Development of a Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay Using a Synthetic Peptide Antigen for Detection of Avian Metapneumovirus Antibodies in Turkey Sera

Science.gov (United States)

Alvarez, Rene; Njenga, M. Kariuki; Scott, Melissa; Seal, Bruce S.

2004-01-01

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes. PMID:15013970

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

Science.gov (United States)

Alcorn, S.W.; Pascho, R.J.

2000-01-01

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

Operating considerations of ultrafiltration in enzyme enhanced carbon capture

DEFF Research Database (Denmark)

Deslauriers, Maria Gundersen; Gladis, Arne; Fosbøl, Philip Loldrup

2017-01-01

capture capacity of 1 MTonn CO2/year, and is here operated for one year continuously. This publication compares soluble enzymes dissolved in a capture solvent with and without the use of ultrafiltration membranes. The membranes used here have an enzyme retention of 90%, 99% and 99.9%. Enzyme retention......Today, enzyme enhanced carbon capture and storage (CCS) is gaining interest, since it can enable the use of energy efficient solvents, and thus potentially reduce the carbon footprint of CCS. However, a limitation of this technology is the high temperatures encountered in the stripper column, which...

Embedded enzymes catalyse capture

Science.gov (United States)

Kentish, Sandra

2018-05-01

Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

International Nuclear Information System (INIS)

Muir, D.; Varon, S.; Manthorpe, M.

1990-01-01

We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

Energy Technology Data Exchange (ETDEWEB)

Muir, D.; Varon, S.; Manthorpe, M. (Univ. of California, San Diego, La Jolla (USA))

1990-03-01

We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

Science.gov (United States)

Muro, Claudio; Gomez-Puerta, Luis A; Flecker, Robert H; Gamboa, Ricardo; Barreto, Percy Vilchez; Dorny, Pierre; Tsang, Victor C W; Gilman, Robert H; Gonzalez, Armando E; Garcia, Hector H; O'Neal, Seth E; For The Cysticercosis Working Group In Peru

2017-12-01

The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena- infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

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

Science.gov (United States)

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.

Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program

DEFF Research Database (Denmark)

Chung, Wen Bin; Sørensen, Karl Johan; Liao, Pei Chih

2002-01-01

Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...... in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased....

Immunoglobulin G1 enzyme-linked Immunosorbent assay for diagnosis of Johne's disease in red deer (Cervus elaphus)

NARCIS (Netherlands)

Griffin, J.F.T.; Spittle, E.; Rodgers, C.R.; Liggett, S.; Cooper, M.; Bakker, D.; Bannantine, J.P.

2005-01-01

This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

Science.gov (United States)

Min, Yuanzeng; Roche, Kyle C.; Tian, Shaomin; Eblan, Michael J.; McKinnon, Karen P.; Caster, Joseph M.; Chai, Shengjie; Herring, Laura E.; Zhang, Longzhen; Zhang, Tian; Desimone, Joseph M.; Tepper, Joel E.; Vincent, Benjamin G.; Serody, Jonathan S.; Wang, Andrew Z.

2017-09-01

Immunotherapy holds tremendous promise for improving cancer treatment. To administer radiotherapy with immunotherapy has been shown to improve immune responses and can elicit the 'abscopal effect'. Unfortunately, response rates for this strategy remain low. Herein we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NP formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent on the NP surface properties. We showed that AC-NPs deliver tumour-specific proteins to antigen-presenting cells (APCs) and significantly improve the efficacy of αPD-1 (anti-programmed cell death 1) treatment using the B16F10 melanoma model, generating up to a 20% cure rate compared with 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+T/Treg and CD8+T/Treg ratios (Treg, regulatory T cells). Our work presents a novel strategy to improve cancer immunotherapy with nanotechnology.

Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

International Nuclear Information System (INIS)

Doinel, C.; Ropars, C.; Salmon, C.

1978-01-01

Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

Evaluation of an Antigen-Antibody “Combination” Enzyme Linked ...

African Journals Online (AJOL)

Conclusion: We conclude that although this assay depicts high sensitivity and specificity in detecting antibodies to HCV, it seems not to add further benefit in our study population to detect HCV infections by enhanced sensitivity due the potential contingency to trace viral capsid antigens. Keywords: Ag-Ab Combination assay ...

