acid during sample processing in the presence of metal ions. We describe a new method for simultaneous analysis of eight catechins in tissue: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCG) (Fig. 1). The new extraction procedure utilized a methanol/ethylacetate/dithionite (2:1:3) mixture during homogenization for simultaneous enzyme precipitation and antioxidant protection. Selective solid phase extraction was used to remove most interfering bio-matrices. Reversed phase HPLC with CoulArray detection was used to determine the eight catechins simultaneously within 25 min. Good linearity (>0.9922) was obtained in the range 20-4000 ng/g. The coefficients of variance (CV) were less than 5%. Absolute recovery ranged from 62 to 96%, accuracy 92.5 +/- 4.5 to 104.9 +/- 6%. The detection limit was 5 ng/g. This method is capable for determining catechins in rat tissues of liver, brain, spleen, and kidney. The method is robust, reproducible, with high recovery, and has been validated for both in vitro and in vivo sample analysis.

Novel electrochemical immunoassay for quantitative monitoring of biotoxin using target-responsive cargo release from mesoporous silica nanocontainers.

PubMed

Zhang, Bing; Liu, Bingqian; Liao, Jiayao; Chen, Guonan; Tang, Dianping

2013-10-01

A novel homogeneous immunoassay protocol was designed for quantitative monitoring of small molecular biotoxin (brevetoxin B, PbTx-2, as a model) by using target-responsive cargo release from polystyrene microsphere-gated mesoporous silica nanocontainer (MSN). Initially, monoclonal mouse anti-PbTx-2 capture antibody was covalently conjugated onto the surface of MSN (mAb-MSN), and the electroactive cargo (methylene blue, MB) was then trapped in the pores of mAb-MSN by using aminated polystyrene microspheres (APSM) based on the electrostatic interaction. Upon addition of target PbTx-2, the positively charged APSM was displaced from the negatively charged mAb-MSN because of the specific antigen-antibody reaction. Thereafter, the molecular gate was opened, and the trapped methylene blue was released from the pores. The released methylene blue could be monitored by using a square wave voltammetry (SWV) in a homemade microelectrochemical detection cell. Under optimal conditions, the SWV peak current increased with the increasing of PbTx-2 concentration in the range from 0.01 to 3.5 ng mL(-1) with a detection limit (LOD) of 6 pg mL(-1) PbTx-2 at the 3Sblank criterion. Intra- and interassay coefficients of variation with identical batches were ≤6% and 9.5%, respectively. The specificity and sample matrix interfering effects were acceptable. The analysis in 12 spiked seafood samples showed good accordance between results obtained by the developed immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method. Importantly, the target-responsive controlled release system-based electrochemical immunoassay (CRECIA) offers a promising scheme for the development of advanced homogeneous immunoassay without the sample separation and washing procedure.

Patterns of fecal gonadal hormone metabolites in the maned wolf (Chrysocyon brachyurus).

PubMed

Songsasen, N; Rodden, M; Brown, J L; Wildt, D E

2006-10-01

Ex situ populations of maned wolves are not viable due to low reproductive efficiency. The objective of this study was to increase knowledge regarding the reproductive physiology of maned wolves to improve captive management. Fecal samples were collected 3-5 d/wk from 12 females of various reproductive age classes (young, prime breeding and aged) and reproductive histories (conceived and raised pups, conceived but lost pups, pseudo-pregnant and unpaired). Ovarian steroids were extracted from feces and assessed by enzyme immunoassay. Concentrations of estrogen metabolites gradually increased, beginning 2-5 d before breeding, and declined to baseline on the day of lordosis and copulation. Fecal progestin metabolite concentrations increased steadily during the periovulatory period, when sexual receptivity was observed, and remained elevated during pregnancy and pseudo-pregnancy. During the luteal phase, young and prime breeding-age females excreted larger amounts of progestins than those of older age classes. Furthermore, progestin concentrations were higher during the luteal phase of pregnant versus pseudo-pregnant bitches. Profiles of fecal progestin metabolites for three singleton females were unchanged throughout the breeding season, suggesting ovulation is induced in this species. However, this finding could be confounded by age, as these females were either young or aged.

Variational transition state theory: theoretical framework and recent developments.

PubMed

Bao, Junwei Lucas; Truhlar, Donald G

2017-12-11

This article reviews the fundamentals of variational transition state theory (VTST), its recent theoretical development, and some modern applications. The theoretical methods reviewed here include multidimensional quantum mechanical tunneling, multistructural VTST (MS-VTST), multi-path VTST (MP-VTST), both reaction-path VTST (RP-VTST) and variable reaction coordinate VTST (VRC-VTST), system-specific quantum Rice-Ramsperger-Kassel theory (SS-QRRK) for predicting pressure-dependent rate constants, and VTST in the solid phase, liquid phase, and enzymes. We also provide some perspectives regarding the general applicability of VTST.

The SOLID (Signs Of LIfe Detector) instrument concept: an antibody microarray-based biosensor for life detection in astrobiology

NASA Astrophysics Data System (ADS)

Parro, V.; Rivas, L. A.; Rodríguez-Manfredi, J. A.; Blanco, Y.; de Diego-Castilla, G.; Cruz-Gil, P.; Moreno-Paz, M.; García-Villadangos, M.; Compostizo, C.; Herrero, P. L.

2009-04-01

Immunosensors have been extensively used since many years for environmental monitoring. Different technological platforms allow new biosensor designs and implementations. We have reported (Rivas et al., 2008) a shotgun approach for antibody production for biomarker detection in astrobiology and environmental monitoring, the production of 150 new polyclonal antibodies against microbial strains and environmental extracts, and the construction and validation of an antibody microarray (LDCHIP200, for "Life Detector Chip") containing 200 different antibodies. We have successfully used the LDCHIP200 for the detection of biological polymers in extreme environments in different parts of the world (e.g., a deep South African mine, Antarctica's Dry valleys, Yellowstone, Iceland, and Rio Tinto). Clustering analysis associated similar immunopatterns to samples from apparently very different environments, indicating that they indeed share similar universal biomarkers. A redundancy in the number of antibodies against different target biomarkers apart of revealing the presence of certain biomolecules, it renders a sample-specific immuno-profile, an "immnuno-fingerprint", which may constitute by itself an indirect biosignature. We will present a case study of immunoprofiling different iron-sulfur as well as phylosilicates rich samples along the Rio Tinto river banks. Based on protein microarray technology, we designed and built the concept instrument called SOLID (for "Signs Of LIfe Detector"; Parro et al., 2005; 2008a, b; http://cab.inta.es/solid) for automatic in situ analysis of soil samples and molecular biomarkers detection. A field prototype, SOLID2, was successfully tested for the analysis of grinded core samples during the 2005 "MARTE" campaign of a Mars drilling simulation experiment by a sandwich microarray immunoassay (Parro et al., 2008b). We will show the new version of the instrument (SOLID3) which is able to perform both sandwich and competitive immunoassays. SOLID3 consists of two separate functional units: a Sample Preparation Unit (SPU), for ten different extractions by ultrasonication, and a Sample Analysis Unit (SAU), for fluorescent immunoassays. The SAU consists of ten different flow cells each of one allocate one antibody microarray (up to 2000 spots), and is equipped with an unique designed optical package for fluorescent detection. We demonstrate the performance of SOLID3 for the detection of a broad range of molecular size compounds, from the amino acid size, peptides, proteins, to whole cells and spores, with sensitivities at the ppb level. References Parro, V., et al., 2005. Planetary and Space Science 53: 729-737. Parro, V., et al., 2008a. Space Science Reviews 135: 293-311 Parro, V., et al., 2008b. Astrobiology 8:987-99 Rivas, L. A., et al., 2008. Analytical Chemistry 80: 7970-7979

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

PubMed

Rasmussen, M; Dahl, M; Buus, S; Djurisic, S; Ohlsson, J; Hviid, T V F

2014-08-01

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

PubMed Central

Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

2011-01-01

Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB. PMID:21966416

Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment.

PubMed

Thal, Johannes; Steffen, Monika; Meier, Bianca; Schneider, Elisabeth; Adriany, Ansgar; Usleber, Ewald

2011-01-01

The aim of this study was to develop and evaluate an enzyme immunoassay (EIA) for the cephalosporin antibiotic in milk, in combination with a new microbiological test system (brilliant black reduction test, BRT-P). Polyclonal antibodies against cefquinome were produced in rabbits, using cefquinome-keyhole limpet hemocyanine as the immunogen. These antibodies and a cefquinome-glucose oxidase conjugate were used in a competitive indirect EIA. The detection limit for cefquinome in milk was 1.5 ng ml(-1), recoveries were 80-128% at 4-40 ng ml(-1). Cross-reactivities with other cephalosporins/penicillins were all <1%. The EIA was used to determine cefquinome in incurred raw milk, the BRT-P (detection limit ≈ 20 ng ml(-1)) and a receptor assay (ßeta-s.t.a.r., detection limit ≈ 15 ng ml(-1)) were used in parallel. Five lactating cows, suffering from clinical mastitis, were treated with cefquinome by simultaneous intramammary and intramuscular injection. Cefquinome residues (maximum 10-27 μg ml(-1)) were most exclusively found in the udder quarter which was treated intramammary, residue levels in the other three quarters were low (<20 ng ml(-1)). Even in milk from intramammary-dosed quarters, residue levels fell below European Union maximum residue level (MRL, 20 μg kg(-1)) 2 days before the end of the withdrawal period. EIA, BRT-P, and ßeta-s.t.a.r. results showed acceptable agreement for milk samples, but the newly developed EIA is superior in aspects of sensitivity. In conclusion, this is the first one description of immunoassay and microbiological tests capable to determine cefquinome in milk at the MRL in incurred sample material.

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources.

PubMed

Beutin, L; Steinrück, H; Krause, G; Steege, K; Haby, S; Hultsch, G; Appel, B

2007-03-01

To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

Determination of alkaline phosphatase based on affinity adsorption solid-substrate room temperature phosphorimetry using rhodamine 6G-dibromoluciferin luminescent nanoparticle to label lectin and prediction of diseases.

PubMed

Liu, Jia-Ming; Liu, Zhen-Bo; Hu, Li-Xiang; He, Hang-Xia; Yang, Min-Lan; Zhou, Ping; Chen, Xin-Hua; Zheng, Min-Min; Zeng, Xiao-Yi; Xu, Yue-Long

2006-10-15

In the presence of heavy atom perturber LiAc, the silicon dioxide nanoparticle containing rhodamine 6G (R) and dibromoluciferin (D) (R-D-SiO(2)) can emit strong and stable solid-substrate room temperature phosphorescence signal of R (lambda(ex)/lambda(em)=481/648 nm) and D (lambda(ex)/lambda(em)=457/622 nm) on the surface of acetyl cellulose membrane (ACM). R-D-SiO(2) is used to label triticum vulgare lectin (WGA). Then two types of affinity adsorption reactions, R-D-SiO(2)-WGA- alkaline phosphatase (ALP) (direct method) and WGA-ALP-WGA-R-D-SiO(2) (sandwich method), are carried out on ACM. The conditions and the analytical characteristics for the determination of ALP using affinity adsorption solid-substrate room temperature phosphorimetry (AA-SS-RTP) were studied. For a 0.40-microl drop of sample, results show that the detection limits of the sandwich method are 0.16 ag spot(-1)(457/622 nm) and 0.17 ag spot(-1)(481/648 nm), and the detection limits of the direct method are 0.41 ag spot(-1) (457/622 nm) and 0.44 ag spot(-1) (481/648 nm). The contents of ALP in human serum correlated well with those obtained by enzyme-linked immunoassay. This study shows that AA-SS-RTP whether by the sandwich method or the direct method, can combine very well the characteristics of both high sensitivity of SS-RTP and specificity of the immunoreaction. Simultaneously, whether the phosphorescence excitation/emission wavelength of either R or D in R-D-SiO(2) is chosen to determine ALP, this can promote the agility and widen the adaptability of AA-SS-RTP.

Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody-coated gold nanoparticles as nanoprobes.

PubMed

Zhou, Yuan; Zhou, Tao; Zhou, Rui; Hu, Yonggang

2014-06-01

A rapid, simple, facile, sensitive and enzyme-amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co-immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle-based nanoprobes. This nanoprobe was employed in a sandwich-type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP-luminol with H2 O2 . Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme-linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme-linked immunosorbent assay results. Copyright © 2013 John Wiley & Sons, Ltd.

Towards an automatic lab-on-valve-ion mobility spectrometric system for detection of cocaine abuse.

PubMed

Cocovi-Solberg, David J; Esteve-Turrillas, Francesc A; Armenta, Sergio; de la Guardia, Miguel; Miró, Manuel

2017-08-25

A lab-on-valve miniaturized system integrating on-line disposable micro-solid phase extraction has been interfaced with ion mobility spectrometry for the accurate and sensitive determination of cocaine and ecgonine methyl ester in oral fluids. The method is based on the automatic loading of 500μL of oral fluid along with the retention of target analytes and matrix clean-up by mixed-mode cationic/reversed-phase solid phase beads, followed by elution with 100μL of 2-propanol containing (3% v/v) ammonia, which are online injected into the IMS. The sorptive particles are automatically discarded after every individual assay inasmuch as the sorptive capacity of the sorbent material is proven to be dramatically deteriorated with reuse. The method provided a limit of detection of 0.3 and 0.14μgL -1 for cocaine and ecgonine methyl ester, respectively, with relative standard deviation values from 8 till 14% with a total analysis time per sample of 7.5min. Method trueness was evaluated by analyzing oral fluid samples spiked with cocaine at different concentration levels (1, 5 and 25μgL -1 ) affording relative recoveries within the range of 85±24%. Fifteen saliva samples were collected from volunteers and analysed following the proposed automatic procedure, showing a 40% cocaine occurrence with concentrations ranging from 1.3 to 97μgL -1 . Field saliva samples were also analysed by reference methods based on lateral flow immunoassay and gas chromatography-mass spectrometry. The application of this procedure to the control of oral fluids of cocaine consumers represents a step forward towards the development of a point-of-care cocaine abuse sensing system. Copyright © 2017 Elsevier B.V. All rights reserved.

Multifunctional picoliter droplet manipulation platform and its application in single cell analysis.

PubMed

Gu, Shu-Qing; Zhang, Yun-Xia; Zhu, Ying; Du, Wen-Bin; Yao, Bo; Fang, Qun

2011-10-01

We developed an automated and multifunctional microfluidic platform based on DropLab to perform flexible generation and complex manipulations of picoliter-scale droplets. Multiple manipulations including precise droplet generation, sequential reagent merging, and multistep solid-phase extraction for picoliter-scale droplets could be achieved in the present platform. The system precision in generating picoliter-scale droplets was significantly improved by minimizing the thermo-induced fluctuation of flow rate. A novel droplet fusion technique based on the difference of droplet interfacial tensions was developed without the need of special microchannel networks or external devices. It enabled sequential addition of reagents to droplets on demand for multistep reactions. We also developed an effective picoliter-scale droplet splitting technique with magnetic actuation. The difficulty in phase separation of magnetic beads from picoliter-scale droplets due to the high interfacial tension was overcome using ferromagnetic particles to carry the magnetic beads to pass through the phase interface. With this technique, multistep solid-phase extraction was achieved among picoliter-scale droplets. The present platform had the ability to perform complex multistep manipulations to picoliter-scale droplets, which is particularly required for single cell analysis. Its utility and potentials in single cell analysis were preliminarily demonstrated in achieving high-efficiency single-cell encapsulation, enzyme activity assay at the single cell level, and especially, single cell DNA purification based on solid-phase extraction.

Optimizing the dosing schedule of l-asparaginase improves its anti-tumor activity in breast tumor-bearing mice.