ANALYSIS OF SOIL AND DUST SAMPLES FOR POLYCHLORINATED BIPHENYLS BY ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

Science.gov (United States)

An inhibition enzyme-linked immunosorbent assay (ELISA) was used to determine polychlorinated biphenyls (PCBs) in house dust and soil. Soil and house dust samples were analyzed for PCB by both gas chromatography/electron capture detection (GC/ECD) and ELISA methods. A correlati...

Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Neiser, Susann; Koskenkorva, Taija S; Schwarz, Katrin; Wilhelm, Maria; Burckhardt, Susanna

2016-07-21

Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may

Diagnosis of selected tropical diseases (Shistosomiasis and Malaria) using the enzyme-linked immunosorbent assays

International Nuclear Information System (INIS)

Chembe, E.

1985-01-01

Immunological reactions are commonly used in diagnostic procedures on the basis of their high levels of specificity and sensitivity. Antibodies or antigens labelled with various markers have been found to be particularly useful for assays of logical substances. The applications of Enzyme-Linked Immunoabsorbent Assays (ELISA) to research on various tropical and non-tropical diseases is now well established. The procedure depends on the labelling of one of the reactants with enzymes which can be detected accurately by an appropriate substrate. The detection mechanism depends on the labelling of one of the reactants in such a way that their their reactivity is not impaired or affected. In the present study, ELISA was applied to sera from kampumbu area of Isoka district in the Northern province of Zambia. The objective of this presentation is to show the relative positivity rate for antigen and antibody and the endemicity of schistosomiasis and malaria as assessed by classical parasitological procedures. (author)

Seroprevalence of Cryptosporidium parvum infection of dairy cows in three northern provinces of Thailand determined by enzyme-linked immunosorbent assay using recombinant antigen CpP23.

Science.gov (United States)

Inpankaew, T; Jittapalapong, S; Phasuk, J; Pinyopanuwut, N; Chimnoi, W; Kengradomkit, C; Sunanta, C; Zhang, G; Aboge, G O; Nishikawa, Y; Igarashi, I; Xuan, X

2009-06-01

Cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in AIDS patients in Thailand. Cryptosporidiosis outbreaks in humans may be attributed to contamination of their drinking water from infected dairy pastures. A 23-kDa glycoprotein of C. parvum (CpP23) is a sporozoite surface protein that is geographically conserved among C. parvum isolates. This glycoprotein is a potentially useful candidate antigen for the diagnosis of cryptosporidiosis by enzyme-linked immunosorbent assay. Therefore, we investigated the seroprevalence of C. parvum infection in dairy cows in northern Thailand using an ELISA based on recombinant CpP23 antigen. Sera were randomly collected from 642 dairy cows of 42 small-holder farmers, which had the top three highest number of the dairy cows' population in Northern Thailand, that included Chiang Mai, Chiang Rai and Lumpang provinces. The overall seroprevalence of the infection was 4.4%, and the seropositive rates for the three provinces were 3.3% in Chiang Mai, 5.1% in Chiang Rai and 3% in Lumpang. These results suggest that cattle could play a role in zoonotic cryptosporidiosis in Thailand.

Thermometric enzyme linked immunosorbent assay: TELISA.

Science.gov (United States)

Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

1977-08-11

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

DEFF Research Database (Denmark)

Klausen, Joan; Andresen, Lars Ole; Barfod, Kristen

2002-01-01

An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis...

Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

Science.gov (United States)

Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

2015-08-25

Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

Immunodiagnosis of Human Fascioliasis by an Enzyme-Linked Immunosorbent Assay (ELISA) and a Micro-ELISA

OpenAIRE

Carnevale, Silvana; Rodríguez, Mónica I.; Santillán, Graciela; Labbé, Jorge H.; Cabrera, Marta G.; Bellegarde, Enrique J.; Velásquez, Jorge N.; Trgovcic, Jorge E.; Guarnera, Eduardo A.

2001-01-01

Enzyme-linked immunosorbent assay (ELISA) and micro-ELISA were evaluated for their ability to detect anti-Fasciola hepatica antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis.

Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

Science.gov (United States)

Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

1998-01-01

Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

Standardization of micro-enzyme-linked immunosorbent assay (ELISA) and Western blot for detection of Trypanosoma cruzi antibodies using extracts from Mexican strains as antigens.

Science.gov (United States)

Sánchez, B; Monteón, V; Reyes, P A; Espinoza, B

2001-01-01

This report describes two assays for the detection of anti-Trypanosoma cruzi antibodies using Mexican strains of the parasite and the concordance with two assays previously evaluated at the Instituto Nacional de Cardiología Ignacio Chávez in Mexico City. Micro-enzyme-linked immunosorbent assay (ELISA) and Western blot were used for the detection of T. cruzi antibodies with a total extract of epimastigote from Ninoa and Queretaro, which are Mexican strains of T. cruzi. To standardize these methods, a total of 246 serum samples was used. In addition, sera from six confirmed Mexican chronic individuals in the asymptomatic phase were also used for comparison with the Argentinean antigen. ELISA was 100% specific in that no false positive results were found with sera of both healthy individuals and non-Chagasic cardiopaths. Sera from individuals infected with Leishmania sp. showed approximately 16% of cross-reaction with ELISA. The test showed a positive predictive value of 90% and a negative predictive value of 100%. Western blot was also a highly sensitive test for detecting chronic Chagasic symptomatic patients from Mexico because no false negative results were obtained. Furthermore, it was possible to use Western blot to detect seven immunodominant antigens of approximately 30, 32, 40, 42, 65, 70, and 83 kDa. Concordance with two previous standardized tests at the Instituto Nacional de Cardiología showed a Kappa index of 0.96, indicating high concordance between the results obtained at these two laboratories. Finally, ELISA using Ninoa antigen extract was more sensitive than ELISA with an Argentinean extract, which failed to detect individuals in the chronic asymptomatic phase (undetermined phase) of infection. This study indicates that ELISA and Western blot using Ninoa and/or Queretaro extracts of T. cruzi as antigens are useful tools in the detection of individuals who have been exposed to T. cruzi both in the undetermined/asymptomatic and symptomatic phases

Diagnosis of Fasciola gigantica infection in cattle using capture-ELISA assay for detecting antigen in faeces

Directory of Open Access Journals (Sweden)

Sarwitri Endah Estuningsih

2006-10-01

Full Text Available Capture-ELISA assay is a diagnosis for antigen detection in the serum or faeces using polyclonal or monoclonal antibodies. The purpose of this study was to determine the sensitivity and specificity of capture-ELISA assay using polyclonal antibody for diagnosing Fasciola gigantica infection in cattle by detecting antigen in the faeces. In this study, faecal samples and livers were collected from 141 cattle slaughtered in the abattoir in Jakarta. From each animal, liver was processed for liver flukes count and the corresponding faecal sample was analysed for coproantigen. The result of capture-ELISA assay for antigen detection showed that from 85 cattle infected with Fasciola gigantica, 83 had OD > 0.52 (range from 0.52-1.39 and 2 cattle had OD < 0.52 (range from 0.17-0.51. The sensitivity and specificity of the assay were 97.6% and 92.8% respectively. The assay also able to detect 50 ng/ml of antigen in faecal supernatant. It suggests that this assay will have the advantage over the other methods on its ability to detect the active infection. Collection of faeces, rather than serum, will allow a more cost-effective and adaptable method.

Novel indirect enzyme-linked immunosorbent assay (ELISA) method to detect Total E. coli in water environment

International Nuclear Information System (INIS)

Wang Na; He Miao; Shi Hanchang

2007-01-01

In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1 x 10 5 . The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 10 4 CFU (colony forming units) L -1 . The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method

Upscaling of enzyme enhanced CO2 capture

DEFF Research Database (Denmark)

Gladis, Arne Berthold

Fossil fuels are the backbone of the energy generation in the coming decades for USA, China, India and Europe, hence high greenhouse gas emissions are expected in future. Carbon capture and storage technology (CCS) is the only technology that can mitigate greenhouse gas emissions from fossil fuel...... the mass transfer of CO2 with slow-capturing but energetically favorable solvents can open up a variety of new process options for this technology. The ubiquitous enzyme carbonic anhydrase (CA), which enhances the mass transfer of CO2 in the lungs by catalyzing the reversible hydration of CO2, is one very...... enhanced CO2 capture technology by identifying the potentials and limitations in lab and in pilot scale and benchmarking the process against proven technologies. The main goal was to derive a realistic process model for technical size absorbers with a wide range of validity incorporating a mechanistic...