PubMed

Shiromizu, Shoya; Kusunose, Naoki; Matsunaga, Naoya; Koyanagi, Satoru; Ohdo, Shigehiro

2018-04-01

Proliferation of acute lymphoblastic leukemic cells is nutritionally dependent on the external supply of asparagine. l-asparaginase, an enzyme hydrolyzing l-asparagine in blood, is used for treatment of acute lymphoblastic leukemic and other related blood cancers. Although previous studies demonstrated that l-asparaginase suppresses the proliferation of cultured solid tumor cells, it remains unclear whether this enzyme prevents the growth of solid tumors in vivo. In this study, we demonstrated the importance of optimizing dosing schedules for the anti-tumor activity of l-asparaginase in 4T1 breast tumor-bearing mice. Cultures of several types of murine solid tumor cells were dependent on the external supply of asparagine. Among them, we selected murine 4T1 breast cancer cells and implanted them into BALB/c female mice kept under standardized light/dark cycle conditions. The growth of 4T1 tumor cells implanted in mice was significantly suppressed by intravenous administration of l-asparaginase during the light phase, whereas its administration during the dark phase failed to show significant anti-tumor activity. Decreases in plasma asparagine levels due to the administration of l-asparaginase were closely related to the dosing time-dependency of its anti-tumor effects. These results suggest that the anti-tumor efficacy of l-asparaginase in breast tumor-bearing mice is improved by optimizing the dosing schedule. Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

[Detection of antibodies against Lamblia antigens in serum from atopic patients].

PubMed

Akhapkina, I G

2004-04-01

The method of solid-phase immune-enzyme assay was used to test the sera of atopic patients for the purpose of detecting IgG to lamblias. A high degree of protozoal invasion was established especially among pre-school and school children. It was proven as expedient to make the serological examinations of atopic patients in cases the clinical finding is unclear.

Determination of 24 personal care products in fish bile using hybrid solvent precipitation and dispersive solid phase extraction cleanup with ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.

PubMed

Yao, Li; Lv, Yin-Zhi; Zhang, Li-Juan; Liu, Wang-Rong; Zhao, Jian-Liang; Liu, You-Sheng; Zhang, Qian-Qian; Ying, Guang-Guo

2018-05-25

Personal care products (PCPs) are ubiquitous in aquatic environments owing to the continuous discharge of domestic wastewater from highly urbanized regions. These PCPs can be adsorbed by fish and thereafter usually enter the bile of the fish through biliary excretion. In this study, a sensitive method based on a combination of hybrid solvent precipitation and dispersive solid phase extraction (d-SPE) purification was developed to simultaneously extract and detect 24 PCPs, namely, 16 biocides, 4 synthetic musks, and 4 benzotriazoles, from fish bile. Hybrid precipitation on solid phase extraction (SPE) tubes was applied to remove phospholipids and proteins, and a d-SPE procedure was used for further purification. The extraction solvents for the hybrid precipitation/SPE tubes and d-SPE materials were optimized. The method performance for bile samples both with and without enzyme hydrolysis using β-glucuronidase/aryl-sulfatase were validated. The 24 PCPs in fish bile were spiked with standard concentrations of 10 ng/mL, 20 ng/mL, 100 ng/mL, and 200 ng/mL to evaluate recoveries, which ranged from 70 to 120% for 16, 16, 22, and 21 analytes with hydrolysis, respectively, and 70-120% for 14, 15, 23, and 23 analytes without hydrolysis, respectively. The quantification limits for target PCPs were in the range 0.26-7.38 ng/mL [excluding musk xylene (MX) and musk ketone (MK)] and 0.20-9.48 ng/mL (excluding MX and MK) for bile samples with and without enzyme hydrolysis, respectively. After enzyme hydrolysis, 12 PCPs were detected in bile from fish collected from the Yangtze River, with a maximum detected concentration of 460 ng/mL, for triclosan (TCS). The hydrolysis reaction indicated that high percentages of glucuronide and sulfate metabolites for some PCPs, i.e. four parabens and TCS, existed in the bile. Copyright © 2018 Elsevier B.V. All rights reserved.

Acrylamide: formation, occurrence in food products, detection methods, and legislation.

PubMed

Arvanitoyannis, Ioannis S; Dionisopoulou, Niki

2014-01-01

This review aims at summarizing the most recent updates in the field of acrylamide (AA) formation (mechanism, conditions) and the determination of AA in a number of foods (fried or baked potatoes, chips, coffee, bread, etc). The methods applied for AA detection [Capillary Electrophoresis-Mass Spectrometry (CE-MS), Liquid Chromatography-Mass Spectrometry (LC-MS), Non-Aqueous Capillary Electrophoresis (NACE), High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS), Pressurized Fluid Extraction (PFE), Matrix Solid-Phase Dispersion (MSPD), Gas Chromatography-Mass Spectrometry (GC-MS), Solid-Phase MicroExtraction-Gas Chromatography (SPME-GC), Enzyme Linked Immunosorbent Assay (ELISA), and MicroEmulsion ElectroKinetic Chromatography (MEEKC) are presented and commented. Several informative figures and tables are included to show the effect of conditions (temperature, time) on the AA formation. A section is also included related to AA legislation in EU and US.

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

PubMed

Lai, Wenqiang; Zeng, Qiao; Tang, Juan; Zhang, Maosheng; Tang, Dianping

2018-01-10

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B 1 (AFB 1 ). It is based on the just-in-time generation of an MnO 2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO 4 ) is converted into manganese dioxide (MnO 2 ) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO 4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO 2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO 4 . Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB 1 and magnetic beads carrying bovine serum albumin conjugated to AFB 1 are used for the determination of AFB 1 . In presence of AFB 1 , it will compete with the BSA-conjugated AFB 1 (on the magnetic beads) for the labeled antibody against AFB 1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB 1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB 1 concentrations in the range from 0.1 to 100 ng mL -1 , with a 0.1 ng mL -1 detection limit (at the 3S blank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO 4 )-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B 1 (AFB 1 ) by coupling with hydrolytic reaction of AOx toward AA and the KMnO 4 -Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].

DEVELOPMENT AND EVALUATION OF AN ENZYME-LINKED IMMUNOASSAY (ELISA) METHOD FOR THE MEASUREMENT OF 2,4-DICHLOROPHENOXYACETIC ACID IN HUMAN URINE

EPA Science Inventory

This paper describes the development of a 96-microwell high sample capacity ELISA method for measuring 2,4-D in urine; the analysis of 2,4-D in real-world urine samples by both ELISA and GC/MS methods; and compares the ELISA and GC/MS results in several key areas: accuracy, preci...

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling

PubMed Central

2013-01-01

Background It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolysis of cellulose was investigated for pretreated corn stover under a variety of recycling conditions. Results It was found that a significant amount of cellulase activity could be recovered by recycling the insoluble biomass fraction, and the enzyme dosage could be decreased by 30% to achieve the same glucose yields under the most favorable conditions. Enzyme productivity (g glucose produced/g enzyme applied) increased between 30 and 50% by the recycling, depending on the reaction conditions. While increasing the amount of solids recycled increased process performance, the methods applicability was limited by its positive correlation with increasing total solids concentrations, reaction volumes, and lignin content of the insoluble residue. However, increasing amounts of lignin rich residue during the recycle did not negatively impact glucose yields. Conclusions To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme usage, and research should be continued for its industrial application. PMID:23336604

Erythema migrans and serodiagnosis by enzyme immunoassay and immunoblot with three borrelia species.

PubMed

Stanek, G; Breier, F; Menzinger, G; Schaar, B; Hafner, M; Partsch, H

1999-12-10

There is wide divergence of opinion between physicians regarding the use of serological measures for the diagnosis and treatment of erythema migrans, the hallmark of Lyme borreliosis. We studied the outcome of an enzyme immunoassay and immunoblot (Western blot) used on the sera of patients who had suffered tick bite and erythema migrans, and had been subsequently treated with various antibiotics. Ninety-nine consecutive patients presenting with erythema migrans after tick bite were prospectively recruited at the outpatient department of two Vienna City hospitals and at the consultation office for Lyme borreliosis of the Institute of Hygiene. University Vienna. Blood samples were taken before antibiotic treatment and 3 and 6 months thereafter. Blood samples from 100 blood donors served as controls. Antibodies against Borrelia burgdorferi sensu lato were determined by enzyme immunoassay (IgG and IgM EIA) and by IgG immunoblot. The latter was performed with isolates of B. alzelii (H2) B. burgdorferi sensu stricto (Le) and B. garinii (W) from Austrian patients. The 4 interpretation criteria for immunoblot results were: A (3 bands out of 8), B (2 bands out of 9), C and D (1 band out of 6). In all patients, the erythema resolved within the treatment period. No complications secondary to the borrelia infection were registered. After treatment there was no significant change in titre, nor was there a difference in the immunoblot pattern between the first, second and third serum samples. Serum antibodies to B. burgdorferi were positive by EIA in 22.9% (IgG) and 2.5% (IgM). Immunoblot results offered by borrelia species and by the interpretation criteria, ranging between 8.3% (criterion A, strain Le) and 44.2% (criterion D, strain H2). By EIA, control samples were IgG and IgM positive in 5% and 1%, respectively. Positive immunoblot results with strain H2 were found in 9%, 13%, 18%, and 20% by the criteria A through D respectively. After antibiotic treatment of erythema migrans the immunological response appears to be abrogated. Thus, serological results are not supportive for the diagnosis of erythema migrans, not will they retrospectively prove successful antibiotic treatment of borrelia infection.

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

Effect of Asian and Siberian ginseng on serum digoxin measurement by five digoxin immunoassays. Significant variation in digoxin-like immunoreactivity among commercial ginsengs.

PubMed

Dasgupta, Amitava; Wu, Sang; Actor, Jeffrey; Olsen, Margaret; Wells, Alice; Datta, Pradip

2003-02-01

Asian and Siberian ginsengs contain glycosides with structural similarities to digoxin. We studied potential interference of ginseng in 5 digoxin immunoassays in 3 Asian (2 liquid extracts, 1 capsule) and 3 Siberian ginseng preparations (1 liquid extract, 2 capsules). With the fluorescence polarization immunoassay (FPIA), we observed apparent digoxin activity in 1 Asian liquid preparation and in the liquid extract and 1 capsule form of Siberian ginseng. In mice fed ginseng, we observed digoxin activities in the serum (Asian, 0.48-0.68 ng/mL [0.6-0.9 nmol/L]; Siberian, 0.20-0.47 ng/mL [0.3-0.6 nmol/L]), indicating that such interferences also occur in vivo. Serum pools prepared from samples from patients receiving digoxin and then supplemented with Asian or Siberian ginseng showed falsely increased digoxin values using the FPIA (e.g., for Asian ginseng, 1.54 ng/mL [2.0 nmol/L] vs control value, 1.10 ng/mL [1.4 nmol/L]) and falsely decreased values using the microparticle enzyme immunoassay (MEIA; 0.73 ng/mL [0.9 nmol/L] vs control value, 1.04 ng/mL [1.3 nmol/L]). Digoxin-like immunoreactive substances (DLISs) showed synergistic effects with ginsengs in interfering with the FPIA and MEIA for digoxin. No interference was observed with 3 other digoxin assays, even in the presence of elevated DLISs.

An organic-inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels.

PubMed

Su, Biling; Tang, Dianping; Tang, Juan; Li, Qunfang; Chen, Guonan

2011-10-01

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic-inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core-shell Fe(3)O(4)-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H(2)O(2) as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001-200 ng mL(-1) AFP with a low detection limit of 0.5 pg mL(-1) at 3s(B). Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL(-1) AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay. Copyright © 2011 Elsevier Inc. All rights reserved.

Multiplexed detection of tumor markers with multicolor quantum dots based on fluorescence polarization immunoassay.

PubMed

Tian, Jianniao; Zhou, Liujin; Zhao, Yanchun; Wang, Yuan; Peng, Yan; Zhao, Shulin

2012-04-15

A multicolor quantum dot (QD)-based nanosensor for multiplex detection of two tumor markers in a homogeneous format based on fluorescence polarization immunoassay was proposed. QDs520 and QDs620 were labeled alpha-fetoprotein(α-AFP) and carcinoembryonic antigen (CEA), respectively. After separated and purified by ultrafiltration, they were used in fluorescence polarization immunoassay for the simultaneous detection of human serum alpha-fetoprotein and carcinoembryonic antigen. Under the optimal conditions, the multi-analyte immunosensor had a wide linear range (from 0.5 ng mL(-1) to 500 ng mL(-1)) for both two tumor markers and good correlation (0.996 for α-AFP and 0.993 for CEA). The detection limits (LOD) were 0.36 ng mL(-1) for CEA and 0.28 ng mL(-1) for α-AFP (S/N=3). The carcinoembryonic antigen and fetoprotein in clinical serum samples were simultaneously detected. The results from 28 serum samples had a good agreement with enzyme-linked immunosorbent assay (ELISA). The relative standard deviation and the recovery suggested that the precision and the accuracy of this analytical method were satisfactory. This strategy with high sensitivity, good specificity, easy procedures and short analysis time shows great promise for clinical diagnoses and basic discovery. The application of QDs with longer fluorescence lifetime and small fluorescence polarization can be used for the determination of high molecular-weight substances which cannot be analyzed using dye fluorescence polarization immunoassay. Copyright © 2012 Elsevier B.V. All rights reserved.

Investigation of hydrazide derivatives inhibitory effect on peroxidase enzyme purified from turnip roots

NASA Astrophysics Data System (ADS)

Almaz, Züleyha; Öztekin, Aykut; Özdemir, Hasan

2017-04-01

Peroxidases (EC: 1.11.1.7) are haem proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group [1]. They are found in all cells and play a critical role in many biological processes, such as the host-defense mechanism [2]. Peroxidases (PODs) are widely used in clinical biochemistry, enzyme immunoassays, synthesis of various aromatic chemicals, treatment of waste water containing phenolic compounds [3, 4]. In this study, peroxidase enzyme was purified with Para amino benzohydrazide (PABH)-L-Tyrosine Sepharose 4B affinity chromatography to investigate the inhibitory effect of hydrazide derivatives on Turnip (Brassica rapa L.). IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzyme and inhibition type of these molecules were determined.

Multi-center Performance Evaluations of Tacrolimus and Cyclosporine Electrochemiluminescence Immunoassays in the Asia-Pacific Region

PubMed Central

Qin, Xuzhen; Rui, Jianzhong; Xia, Yong; Mu, Hong; Song, Sang Hoon; Raja Aziddin, Raja Elina; Miles, Gabrielle; Sun, Yuli

2018-01-01

Background The immunosuppressant drugs (ISDs), tacrolimus and cyclosporine, are vital for solid organ transplant patients to prevent rejection. However, toxicity is a concern, and absorption is highly variable across patients; therefore, ISD levels need to be precisely monitored. In the Asia-Pacific (APAC) region, tacrolimus and cyclosporine concentrations are typically measured using immunoassays. The objective of this study was to assess the analytical performance of Roche Elecsystacrolimus and cyclosporinee electrochemiluminescence immunoassays (ECLIAs). Methods This evaluation was performed in seven centers across China, South Korea, and Malaysia. Imprecision (repeatability and reproducibility), assay accuracy, and lot-to-lot reagent variability were tested. The Elecsys ECLIAs were compared with commercially available immunoassays (Architect, Dimension, and Viva-E systems) using whole blood samples from patients with various transplant types (kidney, liver, heart, and bone marrow). Results Coefficients of variation for repeatability and reproducibility were ≤5.4% and ≤12.4%, respectively, for the tacrolimus ECLIA, and ≤5.1% and ≤7.3%, respectively, for the cyclosporine ECLIA. Method comparisons of the tacrolimus ECLIA with Architect, Dimension, and Viva-E systems yielded slope values of 1.01, 1.14, and 0.897, respectively. The cyclosporine ECLIA showed even closer agreements with the Architect, Dimension, and Viva-E systems (slope values of 1.04, 1.04, and 1.09, respectively). No major differences were observed among the different transplant types. Conclusions The tacrolimus and cyclosporine ECLIAs demonstrated excellent precision and close agreement with other immunoassays tested. These results show that both assays are suitable for ISD monitoring in an APAC population across a range of different transplant types. PMID:29214751

Fullerol-fluorescein isothiocyanate-concanavalin agglutinin phosphorescent sensor for the detection of alpha-fetoprotein and forecast of human diseases

NASA Astrophysics Data System (ADS)

Liu, Jia-ming; Lin, Li-ping; Jiang, Shu-Lian; Cui, Ma Lin; Jiao, Li; Zhang, Xiao Yang; Zhang, Li-hong; Zheng, Zhi Yong; Lin, Xuan; Lin, Shao-qin

2013-11-01

Based on the reaction of the active -OH group in fullerol (F) with the dissociated -COOH group in fluorescein isothiocyanate (FITC) to form an F-FITC and the enhanced effect of N, N-dimethylaniline (DMA) on phosphorescence signal of F-FITC, a new phosphorescent labeling reagent (DMA-F-FITC) was developed. What's more, a phosphorescent sensor for the determination of alpha-fetoprotein variant (AFP-V) has been designed via the coupling technique of the high sensitivity for affinity adsorption-solid substrate-room temperature phosphorimetry (AA-SS-RTP) with the strong specificity reaction between DMA-F-FITC-Con A and AFP-V. The DMA-F-FITC increased the number of luminescent molecules in the biological target which improved the sensitivity of phosphorescent sensor. The proposed sensor was responsive, simple, selective and sensitive, and it has been applied to the determination of trace AFP-V in human serum and the forecast of human diseases using phosphorescence emission wavelength of F or FITC, with the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Meanwhile, the mechanisms for the labeling reaction and the sensing detection of AFP-V were discussed.

Integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching "absolute" zero background in immunoassays.

PubMed

Ma, Hongwei; Wu, Yuanzi; Yang, Xiaoli; Liu, Xing; He, Jianan; Fu, Long; Wang, Jie; Xu, Hongke; Shi, Yi; Zhong, Renqian

2010-08-01

The key to achieve a highly sensitive and specific protein microarray assay is to prevent nonspecific protein adsorption to an "absolute" zero level because any signal amplification method will simultaneously amplify signal and noise. Here, we develop a novel solid supporting material, namely, polymer coated initiator integrated poly(dimethysiloxane) (iPDMS), which was able to achieve such "absolute" zero (i.e., below the detection limit of instrument). The implementation of this iPDMS enables practical and high-quality multiplexed enzyme-linked immunosorbent assay (ELISA) of 11 tumor markers. This iPDMS does not need any blocking steps and only require mild washing conditions. It also uses on an average 8-fold less capture antibodies compared with the mainstream nitrocellulose (NC) film. Besides saving time and materials, iPDMS achieved a limit-of-detection (LOD) as low as 19 pg mL(-1), which is sufficiently low for most current clinical diagnostic applications. We expect to see an immediate impact of this iPDMS on the realization of the great potential of protein microarray in research and practical uses such as large scale and high-throughput screening, clinical diagnosis, inspection, and quarantine.

Chemiluminescence assay for the detection of biological warfare agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Langry, K; Horn, J

A chemiluminescent homogeneous immunoassay and a hand-size multiassay reader are described that could be used for detecting biological materials. The special feature of the assay is that it employs two different antibodies that each bind to a unique epitope on the same antigen. Each group of epitope-specific antibodies has linked to it an enzyme of a proximal-enzyme pair. One enzyme of the pair utilizes a substrate in high concentration to produce a second substrate required by the second enzyme. This new substrate enables the second enzyme to function. The reaction of the second enzyme is configured to produce light. Thismore » chemiluminescence is detected with a charge-coupled device (CCD) camera. The proximal pair enzymes must be in close proximity to one another to allow the second enzyme to react with the product of the first enzyme. This only occurs when the enzyme-linked antibodies are attached to the antigen, whether antigen is a single protein with multiple epitopes or the surface of a cell with a variety of different antigens. As a result of their juxtaposition, the enzymes produce light only in the presence of the biological material. A brief description is given as to how this assay could be utilized in a personal bio-agent detector system.« less

Coupling solid-phase extraction and enzyme-linked immunosorbent assay for ultratrace determination of herbicides in pristine water

USGS Publications Warehouse

Aga, D.S.; Thurman, E.M.

1993-01-01

Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were coupled for automated trace analysis of pristine water samples containing 2-chloro-4-ethylamino-6-isopropylamine-s-triazine (atrazine) and 2-chloro-2???,6???-diethyl-N-(methoxymethyl)acetanilide (alachlor). The isolation of the two herbicides on a C18-resin involved the selection of an elution solvent that both removes interfering substances and is compatible with ELISA. Ethyl acetate was selected as the elution solvent followed by a solvent exchange with methanol/water (20/80, % v/v). The SPE-ELISA method has a detection limit of 5.0 ng/L (5 ppt), >90% recovery, and a relative standard deviation of ??10%. The performance of a microtiter plate-based ELISA and a magnetic particle-based ELISA coupled to SPE was also evaluated. Although the sensitivity of the two ELISA methods was comparable, the precision using magnetic particles was improved considerably (??10% versus ??20%) because of the faster reaction kinetics provided by the magnetic particles. Finally, SPE-ELISA and isotope dilution gas chromatography/ mass spectrometry correlated well (correlation coefficient of 0.96) for lake-water samples. The SPE-ELISA method is simple and may have broader applications for the inexpensive automated analysis of other contaminants in water at trace levels.

Evaluation of reproductive function in Turkana women with enzyme immunoassays of urinary hormones in the field.

PubMed

Leslie, P W; Campbell, K L; Little, M A; Kigondu, C S

1996-02-01

The frequently reported observation that nomadic populations have lower fertility than their settled counterparts is often attributed to what are perceived as harsh, stressful conditions under which the nomads live. But the consequences of the hypothesized stresses for the reproductive biology or demography of these populations have been documented only a little. Traditionally, the Turkana of northwest Kenya are nomadic herders, but increasing numbers have settled on agricultural development schemes. We used an array of hormonal assays along with anthropometric indexes of nutritional status and interviews covering reproductive history, recent menstruation, diet, and health to compare reproductive function in nomadic and settled Turkana women. First morning urine samples were collected for three consecutive days during a series of surveys. Human choriogonadotropin (hCG; a marker for pregnancy), luteinizing hormone (LH; an indicator of ovulation), and pregnanediol glucuronide (PdG; an indicator of postovulatory luteal function) were assessed in the field with commercially available dipstick enzyme immunoassays. These assays along with the interview data allowed us to determine the reproductive status (e.g., pregnant or cycling, and if cycling, which phase of the ovarian cycle) of 166 nomadic and 194 settled Turkana women. The cross-sectional classifications allowed inferences of conception rates and normality of ovarian function. Follow-up surveys provided rates of pregnancy loss. Compared with the settled women, the nomadic women exhibited lower pregnancy rates and cycling nomadic women were less likely to show evidence of ovulation or luteal function. These results suggest that reproductive function of the nomadic women is diminished relative to the settled women. However, the settled women experienced a much higher rate of pregnancy loss, which may mean that their effective fecundability is in fact lower than that of the nomadic women. This study is the first to apply such a wide range of hormonal assays in the field. It demonstrates that field-based assays are feasible and robust and can play an important role in epidemiological and biodemographic studies, even in remote locations under conditions that would ordinarily be considered incompatible with on-site laboratory analysis.

Parallel Workflow for High-Throughput (>1,000 Samples/Day) Quantitative Analysis of Human Insulin-Like Growth Factor 1 Using Mass Spectrometric Immunoassay

PubMed Central

Oran, Paul E.; Trenchevska, Olgica; Nedelkov, Dobrin; Borges, Chad R.; Schaab, Matthew R.; Rehder, Douglas S.; Jarvis, Jason W.; Sherma, Nisha D.; Shen, Luhui; Krastins, Bryan; Lopez, Mary F.; Schwenke, Dawn C.; Reaven, Peter D.; Nelson, Randall W.

2014-01-01

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications. PMID:24664114

Multiple reaction monitoring (MRM) of plasma proteins in cardiovascular proteomics.

PubMed

Dardé, Verónica M; Barderas, Maria G; Vivanco, Fernando

2013-01-01

Different methodologies have been used through years to discover new potential biomarkers related with cardiovascular risk. The conventional proteomic strategy involves a discovery phase that requires the use of mass spectrometry (MS) and a validation phase, usually on an alternative platform such as immunoassays that can be further implemented in clinical practice. This approach is suitable for a single biomarker, but when large panels of biomarkers must be validated, the process becomes inefficient and costly. Therefore, it is essential to find an alternative methodology to perform the biomarker discovery, validation, and -quantification. The skills provided by quantitative MS turn it into an extremely attractive alternative to antibody-based technologies. Although it has been traditionally used for quantification of small molecules in clinical chemistry, MRM is now emerging as an alternative to traditional immunoassays for candidate protein biomarker validation.

Effects of Chronic Alcohol Exposure on Kainate Receptor-Mediated Neurotransmission in the Hippocampus

DTIC Science & Technology

2004-09-01

hippocampus and hypothalamus. J Neurosci Res 63:200-208. Thorpe GH et al. (1989) Chemiluminescent enzyme immunoassay of alpha - fetoprotein based on an...cells. Proc Natl Acad Sci USA 83:6213-6215. Montpied Pet al. (1991) Prolonged ethanol inhalation decreases gamma-aminobutyric acidA receptor alpha ...potentials mediated via alpha -bungarotoxin-sensitive nico- tinic acetylcholine receptors in rat hippocampal interneurons. J Neurosci 18:8228- 8235

Serological Evidence of Dengue Fever Among Refugees, Hargeysa, Somalia

DTIC Science & Technology

1989-01-01

fever, Sindbis, Chikungunya, yellow HISTORY OF THE DISEASE IN THE fever, and Zika viruses . However, antibody reac- DAM CAMP tive to dengue 2 virus was...fever, Crimean-Congo hemorrhagic fever, Sindbis, Chikungunya, yellow fever, and Zika viruses . However, antibody reactive to dengue 2 virus was detected... ZIKA ) viruses . Further testing of sera for evidence of dengue S Barbera S , MOGAISCIO . viral infection was done by the enzyme immunoassay " (EIA

Hedonic Predictors of Tobacco Dependence: A Puff Guide to Smoking Cessation

DTIC Science & Technology

2015-04-07

process may not always be the case. While Perkins et al. (31) showed the potential for a connection between puff volume, considered reinforcement...effect can be attributed to the effects of "acute tolerance" (31 ). Nicotine is thought to acutely desensitize nicotinic receptors ("acute tolerance...company that performs biological assays) for the cotinine assay. Salivary cotinine levels were measured through an enzyme immunoassay conducted by

Determination of relative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX).

PubMed

Samara, Fatin; Gullett, Brian K; Harrison, Robert O; Chu, Andrew; Clark, George C

2009-04-01

Determination of toxic activity requires knowledge of both the concentration and toxicity to evaluate the risk for adverse human health and environmental effects. A chemically-activated luciferase gene expression cell bioassay system (CALUX) and an antibody-based method enzyme immunoassay (EIA) were used to detect the dioxin-like response of several polybrominated, polychlorinated, and polybrominated/chlorinated dibenzo-p-dioxins/furans (PBDDs/Fs, PCDDs/Fs, and PBCDDs/Fs, respectively). It has been suggested that the biological activity of the brominated and mixed bromo/chloro compounds is similar to their chlorinated analogues (measured by binding to the Ah receptor). PBDD/F, PCDD/F, and PBCDD/F laboratory standards exhibited biological activity ranging over three orders of magnitude. The highest relative potency (REP) values from CALUX analysis, when compared to 2,3,7,8-TCDD, were 2,3,7,8-TBDD at 0.99 (+/-0.07), 1,2,3,7,8-PeCDD at 0.69, and 2-Br-3,7,8-TriCDD at 0.72 (+/-0.02). Cross-reactivities were calculated using EIA for several PBDDs/Fs and PBCDDs. The highest percent cross-reactivity was found for 2,3,7,8-TBDD at 138 (+/-34%), and 2,3,7-TriBDD at 84 (+/-36%).

Enzyme immunoassay for tenuazonic acid in apple and tomato products.

PubMed

Gross, Madeleine; Curtui, Valeriu; Ackermann, Yvonne; Latif, Hadri; Usleber, Ewald

2011-12-14

The Alternaria mycotoxin tenuazonic acid was derivatized with succinic anhydride and conjugated to keyhole limpet hemocyanin (KLH) and to horseradish peroxidase (HRP), respectively. The KLH conjugate was used to produce polyclonal antibodies in rabbits. A competitive direct enzyme immunoassay (EIA) for tenuazonic acid was established, which was moderately sensitive for tenuazonic acid [50% inhibition concentration (IC(50)): 320 ± 130 ng/mL] but strongly reacted with tenuazonic acid acetate (IC(50): 23.3 ± 7.5 ng/mL). Therefore, an optimized EIA protocol was established, which employed acetylation of standard and sample extract solutions. The mean standard curve detection limit (IC(30)) for tenuazonic acid acetate was 5.4 ± 2.0 ng/mL, enabling detection limits for tenuazonic acid in apple and tomato products of 25-50 ng/g (150 ng/g in tomato paste). Recoveries in a concentration range of 50-2000 ng/g were 60-130% in apple juice and tomato juice and 40-150% in other tomato products. Tenuazonic acid was detected in apple juice and tomato products from German retail shops at levels of 50-200 ng/g. In conclusion, this novel EIA for tenuazonic acid could be useful within a screening program for Alternaria mycotoxins in food.

Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria.

PubMed

Engler, K H; Efstratiou, A

2000-04-01

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.

Rapid Enzyme Immunoassay for Determination of Toxigenicity among Clinical Isolates of Corynebacteria

PubMed Central

Engler, Kathryn H.; Efstratiou, Androulla

2000-01-01

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day. PMID:10747112

Quantitative measurement of human thyroglobulin-specific antibodies by use of a sensitive enzyme-linked immunoassay.

PubMed

Kuppers, R C; Outschoorn, I M; Hamilton, R G; Burek, C L; Rose, N R

1993-04-01

A quantitative enzyme-linked immunoassay that measures in absolute terms the subclass concentration of human thyroglobulin (huTg)-specific IgG autoantibody was developed. Unique to this study was the use of an affinity-purified anti-huTg standard with a known concentration of the four IgG subclasses. The sensitivity of the ELISA assay was 1-5 ng/ml depending on the IgG subclass being measured. We examined 22 sera of patients with autoimmune thyroid disease. The total huTg-specific antibody concentrations in serum ranged from 0 to nearly 3000 micrograms/ml of IgG. The IgG subclass distribution in individuals with low huTg-specific IgG (< 10 micrograms/ml) was primarily IgG1 and IgG3 Ab. Patients with intermediate levels of huTg IgG (10-600 micrograms/ml) expressed all four subclasses; however, no particular subclass was dominant. Individuals with > 1000 micrograms/ml also showed huTg-Ab in all four subclasses, however, IgG1 and IgG2 were dominant. All four IgG subclasses were used in the response to huTg, although the pattern of usage varied between individuals. There was no dominant subclass usage seen in this patient population.

Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification.

PubMed

Tang, Dianping; Tang, Juan; Su, Biling; Chen, Guonan

2010-10-27

A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9-12 Years Children.

PubMed

Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

2015-01-01

The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9-11 years were determined by two methods of CPBA and HPLC. Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes.

Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme-linked immunosorbent assay for rapid detection of sulfonamides.

PubMed

Liu, Na; Han, Zheng; Lu, Lei; Wang, Lin; Ni, Geng; Zhao, Zhihui; Wu, Aibo; Zheng, Xiaodong

2013-02-01

Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody (RabMAb) against sulfonamides (SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay (ELISA) was then developed and applied to real sample analysis. A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration (IC(50)) values for four SAs were all below 10 ng mL(-1) , with 0.68 ng mL(-1) sulfathiazole (STZ), 1.11 ng mL(-1) sulfadiazine (SD), 1.15 ng mL(-1) sulfapyridine (SP) and 5.27 ng mL(-1) sulfamethoxazole (SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs (STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses. © 2012 Society of Chemical Industry.

A polymerase chain reaction enzyme immunoassay for diagnosing infection caused by Aspergillus fumigatus.

PubMed Central

Golbang, N; Burnie, J P; Matthews, R C

1999-01-01

AIM: To develop a polymerase chain reaction enzyme immunoassay (PCR-EIA) to measure levels of circulating aspergillus DNA in invasive aspergillosis caused by Aspergillus fumigatus. METHODS: The PCR reaction was based on primers from the 18s rRNA gene. Binding of the product to a streptavidin coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR and this was the tag to which antibody was bound in the subsequent EIA. RESULTS: The optical density (OD) endpoint was < 0.1 in 10 sera from neutropenic patients with no evidence of invasive aspergillosis, and in 10 sera from nonneutropenic patients with bacterial pneumonia (group 1). The OD from five of 12 patients with allergic bronchopulmonary aspergillosis (ABPA) (group 2), three with an aspergilloma (group 3), and five with possible invasive aspergillosis (group 4) was > or = 0.1. In 63 sera from 33 cases of proven invasive aspergillosis (group 5) an OD > or = 0.1 was achieved in 48 sera from 30 patients. The maximum OD was 0.510. The level fell in survivors and gradually rose in fatal cases. CONCLUSIONS: This assay validated the concept of diagnosing invasive aspergillosis by measuring levels of circulating fungal DNA in serum. PMID:10562808

The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

PubMed

Hashim, Puziah

2014-03-01

Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid.

PubMed

Piletska, Elena V; Villoslada, Fernando Navarro; Chianella, Iva; Bossi, Alessandra; Karim, Kal; Whitcombe, Michael J; Piletsky, Sergey A; Doucette, Gregory J; Ramsdell, John S

2008-03-03

A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.

Study of the aroma formation and transformation during the manufacturing process of oolong tea by solid-phase micro-extraction and gas chromatography-mass spectrometry combined with chemometrics.

PubMed

Ma, Chengying; Li, Junxing; Chen, Wei; Wang, Wenwen; Qi, Dandan; Pang, Shi; Miao, Aiqing

2018-06-01

Oolong tea is a typical semi-fermented tea and is famous for its unique aroma. The aim of this study was to compare the volatile compounds during manufacturing process to reveal the formation of aroma. In this paper, a method was developed based on head-space solid phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) combined with chemometrics to assess volatile profiles during manufacturing process (fresh leaves, sun-withered leaves, rocked leaves and leaves after de-enzyming). A total of 24 aroma compounds showing significant differences during manufacturing process were identified. Subsequently, according to these aroma compounds, principal component analysis and hierarchical cluster analysis showed that the four samples were clearly distinguished from each other, which suggested that the 24 identified volatile compounds can represent the changes of volatile compounds during the four steps. Additionally, sun-withering, rocking and de-enzyming can influence the variations of volatile compounds in different degree, and we found the changes of volatile compounds in withering step were less than other two manufacturing process, indicating that the characteristic volatile compounds of oolong tea might be mainly formed in rocking stage by biological reactions and de-enzyming stage through thermal chemical transformations rather than withering stage. This study suggested that HS-SPME/GC-MS combined with chemometrics methods is accurate, sensitive, fast and ideal for rapid routine analysis of the aroma compounds changes in oolong teas during manufacturing processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development and validation of a fluorescent microsphere immunoassay for soluble CD30 testing.

PubMed

Pavlov, Igor; Martins, Thomas B; Delgado, Julio C

2009-09-01

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.

Development and Validation of a Fluorescent Microsphere Immunoassay for Soluble CD30 Testing▿

PubMed Central

Pavlov, Igor; Martins, Thomas B.; Delgado, Julio C.

2009-01-01

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported. PMID:19605595

Improving the sensitivity of immunoassay based on MBA-embedded Au@SiO2 nanoparticles and surface enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Wei, Chao; Xu, Min-Min; Fang, Cong-Wei; Jin, Qi; Yuan, Ya-Xian; Yao, Jian-Lin

2017-03-01

Traditional "sandwich" structure immunoassay is mainly based on the self-assembly of "antibody on solid substrate-antigen-antibody with nanotags" architectures, and the sensitivity of this strategy is critically depended on the surface enhanced Raman scattering (SERS) activities and stability of nanotags. Therefore, the rational design and fabrication on the SERS nanotags attracts the common interests to the bio-related detecting and imaging. Herein, silica encapsulated Au with mercaptobenzoic acid (MBA) core-shell nanoparticles (Au-MBA@SiO2) are fabricated instead of the traditional naked Au or Ag nanoparticles for the SERS-based immunoassay on human and mouse IgG antigens. The MBA molecules facilitate the formation of continuous pinhole-free silica shell and are also used as SERS labels. The silica shell is employed to protect MBA labels and to isolate Au core from the ambient solution for blocking the aggregation. This shell also played the similar role to BSA in inhibiting the nonspecific bindings, which allowed the procedures for constructing "sandwich" structures to be simplified. All of these merits of the Au-MBA@SiO2 brought the high performance in the related immunoassay. Benefiting from the introduction of silica shell to encapsulate MBA labels, the detection sensitivity was improved by about 1- 2 orders of magnitude by comparing with the traditional approach based on naked Au-MBA nanoparticles. This kind of label-embedded core-shell nanoparticles could be developed as the versatile nanotags for the bioanalysis and bioimaging.

Validated high-performance liquid chromatographic method utilizing solid-phase extraction for the simultaneous determination of naringenin and hesperetin in human plasma.

PubMed

Kanaze, Feras Imad; Kokkalou, Eugene; Georgarakis, Manolis; Niopas, Ioannis

2004-03-05

Naringenin and hesperetin, the aglycones of the flavanone glucosides naringin and hesperidin occur naturally in citrus fruits. They exert a variety of pharmacological effects such as antioxidant, blood lipid-lowering, anticarcinogenic and inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A specific, sensitive, precise, and accurate solid-phase extraction high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of naringenin and hesperetin in human plasma was developed and validated. After addition of 7-ethoxycoumarin as internal standard, plasma samples were incubated with beta-glucuronidase/sulphatase, and the analytes were isolated from plasma by solid-phase extraction using C(18) cartridges and separated on a C(8) reversed phase column with methanol/water/acetic acid (40:58:2, v/v/v) as the eluent at 45 degrees C. The method was linear in the 10-300 ng/ml concentration range for both naringenin and hesperetin (r>0.999). Recovery for naringenin, hesperetin and internal standard was greater than 76.7%. Intra- and inter-day precision for naringenin ranged from 1.4 to 4.2% and from 1.9 to 5.2%, respectively, and for hesperetin ranged from 1.3 to 4.1% and from 1.7 to 5.1%, respectively. Accuracy was better than 91.5 and 91.3% for naringenin and hesperetin, respectively.

Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

PubMed

Wang, Guochun; Das, Champak; Ledden, Bradley; Sun, Qian; Nguyen, Chien

2017-10-01

Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

Cocaine and opiates use in pregnancy: detection of drugs in neonatal meconium and urine.

PubMed

López, P; Bermejo, A M; Tabernero, M J; Cabarcos, P; Alvarez, I; Fernández, P

2009-09-01

In this study, the case of a newborn with symptoms of hyperexcitability was analyzed. After it was confirmed in the hospital that the mother had consumed drugs during pregnancy using an enzyme multiplied immunoassay technique, samples of the newborn's urine and meconium were sent to our laboratory to observe the evolution in the distribution of cocaine and opiates during the days following birth. For urine analysis, screening was done with an immunoassay technique, and the confirmation was done by gas chromatography-mass spectrometry (GC-MS) according to a published method. A GC-MS method for simultaneous analysis of cocaine, benzoylecgonine, codeine, morphine, and 6-acetylmorphine in meconium is described. GC-MS confirmation of urine and meconium results showed consumption of cocaine and codeine during pregnancy and also showed the levels of drugs gradually declined, totally disappearing by the third day.

Catechol-chitosan redox capacitor for added amplification in electrochemical immunoanalysis.

PubMed

Yan, Kun; Liu, Yi; Guan, Yongguang; Bhokisham, Narendranath; Tsao, Chen-Yu; Kim, Eunkyoung; Shi, Xiao-Wen; Wang, Qin; Bentley, William E; Payne, Gregory F

2018-05-22

Antibodies are common recognition elements for molecular detection but often the signals generated by their stoichiometric binding must be amplified to enhance sensitivity. Here, we report that an electrode coated with a catechol-chitosan redox capacitor can amplify the electrochemical signal generated from an alkaline phosphatase (AP) linked immunoassay. Specifically, the AP product p-aminophenol (PAP) undergoes redox-cycling in the redox capacitor to generate amplified oxidation currents. We estimate an 8-fold amplification associated with this redox-cycling in the capacitor (compared to detection by a bare electrode). Importantly, this capacitor-based amplification is generic and can be coupled to existing amplification approaches based on enzyme-linked catalysis or magnetic nanoparticle-based collection/concentration. Thus, the capacitor should enhance sensitivities in conventional immunoassays and also provide chemical to electrical signal transduction for emerging applications in molecular communication. Copyright © 2018 Elsevier B.V. All rights reserved.

Sensitive, fast, and specific immunoassays for methyltestosterone detection.

PubMed

Kong, Na; Song, Shanshan; Peng, Juan; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

2015-04-29

An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%-100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed.

Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens.

PubMed

Wang, Zhanhui; Zhang, Huiyan; Ni, Hengjia; Zhang, Suxia; Shen, Jianzhong

2014-04-11

In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 μg L(-1) to as low as 0.07 μg L(-1). The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. Copyright © 2014 Elsevier B.V. All rights reserved.

Complement-dependent cytotoxicity (CDC) to detect Anti-HLA antibodies: old but gold.

PubMed

Saito, Patrícia Keiko; Yamakawa, Roger Haruki; Pereira, Lucieni Christina Marques da Silva; da Silva, Waldir Veríssimo; Borelli, Sueli Donizete

2014-07-01

The criterion (gold) standard to detect anti-human leukocyte antigen (HLA) antibodies is the complement-dependent cytotoxicity (CDC) assay. Recently, more sensitive methods have been used for the same purpose. This study analyzed 70 serum samples of patients with end-stage renal disease using CDC, CDC with the addition of anti-human globulin (CDC-AHG), CDC with the addition of dithiothreitol (CDC-DTT), and the recent solid-phase immunoassay (SPI; Labscreen PRA) to detect anti-HLA antibodies. Mean percent panel reactive antibodies (PRA) detected by SPI was 37.5% (±34.2) higher than the values detected by the other methods. Comparative analyses revealed significant difference between CDC and CDC-AHG, and between CDC and SPI (P < 0.0001), but not between CDC-AHG and SPI (P = 0.8026). Although the CDC-AHG method is "old," its performance to detect anti-HLA antibodies in the samples analyzed was comparable to the SPI in the evaluation of percent class I PRA. © 2014 Wiley Periodicals, Inc.

Utility of HLA Antibody Testing in Kidney Transplantation

PubMed Central

Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

The utility of pre-test clinical scoring for clinical diagnosis of heparin-induced thrombocytopenia in cardiac surgery patients of a tertiary care centre in north India.

PubMed

Sachan, D; Gupta, N; Agarwal, P; Chaudhary, R

2011-08-01

Heparin-induced thrombocytopenia (HIT) should be diagnosed clinically as well as by laboratory assays for timely recognition, prevention and management of complications. To evaluate the clinical utility of pre-test clinical scoring system in combination with two immunoassays for the diagnosis of HIT in cardiac surgery patients. A total of 100 consecutive patients undergoing cardiac surgery were studied. Pre-test clinical scoring was carried out in patients with thrombocytopenia and further tested by two immunoassays, i.e., Heparin platelet factor 4 (H-PF4) enzyme-linked immunosorbent assay (ELISA) and particle gel immunoassay (PaGIA). Of the 100 patients studied, 42 patients developed thrombocytopenia post-operatively. On pre-test clinical scoring, low T-score was observed in 6 patients, intermediate in 28 and high score in 8 patients, whereas 19 patients (45.2%) were positive by H-PF4 ELISA and 10 (23.8%) by PaGIA for H-PF4 antibody. The difference in the incidence of clinically significant HIT antibodies in the three categories was statistically significant. A good correlation was also observed with ELISA optical density, T-scoring and PaGIA. Pre-test clinical scoring correlates well with the development of H-PF4 antibodies which are incriminated in the causation of thrombotic complications in patients with HIT. We also propose a protocol for diagnosing patients with clinical suspicion of HIT using pre-test clinical scoring and immunoassay. © 2011 The Authors. Transfusion Medicine © 2011 British Blood Transfusion Society.

Survivability of immunoassay reagents exposed to the space radiation environment on board the ESA BIOPAN-6 platform as a prelude to performing immunoassays on Mars.

PubMed

Derveni, Mariliza; Allen, Marjorie; Sawakuchi, Gabriel O; Yukihara, Eduardo G; Richter, Lutz; Sims, Mark R; Cullen, David C

2013-01-01

The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context.

Electrical immunosensor based on a submicron-gap interdigitated electrode and gold enhancement.

PubMed

Ahn, Junhyoung; Lee, Tae Han; Li, Taihua; Heo, Kwang; Hong, Seunghun; Ko, Jeongheon; Kim, Yongsam; Shin, Yong-Beom; Kim, Min-Gon

2011-08-15

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1μm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations). Copyright © 2011 Elsevier B.V. All rights reserved.

Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

PubMed Central

Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe

2014-01-01

Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103 CFU/ml to 8.8 × 104 CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 105 CFU/ml to 106 CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers.

PubMed

Park, Min Cheol; Kim, Moojong; Lim, Gun Taek; Kang, Sung Min; An, Seong Soo A; Kim, Tae Song; Kang, Ji Yoon

2016-06-21

Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 μL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volumes (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 μL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the μCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

Antigen based detection of cystic echinococcosis in buffaloes using ELISA and Dot-EIA.

PubMed

Sangaran, A; Bino Sundar, S T; Latha, Bhaskaran Ravi

2017-03-01

Cystic echinococcosis is caused by the larval stage of the dog tapeworm, Echinococcus granulosus . The disease is recognized as one of the world's major zoonoses affecting human beings and domestic animals apart from its economic and public health importance. Development of the cysts in the intermediate host such as buffaloes occurs in the lungs, liver and other organs. In this study, detection of circulating antigen in the diagnosis of cystic echinococcosis in buffaloes was done using enzyme linked immunosorbent assay and Dot-Enzyme immunoassay (Dot-EIA). The sensitivity and specificity were determined as 89 and 92 % respectively, whereas those of Dot-EIA were determined as 94 and 96 %.

Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple-Reaction–Monitoring Mass Spectrometry

PubMed Central

Agger, Sean A.; Marney, Luke C.; Hoofnagle, Andrew N.

2011-01-01

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability. PMID:20923952

Validated low-volume aldosterone immunoassay tailored to GCLP-compliant investigations in small sample volumes.

PubMed

Schaefer, J; Burckhardt, B B; Tins, J; Bartel, A; Laeer, S

2017-12-01

Heart failure is well investigated in adults, but data in children is lacking. To overcome this shortage of reliable data, appropriate bioanalytical assays are required. Development and validation of a bioanalytical assay for the determination of aldosterone concentrations in small sample volumes applicable to clinical studies under Good Clinical Laboratory Practice. An immunoassay was developed based on a commercially available enzyme-linked immunosorbent assay and validated according to current bioanalytical guidelines of the EMA and FDA. The assay (range 31.3-1000 pg/mL [86.9-2775 pmol/L]) is characterized by a between-run accuracy from - 3.8% to - 0.8% and a between-run imprecision ranging from 4.9% to 8.9% (coefficient of variation). For within-run accuracy, the relative error was between - 11.1% and + 9.0%, while within-run imprecision ranged from 1.2% to 11.8% (CV). For parallelism and dilutional linearity, the relative error of back-calculated concentrations varied from - 14.1% to + 8.4% and from - 7.4% to + 10.5%, respectively. The immunoassay is compliant with the bioanalytical guidelines of the EMA and FDA and allows accurate and precise aldosterone determinations. As the assay can run low-volume samples, it is especially valuable for pediatric investigations.

Profile of Instrumentation Laboratory's HemosIL® AcuStar HIT-Ab(PF4-H) assay for diagnosis of heparin-induced thrombocytopenia.

PubMed

Nagler, Michael; Cuker, Adam

2017-05-01

Immunoassays play an essential role in the diagnosis of heparin-induced thrombocytopenia (HIT). The objective of this article is to review HemosIL® AcuStar HIT-Ab(PF4-H) (Instrumentation Laboratory, Bedford, MA, USA), a new chemiluminescent immunoassay for HIT. Areas covered: The authors searched the published literature for evaluation studies of HemosIL® AcuStar HIT-Ab(PF4-H) and sought information from the manufacturer. In this paper, the authors discuss the analytical principle and technical aspects of the assay; describe its diagnostic performance in validation studies; report on its reproducibility, cost-effectiveness, and regulatory status; and discuss the implications of the assay on clinical practice and means of integrating it in diagnostic pathways. HemosIL® AcuStar HIT-Ab(PF4-H) is compared with other rapid assays and widely used enzyme-linked immunoassays for the diagnosis of HIT. Expert commentary: HemosIL® AcuStar HIT-Ab(PF4-H) is automatable, can be performed 24 h per day, offers a rapid turnaround time, and appears to have favorable diagnostic accuracy, particularly at thresholds above that listed in the label. These advantages could lead to improved patient outcomes through rapid provision of results at the point of care, enhancing the accuracy of initial diagnosis.

Biological monitoring of 2,4,5-trichlorophenol (I): preparation of antibodies and development of an immunoassay using theoretical models.

PubMed

Nichkova, Mikaela; Galve, Roger; Marco, M-Pilar

2002-11-01

Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular modeling chemical studies of three potential immunizing haptens with the aim to find out the one mimicking best the target analyte. Competitive direct and indirect ELISAs have been developed after screening a battery of haptenized enzyme tracers and coating antigens, respectively. The relation between the degree of heterology of the competitor and the resulting immunoassay detectability has been investigated according to the electronic similarities of the competitor haptens with the target analyte taking in consideration their pK(a) values. These studies have been performed using theoretical and molecular modeling tools to find out their electronic distribution at their minimum energetic levels. The results suggest that the competitors should have a high homology to produced assays with good detectability values. On the other hand detectability improves when lowering the hapten density of the competitors. An indirect competitive ELISA has been finally selected for further investigation. The immunoassay has an IC(50) value of 0.6 microg L(-)(1) and a limit of detection of 0.084 microg L(-)(1). The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples. In contrast, the brominated analogues may also be recognized with this assay.

Motor Nerve Conduction Velocity In Postmenopausal Women with Peripheral Neuropathy.

PubMed

Singh, Akanksha; Asif, Naiyer; Singh, Paras Nath; Hossain, Mohd Mobarak

2016-12-01

The post-menopausal phase is characterized by a decline in the serum oestrogen and progesterone levels. This phase is also associated with higher incidence of peripheral neuropathy. To explore the relationship between the peripheral motor nerve status and serum oestrogen and progesterone levels through assessment of Motor Nerve Conduction Velocity (MNCV) in post-menopausal women with peripheral neuropathy. This cross-sectional study was conducted at Jawaharlal Nehru Medical College during 2011-2013. The study included 30 post-menopausal women with peripheral neuropathy (age: 51.4±7.9) and 30 post-menopausal women without peripheral neuropathy (control) (age: 52.5±4.9). They were compared for MNCV in median, ulnar and common peroneal nerves and serum levels of oestrogen and progesterone estimated through enzyme immunoassays. To study the relationship between hormone levels and MNCV, a stepwise linear regression analysis was done. The post-menopausal women with peripheral neuropathy had significantly lower MNCV and serum oestrogen and progesterone levels as compared to control subjects. Stepwise linear regression analysis showed oestrogen with main effect on MNCV. The findings of the present study suggest that while the post-menopausal age group is at a greater risk of peripheral neuropathy, it is the decline in the serum estrogen levels which is critical in the development of peripheral neuropathy.

Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues.

PubMed

Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso

2016-11-01

A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification

NASA Astrophysics Data System (ADS)

Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong

2014-12-01

The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development. Electronic supplementary information (ESI) available: Preparation of the chemically modified multi-walled carbon nanotubes (CNTs), characterization of the CNTs and modified CNTs, preparation of the circular probe, gel electrophoresis of the RCA products, and DNA probes as noted in the text. See DOI: 10.1039/c4nr05243a

Pilot-scale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum.

PubMed

Postemsky, P D; Bidegain, M A; González-Matute, R; Figlas, N D; Cubitto, M A

2017-05-01

Solid-state fermentation was evaluated at the pilot-scale for the bioconversion and valorization of rice husks and straw (RSH), or sunflower seed hulls (SSH), into medicinal mushrooms and crude extracts, with laccase activity. The average mushroom yield was 56kg dry weight per ton of agro-residues. Laccase activity in crude aqueous extracts showed its maximum value of 10,927Ukg -1 in RSH (day 10, Exudate phase) and 16,442Ukg -1 in SSH (day 5, Full colonization phase), the activity at the Residual substrate phase being 511Ukg -1 in RSH and 803Ukg -1 in SSH, respectively. Crude extracts obtained with various protocols revealed differences in the extraction yields. Lyophilization followed by storage at 4°C allowed the preservation of laccase activity for more than one month. It is proposed that standard mushroom farms could increase their profits by obtaining laccase as a byproduct during the gaps in mycelium running. Copyright © 2017 Elsevier Ltd. All rights reserved.

Performance of a Limiting-Antigen Avidity Enzyme Immunoassay for Cross-Sectional Estimation of HIV Incidence in the United States

PubMed Central

Konikoff, Jacob; Brookmeyer, Ron; Longosz, Andrew F.; Cousins, Matthew M.; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Kirk, Gregory D.; Moore, Richard D.; Mehta, Shruti H.; Margolick, Joseph B.; Brown, Joelle; Mayer, Kenneth H.; Koblin, Beryl A.; Justman, Jessica E.; Hodder, Sally L.; Quinn, Thomas C.; Eshleman, Susan H.; Laeyendecker, Oliver

2013-01-01

Background A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay) was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs) that included other biomarkers. Methods and Findings Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count), varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA. Conclusions The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence estimates. PMID:24386116

A bleached-kraft mill effluent fraction causing induction of a fish mixed-function oxygenase enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burnison, B.K.; Hodson, P.V.; Nuttley, D.J.

1996-09-01

Pulp mill effluents contain a myriad of chemicals that have the potential to cause deleterious effects on aquatic biota in receiving waters. Some of these chemicals evoke an acute lethal response of exposed biota while others evoke sublethal responses. One such sublethal response is the induction of mixed-function oxygenases (MFO) in fish, specifically the CYP1A1 enzyme ethoxy-resorufin-o-deethylase (EROD). Compounds causing MFO induction include congeners of polychlorinated biphenyls (PCBs), dioxins, furans, and polycyclic aromatic hydrocarbons (PAHs). The authors followed the partitioning of the inducing chemicals in pulp mill effluent fractions by Toxicity Identification Evaluation (TIE), or bioassay-driven chemical analysis. This proceduremore » was eventually modified to a more direct technique involving centrifugation, filtration, cleanup procedures, and C{sub 18} solid-phase adsorption. The extracts from the fractionation of two pulp mill effluents after secondary treatment were tested for EROD-inducing activity in a 4-d rainbow trout bioassay. The methanol extracts of particulates/colloids showed significant inducing capacity in Mill A effluent but not in Mill B effluent. The C{sub 18} methanol extracts induced activity from both effluents, with extracts from Mill A causing the greatest response. The particulate/colloidal extract (Mill A) was used as the source material for chemicals which caused EROD induction. The fraction was purified by solid-phase extraction techniques and reverse-phase high-performance liquid chromatography. The majority of the EROD activity was found in the moderately nonpolar region of the chromatogram (K{sub ow} = 4.6 to 5.1).« less

Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

PubMed

Basketter, David; Berg, Ninna; Broekhuizen, Cees; Fieldsend, Mark; Kirkwood, Sheila; Kluin, Cornelia; Mathieu, Sophie; Rodriguez, Carlos

2012-10-01

Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products. Copyright © 2012 Elsevier Inc. All rights reserved.

A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

PubMed Central

Robinet, Peggy; Wang, Zeneng; Hazen, Stanley L.; Smith, Jonathan D.

2010-01-01

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. PMID:20688754

Does the Alpha-defensin Immunoassay or the Lateral Flow Test Have Better Diagnostic Value for Periprosthetic Joint Infection? A Systematic Review.

PubMed

Eriksson, Hannah K; Nordström, Jakob; Gabrysch, Katja; Hailer, Nils P; Lazarinis, Stergios

2018-05-01

Measuring alpha-defensin concentrations in synovial fluid may help to diagnose periprosthetic joint infection (PJI). There are two commercially available methods for measuring alpha-defensin in synovial fluid: the enzyme-linked immunosorbent assay-based Synovasure® alpha-defensin immunoassay, which gives a numeric readout within 24 hours, and the Synovasure lateral flow test, which gives a binary readout within 20 minutes. There is no compilation of the existing literature to support the use of one of these two tests over the other. Does the immunoassay or the lateral flow test have better diagnostic value (sensitivity and specificity) in diagnosing PJI? We followed PRISMA guidelines and identified all studies on alpha-defensin concentration in synovial fluid as a PJI diagnostic marker, indexed to April 14, 2017, in PubMed, JSTOR, Google Scholar, and OVID databases. The search retrieved 1578 records. All prospective and retrospective studies on alpha-defensin as a PJI marker (PJI classified according to the criteria of the Musculoskeletal Infection Society) after THA or TKA were included in the analysis. All studies used only one of the two commercially available test methods, but none of them was comparative. After excluding studies with overlapping patient populations, four studies investigating the alpha-defensin immunoassay and three investigating the lateral flow test remained. Alpha-defensin immunoassay studies included 482 joints and lateral flow test studies included 119. The quality of the trials was assessed according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. The heterogeneity among studies was evaluated by the I index, indicating that the heterogeneity of the included studies was low. Pooled sensitivity, specificity, positive and negative likelihood ratios, and receiver operating curves were calculated for each method and compared with each other. The alpha-defensin immunoassay had superior overall diagnostic value compared with the lateral flow test (area under the curve, 0.98 versus 0.75) with higher sensitivity (96% [90%-98%] versus 71% [55%-83%], p < 0.001), but no difference in specificity with the numbers available (96% [93%-97%] versus 90% [81%-95%], p = 0.060). Measurement of alpha-defensin in synovial fluid is a valuable complement to existing diagnostic criteria, and the immunoassay test detects PJI more accurately than the lateral flow test. The lateral flow test has lower sensitivity, making it difficult to rule out infection, but its relatively high specificity combined with the advantage of a quick response time can make it useful to rule in infection perioperatively. Level III, diagnostic study.

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics.

PubMed

Wang, Yang; Ruan, Qingyu; Lei, Zhi-Chao; Lin, Shui-Chao; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

2018-04-17

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Comparison of the Farr radioimmunoassay, 3 commercial enzyme immunoassays and Crithidia luciliae immunofluorescence test for diagnosis and activity assessment of systemic lupus erythematosus.

PubMed

Launay, David; Schmidt, Jean; Lepers, Sébastien; Mirault, Tristan; Lambert, Marc; Kyndt, Xavier; Reumaux, Dominique; Duhamel, Alain; Hachulla, Eric; Hatron, Pierre-Yves; Prin, Lionel; Dubucquoi, Sylvain

2010-07-04

Among anti-double-strand (ds)DNA antibody assays, Farr radioimmunoassay is decreasingly used because it requires radioactive material and is labor intensive. We evaluated the performance of Farr, three commercial enzyme immunoassays (EIAs) and the Crithidia luciliae immunofluorescence test (CLIFT) in systemic lupus erythematosus (SLE). Anti-dsDNA antibodies were determined in 99 SLE patients, 101 healthy subjects, and 53 patients with autoimmune rheumatic diseases. Farr performed better than the 3 EIAs and CLIFT for the diagnosis of SLE at the manufacturer's cut off and at the cut off set to achieve a specificity of 95%. To achieve a similar level of specificity, some EIAs had a decrease in sensitivity which was dramatic for some tests. Farr was also the best at distinguishing patients with quiescent to mildly active disease from patients with more active disease at the cut off value of 93 IU/ml. Using manufacturer's cut off did not allow distinguishing between patients with quiescent and active SLE. Farr was the best global test to assess the level of anti-dsDNA antibodies for both diagnosis and disease activity evaluation in SLE with adequately determined cut off values. Some EIA had low performances limiting their use in decision-making regarding diagnosis and/or treatment. Copyright 2010 Elsevier B.V. All rights reserved.

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

PubMed Central

Khan, A S; Moe, C L; Glass, R I; Monroe, S S; Estes, M K; Chapman, L E; Jiang, X; Humphrey, C; Pon, E; Iskander, J K

1994-01-01

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis. Images PMID:8150941

Ovine echinococcosis I. Immunological diagnosis by enzyme immunoassay.

PubMed

Gatti, Antonio; Alvarez, Angela Rosa; Araya, Daniel; Mancini, Sergio; Herrero, Eduardo; Santillan, Graciela; Larrieu, Edmundo

2007-01-31

Immunodiagnosis in sheep presents problems of sensitivity and specificity, limiting its applicability in surveillance systems. The objective of this study was to develop a sensitive, specific and accessible technique for diagnosing cystic echinococcosis in naturally infected sheep and to evaluate the validity of necropsy as a reference test. A total of 247 sheep were studied at slaughterhouses, confirming the parasitological diagnosis with histology. Serum was processed with enzyme immunoassay (EIA) using three antigen preparations: total hydatid liquid (LHT), purified fraction of LHT (S2B) and purified lipoprotein (B). Western Blot (WB) was used as a control. EIA proved effective for differentiating Echinococcus granulosus from larval stage of Taenia hydatigena and intestinal cestodes in all three antigen preparations. Serums from macroscopically negative sheep were reactive to EIA and positive with WB. In the whole flock, sensitivity was 89.2% for LHT, 80.0% for S2B and 86.4% for B. Sensitivity in lambs was 78.6% for LHT, 75.0% for S2B and 64.3% for B. Macroscopic diagnosis at the time of slaughter was found to have limitations as a reference test for immunodiagnosis of cystic equinococcosis in sheep, so it was necessary to include histology and WB as reference tests. LHT was the antigen preparation of greatest value and EIA proved to be a sensitive and specific technique, adequate for surveillance systems and for evaluating control programmes.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

PubMed

Gaafar, Maha R

2011-08-01

This study was designed to evaluate the antigen capture enzyme immunoassays (EIAs) Triage parasite panel and TechLab Entamoeba histolytica II in detecting Giardia intestinalis, Cryptosporidium sp, and Entamoeba histolytica in fecal samples in comparison to microscopy, and in differentiating Entamoeba histolytica from Entamoeba dispar. The Triage EIA was evaluated using 100 stool specimens that were tested by standard ova and parasite examination, including staining with both trichrome and modified acid-fast stains. Differentiation between E. histolytica and E. dispar was performed using TechLab. Microscopic examination revealed that 19% of the samples were positive for Giardia, 4% for Cryptosporidium, and 1% for E. histolytica/E. dispar, and other parasites were found in 5%. By Triage, 23% of the samples were infected with Giardia, 5% with Cryptosporidium, and 2% with E. histolytica/E. dispar. Triage showed a sensitivity of 100% and specificity of 91.5%. The TechLab assay was negative for both samples diagnosed as E. histolytica/E. dispar by Triage, which suggested that they were E. dispar. Both tests showed no cross-reactivity with other intestinal protozoa. These results indicate that antigen detection by EIA has the potential to become a valuable tool, capable of making stool diagnostics more effective. Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Seroprevalence of cytomegalovirus infection in France in 2010.

PubMed

Antona, D; Lepoutre, A; Fonteneau, L; Baudon, C; Halftermeyer-Zhou, F; LE Strat, Y; Lévy-Bruhl, D

2017-05-01

Cytomegalovirus (CMV) infection remains the leading cause of congenital virus infection in developed countries. Measuring the national prevalence of this infection, especially among women of childbearing age, is of great value to estimate the risk of congenital CMV infection, as well as to identify risk groups that should be targeted for behavioural interventions and/or vaccination once a CMV vaccine finally becomes available. In order to fulfil these objectives, a seroprevalence survey was conducted in 2010, using a nationally representative, population-based sample of 2536 people aged between 15 and 49 years, living in metropolitan France and attending private microbiological laboratories for blood testing. All blood samples were analysed in the same laboratory and screened for CMV-specific IgG using an enzyme-linked immunoassay technique (Elisa PKS Medac Enzyme immunoassay). The overall point estimate of CMV infection seroprevalence for individuals aged 15-49 years was 41.9%. The estimates were higher in women than in men (respectively 45.6% and 39.3%), and people born in a non-Western country were more likely to be CMV seropositive than those born in France or in another Western country (93.7% vs. 37.7%). Our results showed that a substantial percentage of women of childbearing age in France are CMV seronegative and therefore at risk of primary CMV infection during pregnancy. Educational measures and future vaccine are key issues to prevent infection in pregnant women and congenital CMV disease.

Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

PubMed Central

Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

2015-01-01

Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

Clostridium difficile infection diagnostics - evaluation of the C. DIFF Quik Chek Complete assay, a rapid enzyme immunoassay for detection of toxigenic C. difficile in clinical stool samples.

PubMed

Johansson, Karin; Karlsson, Hanna; Norén, Torbjörn

2016-11-01

Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician. © 2016 APMIS. Published by John Wiley & Sons Ltd.

[A system review on the application of BED-capture enzyme immunoassay in detecting new HIV-1 infection].

PubMed

Tao, Jun; Zhao, Jing; Liu, Yong; Meng, Ling-zhang; Yu, Shi-cheng; Jiang, Yan; Xiao, Yao

2011-02-01

To study whether BED-capture enzyme immunoassay (CEIA) is feasible to be used in wide-ranging population, we collect papers and conference abstracts related to BED-CEIA and HIV-1 incidence. 10 papers are included for the discussion, regarding the concordance between the estimated HIV-1 incidence from BED-CEIA and the results from a cohort studies; and 11 papers are selected to discuss the related misclassification on the estimation of HIV-1 incidence. Concordance between the two sets is related to the districts and design of research. Results from Africa are not so satisfactory, but researches, those BED-CEIA samples are collected during the follow-up of the cohort study, have shown better outcomes than other ones. There are totally 7303 samples of LTI (long-term infections) collected for analyzing the misclassification of BED-CEIA. 432 LTI are misclassified as new infections, making the raw rate of misclassification as 5.9% with 95% confidential interval between 5.36 and 6.44. Data from systematic review shows that the BED-CEIA's misclassification rate relates to the count of CD4(+)T lymphocytes and time after the infection but has no relation to the classification of sub-populations (female sexual workers and intravenous drug users in China) and districts (China and Africa). Our results reveal that the misclassification is relevant to the immune-status of the infected persons.

A non-toxic enzyme-linked immunosorbent assay for aflatoxin B1 using anti-idiotypic antibodies as substitutes.

PubMed

Hu, Li; Liu, Aiping; Chen, Weifeng; Yang, Hongxiu; Wang, Xiaohong; Chen, Fusheng

2017-03-01

Immunoassays are widely employed techniques to detect aflatoxins since they are rapid, selective and sensitive. One common disadvantage of them is using aflatoxins as standard substances, which may trigger exposure risks to operators and environmental contamination without proper handling. Anti-idiotypic antibodies (anti-Ids or Ab2s), also named as internal-image anti-Ids, are able to mimic and function as antigens, so a non-toxic enzyme-linked immunosorbent assay (ELISA) for aflatoxin B 1 (AFB 1 ) is developed and validated using anti-Ids as substitutes. Mouse monoclonal anti-idiotypic antibody (McAb2) to AFB 1 was generated by the hybridoma technique using Fab fragments of rabbit anti-AFB 1 idiotype antibody (Ab1) as immunogen. As indicated by indirect competitive ELISA, McAb2, represented an internal-image of antigen AFB 1 , was able to bind Fab with competition to AFB 1 . Then, analysis of AFB 1 in spiked samples by non-toxic ELISA using anti-Ids as substitutes was developed, and it showed no significant differences with comparison to AFB 1 as competitive antigens. Our work demonstrated that anti-Ids could be used as internal-image mimicry of AFB 1 , and it had potential applications in immunoassays for antigen substitution to reduce operational risk for operators and environmental contamination. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard.

PubMed

Wang, Yanru; Li, Peiwu; Zhang, Qi; Hu, Xiaofeng; Zhang, Wen

2016-09-01

A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2-5 as surrogate standard. Anti-idiotype nanobody VHH 2-5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2-5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL(-1), which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R (2) = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds. Graphical Abstract ᅟ.

Reagentless chemiluminescence-based fiber optic sensors for regenerative life support in space

NASA Astrophysics Data System (ADS)

Atwater, James E.; Akse, James R.; DeHart, Jeffrey; Wheeler, Richard R., Jr.

1995-04-01

The initial feasibility demonstration of a reagentless chemiluminescence based fiber optic sensor technology for use in advanced regenerative life support applications in space and planetary outposts is described. The primary constraints for extraterrestrial deployment of any technology are compatibility with microgravity and hypogravity environments; minimal size, weight, and power consumption; and minimal use of expendables due to the great expense and difficulty inherent to resupply logistics. In the current research, we report the integration of solid state flow through modules for the production of aqueous phase reagents into an integrated system for the detection of important analytes by chemiluminescence, with fiber optic light transmission. By minimizing the need for resupply expendables, the use of solid phase modules makes complex chemical detection schemes practical. For the proof of concept, hydrogen peroxide and glucose were chosen as analytes. The reaction is catalyzed by glucose oxidase, an immobilized enzyme. The aqueous phase chemistry required for sensor operation is implemented using solid phase modules which adjust the pH of the influent stream, catalyze the oxidation of analyte, and provide the controlled addition of the luminophore to the flowing aqueous stream. Precise control of the pH has proven essential for the long-term sustained release of the luminophore. Electrocatalysis is achieved using a controlled potential across gold mesh and gold foil electrodes which undergo periodic polarity reversals. The development and initial characterization of performance of the reagentless fiber optic chemiluminescence sensors are presented in this paper.

Antenatal screening for Toxoplasma gondii, Cytomegalovirus, rubella and Treponema pallidum infections in northern Benin.

PubMed

De Paschale, Massimo; Ceriani, Cristina; Cerulli, Teresa; Cagnin, Debora; Cavallari, Serena; Cianflone, Annalisa; Diombo, Kouma; Ndayaké, Joseph; Aouanou, Guy; Zaongo, Dieudonné; Priuli, Gianbattista; Viganò, Paolo; Clerici, Pierangelo

2014-06-01

Toxoplasma gondii, cytomegalovirus (HCMV) and rubella virus infections are among the most serious of those contracted during pregnancy in terms of foetal consequences. Toxoplasma, HCMV and rubella antibody screening is unusual in Africa, and there are few published data. The aim of this study was to evaluate the prevalence of these markers among pregnant women in northern Benin on the occasion of routine syphilis screening. Toxoplasma, HCMV and rubella IgG and IgM antibodies were determined in the serum of 283 women attending Saint Jean de Dieu de Tanguiéta hospital, using an enzyme immunoassay, and IgM were confirmed using an enzyme-linked fluorescent assay (ELFA). In the case of IgM positivity, the avidity of anti-HCMV and anti-Toxoplasma IgG was measured. Total anti-Treponema pallidum antibodies were determined using an enzyme immunoassay and confirmed by immunoblotting. In the case of positivity, the Venereal Disease Research Laboratory (VDRL) test was used. The prevalence of anti-Toxoplasma, anti-HCMV, anti-rubella IgG and total anti-Treponema antibodies was, respectively, 30.0%, 100%, 94% and 2.5%. The VDRL test was positive in 62.5% of the anti-Treponema-positive samples. The prevalence of anti-Toxoplasma, anti-HCMV and anti-rubella IgM was, respectively, 0.4%, 1.4% and 0%. There were no statistically significant differences in terms of age class or trimester of pregnancy. Anti-Toxoplasma and anti-HCMV IgG avidity was always high. The prevalence of HCMV and rubella antibodies is high in northern Benin, whereas that of Toxoplasma antibodies is lower. As nearly two-thirds of the pregnant women were anti-Toxoplasma seronegative, antibody screening should be introduced. © 2014 John Wiley & Sons Ltd.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate).

PubMed

Nascimento, Thiago Pajeú; Sales, Amanda Emmanuelle; Porto, Camila Souza; Brandão, Romero Marcos Pedrosa; de Campos-Takaki, Galba Maria; Teixeira, José Antônio Couto; Porto, Tatiana Souza; Porto, Ana Lúcia Figueiredo; Converti, Attilio

2016-07-01

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification. Copyright © 2016 Elsevier B.V. All rights reserved.

Implementation of a rapid HIT immunoassay at a university hospital - Retrospective analysis of HIT laboratory orders in patients with thrombocytopenia.

PubMed

Black, Anne; Heimerl, Susanne; Oertli, Linnéa; Wilczek, Wolf; Greinacher, Andreas; Spannagl, Michael; Herr, Wolfgang; Hart, Christina

2017-10-01

Heparin-induced thrombocytopenia (HIT) is a rare cause of thrombocytopenia and a potentially life-threatening adverse drug reaction. Clinical overdiagnosis of HIT results in costly laboratory tests and anticoagulation. Criteria and algorithms for diagnosis are established, but their translation into clinical practice is still challenging. In a retrospective approach we studied all HIT related laboratory test requests within four years and evaluated data before (1st period, 24month) and after (2nd period, 24month) replacing particle gel immunoassay (PaGIA) and enzyme-linked immunosorbent assay (ELISA) by a chemiluminescent immunoassay (CLIA). HIT was confirmed by heparin-induced platelet activation (HIPA) test. Clinical pretest probability for HIT using an implemented simplified 4Ts score and platelet count were evaluated. Costs for laboratory tests and alternative anticoagulation were calculated. In 1850 patients with suspected HIT, 2327 laboratory orders were performed. In 87.2% of these orders an intermediate/high simplified 4Ts score was found. Thrombocytopenia was present in 87.1%. After replacing PaGIA and ELISA by CLIA the number of immunological and functional laboratory tests was reduced by 38.2%. The number of positive HIT immunoassays declined from 22.6% to 6.0%, while the number of positive HIPA tests among positive immunological tests increased by 19%. Altogether, acute HIT was confirmed in 59 patients. A decline in the use of alternative anticoagulants was observed in the 2nd period. Our study shows that in a university hospital setting HIT is well-known, but diagnosis requires a precise laboratory confirmation. Replacing PaGIA and ELISA by CLIA did not influence laboratory order behavior but results in reduced overall costs for laboratory diagnostics and alternative anticoagulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

PubMed

Okamoto-Uchida, Yoshimi; Nakamura, Ryosuke; Matsuzawa, Yumiko; Soma, Megumi; Kawakami, Hiroshi; Ishii-Watabe, Akiko; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Saito, Yoshiro

2016-01-01

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

Self-organization of glucose oxidase-polymer surfactant nanoconstructs in solvent-free soft solids and liquids.

PubMed

Sharma, Kamendra P; Zhang, Yixiong; Thomas, Michael R; Brogan, Alex P S; Perriman, Adam W; Mann, Stephen

2014-10-02

An anisotropic glucose oxidase-polymer surfactant nanoconjugate is synthesized and shown to exhibit complex temperature-dependent phase behavior in the solvent-free state. At close to room temperature, the nanoconjugate crystallizes as a mesolamellar soft solid with an expanded interlayer spacing of ca. 12 nm and interchain correlation lengths consistent with alkyl tail-tail and PEO-PEO ordering. The soft solid displays a birefringent spherulitic texture and melts at 40 °C to produce a solvent-free liquid protein without loss of enzyme secondary structure. The nanoconjugate melt exhibits a birefringent dendritic texture below the conformation transition temperature (Tc) of glucose oxidase (58 °C) and retains interchain PEO-PEO ordering. Our results indicate that the shape anisotropy of the protein-polymer surfactant globular building block plays a key role in directing mesolamellar formation in the solvent-free solid and suggests that the microstructure observed in the solvent-free liquid protein below Tc is associated with restrictions in the intramolecular motions of the protein core of the nanoconjugate.

Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay

PubMed Central

Logue, James; Tuznik, Kaylie; Follmann, Dean; Grandits, Greg; Marchand, Jonathan; Reilly, Cavan; Sarro, Yeya dit Sadio; Pettitt, James; Stavale, Eric J.; Fallah, Mosoka; Olinger, Gene G.; Bolay, Fatorma K.; Hensley, Lisa E.

2018-01-01

As part of the scientific community’s development of medical countermeasures against Ebola virus disease, optimization of standardized assays for product evaluation is paramount. The recent outbreak heightened awareness to the scarcity of available assays and limited information on performance and reproducibility. To evaluate the immunogenicity of vaccines entering Phase I–III trials and to identify survivors, two enzyme-linked immunosorbent assays, the Filovirus Animal Non-Clinical Group assay and the Alpha Diagnostics International assay, were evaluated for detection of immunoglobulin G against Ebola virus glycoprotein. We found that the Filovirus Animal Nonclinical Group assay produced a wider range of relative antibody concentrations, higher assay precision, larger relative accuracy range, and lower regional background. Additionally, to sufficiently power a vaccine trial, use of the Filovirus Animal Nonclinical Group assay would require one third the number of participants than the Alpha Diagnostics International assay. This reduction in needed study participants will require less money, fewer man hours, and much less time to evaluate vaccine immunogenicity. PMID:29481881

Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes.

PubMed

He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

2017-07-15

Bovine β-lactoglobulin (BLG) is the main allergen in cows' milk, and the most commonly used method for detecting BLG is enzyme-linked immunosorbent assay (ELISA). However, antibodies used in commercial ELISA kits do not recognize specifically BLG IgE epitopes. Here, an antibody specific to IgE linear epitopes for BLG was used to develop a sandwich ELISA using a rabbit anti-BLG polyclonal antibody. The linear range for BLG detection was 31.25-8000ng/mL and limit of detection was 1.96ng/mL. BLG content in dairy samples was determined, and there was a good agreement between this immunoassay and reversed-phase high-performance liquid chromatography with high recovery. Additionally, BLG content in food samples had an average recovery of 104.25%. Allergenic residues were also detected in hydrolyzed infant formulas. The method developed could be a practical approach to determine BLG and its allergenic residues in food with a high degree of sensitivity, reliability and recovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methodological issues in assessing plasma 25-hydroxyvitamin D concentration in newborn infants.

PubMed

Gallo, Sina; Comeau, Kathryn; Agellon, Sherry; Vanstone, Catherine; Sharma, Atul; Jones, Glenville; L'abbé, Mary; Khamessan, Ali; Weiler, Hope; Rodd, Celia

2014-04-01

Although no gold standard exists, liquid chromatography tandem mass spectrometry (LC-MS/MS) is a precise and accurate method for the analysis of plasma 25-hydroxyvitamin D (25(OH)D). Immunoassays are more readily available and require small volume sampling, ideal for infant testing. The objective was to compare two commercially available immunoassays for measuring circulating 25(OH)D concentration in infant plasma against LC-MS/MS. Capillary blood samples from 103 infants were analyzed for plasma 25(OH)D using an enzyme immunoassay (EIA, Octeia, IDS Ltd.) and radioimmunoassay (RIA, DiaSorin). Plasma 25(OH)D(3), C-3 epimer of 25(OH)D(3) (3-epi-25(OH)D(3)) and 24,25-dihydroxyvitamin D (24,25(OH)(2)D(3)) were measured on the same samples using LC-MS/MS. To establish whether plasma 24,25(OH)(2)D(3) or 3-epi-25(OH)D(3) interferes with these immunoassay results, the zero 25(OH)D calibrator from each assay kit was spiked with increasing amounts of 24,25(OH)(2)D(3) or 3-epi-25(OH)D(3). Classifying infants below the common vitamin D status targets of 50 nmol/L and 75 nmol/L respectively, 58% and 99% fell below using the RIA, 19% and 56% with the EIA and 31% and 76% with LC-MS/MS. Compared to LC-MS/MS, both immunoassays showed poor Bland-Altman limits of agreement for 25(OH)D concentrations (RIA: limits of agreement -27 to +13%; EIA: -12 to +41%), and mountain plots (folded cumulative distribution) depicted significant skew and bias. Spiked 24,25(OH)2D3 concentrations, but not 3-epi-25(OH)D3, appeared as >100% of known values on the EIA but not on the RIA thus, suggesting that the EIA may cross-react with 24,25(OH)(2)D(3) to a greater extent than 3-epi-25(OH)D(3). Two common immunoassays resulted in very different classifications of vitamin D status possibly related to the interference of other vitamin D metabolites. Based on these data, LC-MS/MS assessment of vitamin D status is recommended in young infants (4-6 weeks of age). Copyright © 2014 Elsevier Inc. All rights reserved.

A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

PubMed

Takahashi, Nozomi; Harada, Miyuki; Hirota, Yasushi; Zhao, Lin; Azhary, Jerilee M K; Yoshino, Osamu; Izumi, Gentaro; Hirata, Tetsuya; Koga, Kaori; Wada-Hiraike, Osamu; Fujii, Tomoyuki; Osuga, Yutaka

2017-01-01

Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells. Copyright © 2017 by the Endocrine Society.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

PubMed

Spieler, Valerie; Valldorf, Bernhard; Maaß, Franziska; Kleinschek, Alexander; Hüttenhain, Stefan H; Kolmar, Harald

2016-07-01

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electron spin resonance spectroscopy for immunoassay using iron oxide nanoparticles as probe.

PubMed

Jiang, Jia; Tian, Sizhu; Wang, Kun; Wang, Yang; Zang, Shuang; Yu, Aimin; Zhang, Ziwei

2018-02-01

With the help of iron oxide nanoparticles, electron spin resonance spectroscopy (ESR) was applied to immunoassay. Iron oxide nanoparticles were used as the ESR probe in order to achieve an amplification of the signal resulting from the large amount of Fe 3+ ion enclosed in each nanoparticle. Rabbit IgG was used as antigen to test this method. Polyclonal antibody of rabbit IgG was used as antibody to detect the antigen. Iron oxide nanoparticle with a diameter of either 10 or 30 nm was labeled to the antibody, and Fe 3+ in the nanoparticle was probed for ESR signal. The sepharose beads were used as solid phase to which rabbit IgG was conjugated. The nanoparticle-labeled antibody was first added in the sample containing antigen, and the antigen-conjugated sepharose beads were then added into the sample. The nanoparticle-labeled antibody bound to the antigen on sepharose beads was separated from the sample by centrifugation and measured. We found that the detection ranges of the antigen obtained with nanoparticles of different sizes were different because the amount of antibody on nanoparticles of 10 nm was about one order of magnitude higher than that on nanoparticles of 30 nm. When 10 nm nanoparticle was used as probe, the upper limit of detection was 40.00 μg mL -1 , and the analytical sensitivity was 1.81 μg mL -1 . When 30 nm nanoparticle was used, the upper limit of detection was 3.00 μg mL -1 , and the sensitivity was 0.014 and 0.13 μg mL -1 depending on the ratio of nanoparticle to antibody. Graphical abstract Schematic diagram of procedure and ESR spectra.

A solid-phase assay for studying direct binding of progranulin to TNFR and progranulin antagonism of TNF/TNFR interactions.

PubMed

Tian, Qingyun; Zhao, Shuai; Liu, Chuanju

2014-01-01

The discovery that TNF receptors (TNFR) serve as the binding receptors for progranulin (PGRN) reveals the significant role of PGRN in inflammatory and autoimmune diseases, including inflammatory arthritis. Herein we describe a simple, antibody-free analytical assay, i.e., a biotin-based solid-phase binding assay, to examine the direct interaction of PGRN/TNFR and the PGRN inhibition of TNF/TNFR interactions. Briefly, a 96-well high-binding microplate is first coated with the first protein (protein A), and after blocking, the coated microplate is incubated with the biotin-labeled second protein (protein B) in the absence or presence of the third protein (protein C). Finally the streptavidin conjugated with a detecting enzyme is added, followed by a signal measurement. Also discussed in this chapter are the advantages of the strategy, key elements to obtain reliable results, and discrepancies among various PGRN proteins in view of the binding activity with TNFR.

Change in enzyme production by gradually drying culture substrate during solid-state fermentation.

PubMed

Ito, Kazunari; Gomi, Katsuya; Kariyama, Masahiro; Miyake, Tsuyoshi

2015-06-01

The influence of drying the culture substrate during solid-state fermentation on enzyme production was investigated using a non-airflow box. The drying caused a significant increase in enzyme production, while the mycelium content decreased slightly. This suggests that changes in the water content in the substrate during culture affect enzyme production in fungi. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

[Effect of haw leaf extract and its preparation on polymorphonuclear leucocyte adhesion during HUVEC anoxia/reoxygenation injury].

PubMed

Li, Peng; Fu, Jian-hua; Li, Xin-zhi

2008-08-01

To study the effect and molecular mechanism of two haw leaf extracts, Vitexin-rhamnoside (VR) and Vitexin-glucoside (VG), and their preparation, Aoshaen injection (AI), on the polymorphonuclear leucocyte (PMN) adhesion during human umbilical vein endothelial cell (HUVEC) anoxia/reoxygenation (A/R) injury. The cell model of A/R injury duplicated by breaking off the oxygen supplying of HUVEC for 60 min followed with reoxygenating for 30 min (phase 1) or 240 min (phase 2) was taken as the experimental objective. The effects of testing drugs (VR, VG and AI) on PMN adhesion in the model cells were measured by enzyme immunoassay, and their effects on PMN superficial adhesion molecule CD11/CD18 expression were measured by flow cytometer respectively. After 60 min of anoxia, HUVEC was shrunk and deformed. The adhesion between PMN and HUVEC significantly revealed at phase 1 in the model group, but it was fewer in the normal cell group, and also lesser in the groups treated with various drugs. The condition of cell adhesion revealed at phase 2 was the similar to that at phase 1. All testing drugs, VR, VG and AI, showed inhibitory effect on the cell adhesion at either phase 1 or phase 2, showing a certain dose-effect relationship. The expression of CD11/ CD18 was also inhibited by the testing drugs, and a good dose-effect relation was shown by VG and AI. At the resting condition, there are almost no expression of CD11/CD18 molecule, but it could be enhanced by incubating PMN with supernate of A/R injured HUVEC culture, and more marked at phase 1. Adding the test drugs into the supernate could inhibit the enhancing of CD11/CD18 molecule expression and reduce the PMN-HUVEC adhesion, which may be one of the molecular mechanisms of haw leaf extracts and their preparation in protecting heart against A/R injury.

Molecular Diagnosis in Autoimmune Skin Blistering Conditions

PubMed Central

Otten, J.V.; Hashimoto, T.; Hertl, M.; Payne, A.S.; Sitaru, C.

2014-01-01

Blister formation in skin and mucous membranes results from a loss of cell-cell or cell-matrix adhesion and is a common outcome of pathological events in a variety of conditions, including autoimmune and genetic diseases, viral and bacterial infections, or injury by physical and chemical factors. Autoantibodies against structural components maintaining cell-cell and cell-matrix adhesion induce tissue damage in autoimmune blistering diseases. Detection of these autoantibodies either tissue-bound or circulating in serum is essential to diagnose the autoimmune nature of disease. Various immunofluorescence methods as well as molecular immunoassays, including enzyme-linked immunosorbent assay and immunoblotting, belong to the modern diagnostic algorithms for these disorders. There is still a considerable need to increase awareness of the rare autoimmune blistering diseases, which often show a severe, chronic-relapsing course, among physicians and the public. This review article describes the immunopathological features of autoimmune bullous diseases and the molecular immunoassays currently available for their diagnosis and monitoring. PMID:24160488

A new paper-based platform technology for point-of-care diagnostics.

PubMed

Gerbers, Roman; Foellscher, Wilke; Chen, Hong; Anagnostopoulos, Constantine; Faghri, Mohammad

2014-10-21

Currently, the Lateral flow Immunoassays (LFIAs) are not able to perform complex multi-step immunodetection tests because of their inability to introduce multiple reagents in a controlled manner to the detection area autonomously. In this research, a point-of-care (POC) paper-based lateral flow immunosensor was developed incorporating a novel microfluidic valve technology. Layers of paper and tape were used to create a three-dimensional structure to form the fluidic network. Unlike the existing LFIAs, multiple directional valves are embedded in the test strip layers to control the order and the timing of mixing for the sample and multiple reagents. In this paper, we report a four-valve device which autonomously directs three different fluids to flow sequentially over the detection area. As proof of concept, a three-step alkaline phosphatase based Enzyme-Linked ImmunoSorbent Assay (ELISA) protocol with Rabbit IgG as the model analyte was conducted to prove the suitability of the device for immunoassays. The detection limit of about 4.8 fm was obtained.

A retrospective analysis of sera collected by the Hemorrhagic Fever Commission during the Korean Conflict.

PubMed

LeDuc, J W; Ksiazek, T G; Rossi, C A; Dalrymple, J M

1990-11-01

More than 600 sera from 245 patients with a clinical diagnosis of hemorrhagic fever were preserved by the Hemorrhagic Fever Commission during the Korean Conflict, 1951-1954. These sera were tested for IgM- and IgG-specific antibodies to Hantaan virus by enzyme immunoassay and for hantaviral antigen by immunoassay; one serum from each patient was tested by plaque reduction neutralization using both Hantaan and Seoul viruses. Only 15 patients failed to develop antihantaviral antibodies; most sera contained high titered IgM antibody on admission, and all were IgM-seropositive by day 7 after onset. Attempts to detect hantaviral antigen were unsuccessful. All seropositive patients had highest plaque reduction neutralization titers to Hantaan virus, suggesting that this virus was responsible for the disease seen. These results confirm that hemorrhagic fever of the Korean Conflict was due to Hantaan virus and demonstrate that measurement of specific IgM antibody is the method of choice for diagnosis of acute disease.

Detection of Serum microRNAs From Department of Defense Serum Repository

PubMed Central

Woeller, Collynn F.; Thatcher, Thomas H.; Van Twisk, Daniel; Pollock, Stephen J.; Croasdell, Amanda; Kim, Nina; Hopke, Philip K.; Xia, Xiaoyan; Thakar, Juilee; Mallon, COL Timothy M.; Utell, Mark J.; Phipps, Richard P.

2017-01-01

Objective The aim of this study was to investigate whether serum samples from the Department of Defense Serum Repository (DoDSR) are of sufficient quality to detect microRNAs (miRNAs), cytokines, immunoglobulin E (IgE), and polycyclic aromatic hydrocarbons (PAHs). Methods MiRNAs were isolated and quantified by polymerase chain reaction (PCR) array. Cytokines and chemokines related to inflammation were measured using multiplex immunoassays. Cotinine and IgE were detected by enzyme-linked immunoassay (ELISA) and PAHs were detected by Liquid Chromatography/Mass Spectroscopy. Results We detected miRNAs, cytokines, IgE, and PAHs with high sensitivity. Eleven of 30 samples tested positive for cotinine suggesting tobacco exposure. Significant associations between serum cotinine, cytokine, IgE, PAHs, and miRNA were discovered. Conclusion We successfully quantified over 200 potential biomarkers of occupational exposure from DoDSR samples. The stored serum samples were not affected by hemolysis and represent a powerful tool for biomarker discovery and analysis in retrospective studies. PMID:27501106

Protein detection using biobarcodes.

PubMed

Müller, Uwe R

2006-10-01

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.

Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

PubMed Central

Kong, Na; Song, Shanshan; Peng, Juan; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

2015-01-01

An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed. PMID:25938198

Effect of enzymes on anaerobic digestion of primary sludge and septic tank performance.

PubMed

Diak, James; Örmeci, Banu; Kennedy, Kevin J

2012-11-01

Enzyme additives are believed to improve septic tank performance by increasing the hydrolysis and digestion rates and maintaining a healthy microbial population. Previous studies reported mixed results on the effectiveness of enzymes on mesophilic and thermophilic digestion, and it is not clear whether enzymes would be effective under septic tank conditions where there is no heating or mixing, quantities of enzymes added are small, and they can be washed out quickly. In this study, batch reactors and continuous-flow reactors designed and operated as septic tanks were used to evaluate whether enzymatic treatment would increase the hydrolysis and digestion rates in primary sludge. Total solids, volatile solids, total suspended solids, total and soluble chemical oxygen demand, concentrations of protein, carbohydrate, ammonia and volatile acids in sludge and effluent samples were measured to determine the differences in digestion rates in the presence and absence of enzymes. Overall, no significant improvement was observed in enzyme-treated reactors compared with the control reactors.

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Ku??tzing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L-1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method. ?? Springer 2005.

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Kützing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L−1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method.

Preparation of peanut butter suspension for determination of peanuts using enzyme-linked immunoassay kits.

PubMed

Trucksess, Mary W; Brewer, Vickery A; Williams, Kristina M; Westphal, Carmen D; Heeres, James T

2004-01-01

Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 microg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 microg/g.

Evaluation of Non-Lethal Effects of N2 Bubbles on Marine Mammal Health and the Potential Role of Immune Activity in Facilitating the Development of Dive Related Injury

DTIC Science & Technology

2015-09-30

control for confounding effects of changing physiological status. Cortisol will be measured using a commercial enzyme immunoassay ( EIA ) and plasma...complement activity in plasma and serum has been tested using zymosan (Sigma Aldrich) which is a known activator of complement. Validation of the EIA ...expected to be underway by November 2015. Data collection for objective 3 will occur during project year 2. RESULTS EIA kits for seal

Immunoassays for Identification of Biological Agents in Sample Unknowns: NATO SlBCA Exercise VI

DTIC Science & Technology

2005-12-01

Yersiniapestis 103 cfu/mL Enzyme-linked immunosorbent assays Antibodies Unlabelled antibodies Antibody stocks developed under DRES contract by SciLab Consulting...goat anti-rabbit IgG, (whole molecule, lot no. 90H8990). Antibody purification Antibodies produced by Scilab Consulting Inc. were purified on a Bio...No. W7702-4-R430, Final Report. Scilab Consulting Inc. DRDC Suffield TM 2005-223 17 14. Fulton, R.E. and Thompson, H.G. Evaluation of the Rapid

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

Acquisition of Heat Stable Enzymes from Thermophilic Microorganisms: Peroxidases, Ureases, and Glucose Oxidases.

DTIC Science & Technology

1992-04-01

foetus antibody (approx. 43.8 mg/ mL) (Nakane). After conjugation, unconjugated antibody and urease were separated from the IgG-Urease conjugate by...using fixed T. foetus organisms were incubated in 10 mM citrate buffer, pH 7, as a control or IgG-Urease in the same citrate buffer for 15 min. Organisms...Table 15. T. foetus - Anti T. foetus Model Immunoassay Relative Urease Activity Reaction (Absofbances @590 nm) T. foetus + Buffer Control 0.501 IgG-Urease

Risks Associated with Fresh Whole Blood and Red Blood Cell Transfusions in a Combat Support Hospital

DTIC Science & Technology

2007-01-01

collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 2007 2 . REPORT TYPE 3 . DATES COVERED 00-00-2007...coagulation function [13, 14] Increased RBC aggregation [28] Increased free hemoglobin [29] Decreased 2 , 3 DPG and tissue perfusion [30] Decreased oxygen...0.07%) were positive for human lymphotropic virus enzyme immunoassay, and none of 2,831 were positive for both human immunodeficiency virus 1/ 2 and

Optical fiber-based biosensors.

PubMed

Monk, David J; Walt, David R

2004-08-01

This review outlines optical fiber-based biosensor research from January 2001 through September 2003 and was written to complement the previous review in this journal by Marazuela and Moreno-Bondi. Optical fiber-based biosensors combine the use of a biological recognition element with an optical fiber or optical fiber bundle. They are classified by the nature of the biological recognition element used for sensing: enzyme, antibody/antigen (immunoassay), nucleic acid, whole cell, and biomimetic, and may be used for a variety of analytes ranging from metals and chemicals to physiological materials.

Possible Mechanism for Denervation Effect on Wound Healing.

DTIC Science & Technology

1990-12-14

investigation was the regenerating limb of the axolotl , in which growth is strictly dependent on unknown factors from peripheral nerves. The rationale of the...that axons transport transferrin to cells of the regenerating tissues. Before experiments of this nature could be undertaken, axolotl transferrin and...other tissues from axolotls . These goals were accomplished in the first phase of the project and during the last phase the immunoassay was used to

Investigation on the Influence of Bio-catalytic Enzyme Produced from Fruit and Vegetable Waste on Palm Oil Mill Effluent

NASA Astrophysics Data System (ADS)

Rasit, Nazaitulshila; Chee Kuan, Ooi

2018-04-01

Pre-consumer waste from supermarkets, such as vegetables and fruits dreg are always discarded as solid waste and disposed to landfill. Implementing waste recovery method as a form of waste management strategy will reduce the amount of waste disposed. One of the ways to achieve this goal is through fermentation of the pre-consumer supermarket waste to produce a solution known as garbage enzyme. This study has been conducted to produce and characterize biocatalytic garbage enzyme and to evaluate its influence on palm oil mill effluent as a pre-treatment process before further biological process takes place. Garbage enzyme was produced by three-month long fermentation of a mixture of molasses, pre-consumer supermarket residues, and water in the ratio of 1:3:10. Subsequently, the characterization of enzyme was conducted based on pH, total solids (TS), total suspended solids (TSS), total dissolved solids (TDS), chemical oxygen demand (COD), and enzyme activities. The influence of produced enzyme was evaluated on oil & grease (O&G), TSS and COD of palm oil mill effluent (POME). Different levels of dilution of garbage enzyme to POME samples (5%, 10%, 15%) were explored as pre-treatment (duration of six days) and the results showed that the garbage enzyme contained bio-catalytic enzyme such as amylase, protease, and lipase. The pre-treatment showed removal of 90% of O&G in 15% dilution of garbage enzyme. Meanwhile, reduction of TSS and COD in dilution of 10% garbage enzyme were measured at 50% and 25% respectively. The findings of this study are important to analyse the effectiveness of pre-treatment for further improvement of anaerobic treatment process of POME, especially during hydrolysis stage.

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system.

PubMed

de Albuquerque Wanderley, Maria Carolina; Wanderley Duarte Neto, José Manoel; Campos Albuquerque, Wendell Wagner; de Araújo Viana Marques, Daniela; de Albuquerque Lima, Carolina; da Cruz Silvério, Sara Isabel; de Lima Filho, José Luiz; Couto Teixeira, José António; Porto, Ana Lúcia Figueiredo

2017-05-01

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method. Copyright © 2017 Elsevier Inc. All rights reserved.

Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports.

PubMed

Barig, Susann; Funke, Andreas; Merseburg, Andrea; Schnitzlein, Klaus; Stahmann, K-Peter

2014-06-10

Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports. As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5'-phosphate binding homotetramer with a mass of 180kDa. The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values. Copyright © 2014 Elsevier Inc. All rights reserved.

Concomitant production of cellulase and xylanase by thermophilic mould Sporotrichum thermophile in solid state fermentation and their applicability in bread making.

PubMed

Bala, Anju; Singh, Bijender

2017-06-01

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45 °C, pH 5.0 after 72 h inoculated with 2.9 × 10 7  CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.

An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

PubMed Central

Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

2014-01-01

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

Ultrasensitive fluorescence immunoassay for detection of ochratoxin A using catalase-mediated fluorescence quenching of CdTe QDs

NASA Astrophysics Data System (ADS)

Huang, Xiaolin; Zhan, Shengnan; Xu, Hengyi; Meng, Xianwei; Xiong, Yonghua; Chen, Xiaoyuan

2016-04-01

Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring.Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01136e

Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse.

PubMed

Zhang, Teng; Zhu, Ming-Jun

2017-04-01

A study on the fed-batch simultaneous saccharification and co-fermentation (SSCF) of Fenton reaction combined with NaOH pretreated sugarcane bagasse (SCB) at a high solid loading of 10-30% (w/v) was investigated. Enzyme feeding mode, substrate feeding mode and combination of both were compared with the batch mode under respective solid loadings. Ethanol concentrations of above 80g/L were obtained in batch and enzyme feeding modes at a solid loading of 30% (w/v). Enzyme feeding mode was found to increase ethanol productivity and reduce enzyme loading to a value of 1.23g/L/h and 9FPU/g substrate, respectively. The present study provides an economically feasible process for high concentration bioethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Hepatitis C prevalence and the significance of liver enzyme elevations in the insurance population.

PubMed

Stout, R L

1997-01-01

Liver enzyme elevation(s) are a common finding in the insurance applicant population. Hepatitis C infection results in histological and functional changes in the liver with both short and long term changes in serum liver enzyme levels. The prevalence of antibodies to HCV in the general population is estimated to be 4%. This paper reports on the prevalence of antibodies to HCV in the insurance applicant population and their relationship to the liver enzyme(s). Antibodies to HCV are present in 1.8% of a random sampling of insurance applicants. Alanine aminotransferase (ALT) elevations occur in 95.4% of all samples positive for antibodies to HCV. More than half of positive samples (56.7%) have ALT elevations of less than two time the upper range of normal. Antibody prevalence is lowest in samples with single enzyme elevation, 4.2%. In comparison, the prevalence is 16.4% in samples with all three enzymes, ALT, AST, and GGT, elevated. For maximal specificity two immunoassays, configured with different HCV antigens, should be performed sequentially on all positive applicant samples. HCV is the most prevalent, chronic viral infection in the insurance population. HCV prevalence is 40 times HIV prevalence. In an evaluation of enzyme reflex markers ALT was positive for antibodies to HCV 8.6% of the time while identifying 95.4% of HCV antibody positive applicants.

Rapid immunoassays for diagnosis of heparin-induced thrombocytopenia: Comparison of diagnostic accuracy, reproducibility, and costs in clinical practice

PubMed Central

Bankova, Andriyana; Andres, Yvonne; Horn, Michael P.; Alberio, Lorenzo

2017-01-01

Background Immunoassays are crucial in the work-up of patients with suspected heparin-induced thrombocytopenia (HIT) and rapid tests have been recently developed. However, comparative data on diagnostic accuracy, reproducibility, and analytical costs of different immunoassays in clinical practice are limited. Methods Samples of 179 consecutive patients evaluated for suspected HIT in clinical practice using a polyspecific enzyme-linked immunoabsorbent assay (GTI diagnostics; ELISA) and a rapid particle gel immunoassay (PaGIA), were additionally analysed with a IgG-specific chemiluminescent immunoassay (AcuStar HIT-IgG). Presence of HIT was defined as a positive functional heparin-induced platelet aggregation test. Diagnostic accuracy was determined for low, intermediate and high thresholds as previously established (ELISA: optical density 0.4, 1.3, and 2.0 respectively; PaGIA: positive/negative, titre of 4, titre of 32; AcuStar HIT-IgG: 1.0 U/ml, 2.8, 9.4) and reproducibility was assessed by repeated measurements. Costs of test determination were calculated taking reagents, controls, and working time of technicians according to Swiss health care system into account. Results Data on PaGIA results were available for 171 patients (95.5%), ELISA for 144 patients (80.4%), and AcuStar HIT-IgG for 179 patients (100%). Sensitivity was above 95% for all assays at low and intermediate thresholds. Specificity increased with higher thresholds and was above 90% for all assays with intermediate and high thresholds. Specificity of AcuStar HIT-IgG (92.8%; 95% CI 87.7, 96.2) was significantly higher than PaGIA (83.0%; 95% CI 76.3, 88.5) and higher than ELISA (81.8%, 95% CI 74.2, 88.0) at low threshold (p<0.05). Reproducibility was adequate for all assays. Total costs per test were CHF 51.02 for ELISA, 117.70 for AcuStar HIT-IgG, and 83.13 for PaGIA. Conclusions We observed favourable diagnostic accuracy measures and a high reproducibility for PaGIA and AcuStar HIT-IgG. Implementation into 24-hours-service might improve patient care but the results must be confirmed in other settings and larger populations as well. PMID:28594835

Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests

PubMed Central

Santos, Fred Luciano Neves; de Souza, Wayner Vieira; da Silva Barros, Michelle; Nakazawa, Mineo; Krieger, Marco Aurélio; de Miranda Gomes, Yara

2016-01-01

There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic. PMID:26976886

Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.

Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production frommore » cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. In conclusion, the carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.« less

Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

DOE PAGES

Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; ...

2015-11-26

Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production frommore » cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. In conclusion, the carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.« less

The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

PubMed Central

Dimitrov, S I; Russanova, V R; Pashev, I G

1987-01-01

The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber. Images Fig. 1. Fig. 2. PMID:2444434

On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

PubMed

Kongsuphol, Patthara; Liu, Yunxiao; Ramadan, Qasem

2016-10-01

Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

n/a

NASA Image and Video Library

2003-06-10

Cadmium selenium Quantum Dots (QDs) are metal nanoparticles that fluoresce in a variety of colors determined by their size. QDs are solid state structures made of semiconductors or metals that confine a countable, small number of electrons into a small space. The confinement of electrons is achieved by the placement of some insulating material(s) around a central, well conducted region. Coupling QDs with antibodies can be used to make spectrally multiplexed immunoassays that test for a number of microbial contaminants using a single test.

Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE) Coupled with Enzyme-Linked Immunosorbent Assay (ELISA)

PubMed Central

Pschenitza, Michael; Hackenberg, Rudolf; Niessner, Reinhard; Knopp, Dietmar

2014-01-01

This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q) of ∼32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v)) was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v)) for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values. PMID:24887045

The Laboratory’s Role in Opioid Pain Medication Monitoring

PubMed Central

2012-01-01

Opioid analgesics are the most potent pain medications therefore they are often used for the treatment of chronic malignant and non-malignant pain. Their strong addictive potential requires close monitoring of patients on opioid therapy for possible non-compliance with prescriptions, for drug diversion, and for proof of avoidance of non-prescribed or illicit opioids. Monitoring can be performed by urine drug screens or qualitative or quantitative drug confirmation assays. Natural, semi-synthetic and synthetic opioids have dissimilar chemical structures and they undergo extensive metabolism. Phase one metabolic reactions of opioids can produce other opioids with similar structures to other, non-prescribed medications. Only detailed and concurrent analysis of parent drugs and metabolites can provide accurate clinical information regarding patient compliance. Traditional immunoassays, often used for urine drug screening, react with only a small number of opioids or only with a single medication and they exhibit variable cross reactivity with their phase two metabolites. Additionally the limit of detection of these immunoassays may not be sufficient for medical purposes, therefore clinical interpretation of immunoassay test results can be challenging. Recently liquid chromatography, mass spectrometry (LCMSMS) based assays have been adapted by many clinical laboratories. These LCMSMS tests can provide information about the presence of several opioids and their metabolites in a single sample at clinically meaningful detection limits, allowing accurate assessment of patient compliance. This review article will investigate in details the various opioids, their metabolism and the challenges the testing laboratories and ordering clinicians face. PMID:27683413

Selective biotinylation of Neisseria meningitidis group B capsular polysaccharide and application in an improved ELISA for the detection of specific antibodies.

PubMed

Diaz Romero, J; Outschoorn, I

1993-03-15

A method is described for the selective biotinylation of meningococcal capsular polysaccharide from Neisseria meningitidis group B and its application to an enzyme-linked immunoabsorbent assay (ELISA) to detect specific antibodies by immobilization on streptavidin-coated microtiter wells. Capsular polysaccharide from Neisseria meningitidis B has been biotinylated by specific periodate oxidation of terminal residues and condensation of the resulting aldehydes with biotin hydrazide, using a spin-column technique in the intermediate purification steps. The ELISA was optimized employing an extended reaction time between the label alkaline phosphatase and its most common substrate, p-nitrophenyl phosphate, together with evaluation of blocking agents to minimize non-specific binding. Specificity was demonstrated by a direct competitive enzyme immunoassay (EIA).

Basic analytical methods for identification of erythropoiesis-stimulating agents in doping control

NASA Astrophysics Data System (ADS)

Postnikov, P. V.; Krotov, G. I.; Efimova, Yu A.; Rodchenkov, G. M.

2016-02-01

The design of new erythropoiesis-stimulating agents for clinical use necessitates constant development of methods for detecting the abuse of these substances, which are prohibited under the World Anti-Doping Code and are included in the World Anti-Doping Agency (WADA) prohibited list. This review integrates and describes systematically the published data on the key methods currently used by WADA-accredited anti-doping laboratories around the world to detect the abuse of erythropoiesis-stimulating agents, including direct methods (various polyacrylamide gel electrophoresis techniques, enzyme-linked immunosorbent assay, membrane enzyme immunoassay and mass spectrometry) and indirect methods (athlete biological passport). Particular attention is given to promising approaches and investigations that can be used to control prohibited erythropoietins in the near future. The bibliography includes 122 references.

Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

PubMed

Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

2015-01-01

Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry.

Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Konstantinou, George N

2017-01-01

Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

[Detecting the markers of HIV infection with the new enzyme immunoassay diagnostic kit "DS-EIA-HIV-AB-AG-SPECTRUM" at the laboratories of AIDS prevention and control centers in the Volga Federal District].

PubMed

Ivanova, N I; Peksheva, O Iu

2009-03-01

A possibility of simultaneously detecting specific antibodies to HIV-1 and HIV-2 by enzyme immunoassay (EIA) at lower concentrations than those by immunoblotting (IB), and well as an additional possibility of earlier diagnosis of HIV infection, by identifying the HIV-1 antigen p24 lay the foundation of the "DS-EIA-HIV-AB-AG-SPECTRUM" test system made by OOO "Research-and-Production Association "Diagnosticheskiye Sistemy" (Diagnostic Systems). These peculiarities were compared with those of IB at a number of laboratories of AIDS prevention and control centers in the Volga Federal District, by using native serum/plasma samples and a specially designed control panel. The analysis of the conducted studies to identify HIV-1 and HIV-2 antibodies and HIV-1 antigen p24 in 65 plasma/serum samples in the "DS-EIA-HIV-AB-AG-SPECTRUM" and "LIA-HIV-1/2" (OOO "Niarmedik plus") test systems while confirming the positive result indicated agreement in 57 (87.7%) cases. The diagnostic possibilities of the "DS-EIA-HIV-AB-AG-SPECTRUM" test system versus the "New Lav-Blot I" one to make a laboratory diagnosis of HIV infection were studied. Irrefragable answers as to the availability of HIV-1 markers in the study serum samples on the enciphered panel were provided by IB in 73.3% of cases and EIA in 92%.

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

PubMed

Pribbenow, Susanne; Wachter, Bettina; Ludwig, Carsten; Weigold, Annika; Dehnhard, Martin

2016-03-01

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs. Copyright © 2016 Elsevier Inc. All rights reserved.

ELISA analysis of soybean trypsin inhibitors in processed foods.

PubMed

Brandon, D L; Bates, A H; Friedman, M

1991-01-01

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

Bacterial Expression of a Single-Chain Variable Fragment (scFv) Antibody against Ganoderic Acid A: A Cost-Effective Approach for Quantitative Analysis Using the scFv-Based Enzyme-Linked Immunosorbent Assay.

PubMed

Yusakul, Gorawit; Nuntawong, Poomraphie; Sakamoto, Seiichi; Ratnatilaka Na Bhuket, Pahweenvaj; Kohno, Toshitaka; Kikkawa, Nao; Rojsitthisak, Pornchai; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

2017-01-01

Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle ® strain. Purified anti-GAA scFv, which yielded 2.56 mg from 1 L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078-1.25 µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1-101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.

High Seroprevalence of Toxoplasma Gondii Infection in Female Sex Workers: A Case-Control Study

PubMed Central

Alvarado-Esquivel, Cosme; Sánchez-Anguiano, Luis Francisco; Hernández-Tinoco, Jesús; Arreola-Cháidez, Emilio; López, Juan; Salcido-Meraz, Karla Itzel; Estrada-Martínez, Sergio; Navarrete-Flores, José Antonio; Pérez-Álamos, Alma Rosa; Hernández-Ochoa, Marcia; Rábago-Sánchez, Elizabeth; Liesenfeld, Oliver

2015-01-01

Through an age- and sex-matched case-control study, we sought to determine whether female sex workers have an increased risk of Toxoplasma gondii exposure and to determine the sociodemographic, work, clinical, and behavioral characteristics of these workers associated with T. gondii exposure. Female workers (n = 136) and controls (n = 272) were examined with enzyme-linked immunoassays (EIA) for the presence of anti-Toxoplasma IgG and IgM antibodies. IgM positive sera were additionally tested with enzyme linked-fluorescence immunoassay (ELFA). Anti-T. gondii IgG antibodies were found in 21 (15.44%) of 136 cases and in 10 (3.67%) of 272 controls (OR = 4.05; 95% CI: 1.84–8.89; P = 0.0001). Anti-T. gondii IgG levels higher than 150 IU/ml were found in 13 (9.6%) of 136 cases and in 8 (2.9%) of 272 controls (P = 0.007). Anti-T. gondii IgM antibodies were found in two cases and in six controls by EIA, but all were negative by ELFA. T. gondii seropositivity was associated with being born out of Durango State (OR = 10.47; 95% CI: 2.9–36.8; P < 0.01), injuries during sex work (OR = 6.30; 95% CI: 1.1–33.7; P = 0.03), and soil contact (OR = 4.11; 95% CI: 1.2–14.0; P = 0.02). This is the first report of an association of T. gondii infection and female sex workers. PMID:26716017

Detection of Total Ergot Alkaloids in Cereal Flour and in Bread by a Generic Enzyme Immunoassay Method.

PubMed

Gross, Madeleine; Curtui, Valeriu; Usleber, Ewald

2018-05-01

Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, α-ergocryptine, and ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions

PubMed Central

TOISHI, Yuko; TSUNODA, Nobuo; NAGATA, Shun-ichi; KIRISAWA, Rikio; NAGAOKA, Kentaro; WATANABE, Gen; YANAGAWA, Yojiro; KATAGIRI, Seiji; TAYA, Kazuyoshi

2017-01-01

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs. PMID:29129877