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

Science.gov (United States)

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.

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

Science.gov (United States)

Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

2016-02-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

Science.gov (United States)

Borrebaeck, C; Börjeson, J; Mattiasson, B

1978-06-15

Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

Detection of Serum Thermolabile β-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans

Science.gov (United States)

Tsujimura, Mitsushi; Ishida, Chuzo; Sagara, Yasuko; Miyazaki, Takashi; Murakami, Koichi; Shiraki, Hiroshi; Okochi, Kazuo; Maeda, Yoshiaki

2001-01-01

Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose, N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG. PMID:11238239

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Bergmann, S M; Wang, Q; Zeng, W; Li, Y; Wang, Y; Matras, M; Reichert, M; Fichtner, D; Lenk, M; Morin, T; Olesen, N J; Skall, H F; Lee, P-Y; Zheng, S; Monaghan, S; Reiche, S; Fuchs, W; Kotler, M; Way, K; Bräuer, G; Böttcher, K; Kappe, A; Kielpinska, J

2017-11-01

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs. © 2017 John Wiley & Sons Ltd.

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus.

Science.gov (United States)

Kardi, V; Szegletes, E; Perényi, T; Pergel, I; Smal, Z

1990-01-01

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.

Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens.

OpenAIRE

Ricke, S C; Schaefer, D M; Cook, M E; Kang, K H

1988-01-01

Cross-reactivity among four species of ruminal bacteria was examined by using egg yolk antibodies from immunized Leghorn laying hens and an enzyme-linked-immunosorbent assay. The effects of the four species on the hens were compared on various days postimmunization. Hens injected with the same bacterial species had similar apparent antibody levels over the entire postimmunization period, but only Bacteroides ruminicola B1(4) and Selenomonas ruminantium D antigens elicited early increases in a...

Detecting West Nile virus in owls and raptors by an antigen-capture assay.

Science.gov (United States)

Gancz, Ady Y; Campbell, Douglas G; Barker, Ian K; Lindsay, Robbin; Hunter, Bruce

2004-12-01

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%-95.2% for northern owl species but raptors.

Detecting West Nile Virus in Owls and Raptors by an Antigen-capture Assay

OpenAIRE

Gancz, Ady Y.; Campbell, Douglas G.; Barker, Ian K.; Lindsay, Robbin; Hunter, Bruce

2004-01-01

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%–95.2% for northern owl species but

Dot enzyme-linked immunosorbent assay (ELISA) for the detection of Toxocara infection using a rat model.

Science.gov (United States)

Paller, Vachel Gay V; Besana, Cyrelle M; Valdez, Isabel Kristine M

2017-12-01

Toxocariasis is a zoonotic disease usually caused by dog and cat roundworms, Toxocara canis and T. cati. Detection and diagnosis is difficult in paratenic and accidental hosts, including humans, as they cannot be detected through conventional methods such as fecal examination. Diagnosis therefore relies on immunological methods and molecular methods such as enzyme-linked immunosorbent assay (ELISA) and Western Blot, which are both time-consuming and requires sophisticated equipment. In the Philippines, only a few studies are available on Toxocara seroprevalence. Therefore, there is a need to adapt methods for serodiagnosis of Toxocara infection in humans for the Philippine setting. A dot enzyme linked immunosorbent assay (dot-ELISA) was standardized using T. canis excretory-secretory antigens. Test sera were collected from laboratory rats (Sprague-Dawley strain) experimentally infected with embryonated eggs of T. canis and Ascaris suum as well as rice field rats naturally infected with Taenia taeniaeformis and Nippostrongylus sp. Optimum conditions used were 20 µg/ml antigen concentration and 1:10 serum dilution. The sensitivity, specificity, positive, and negative predictive values were 90% (95% CI 55.5-99.7%), 100% (95% CI 69.2-100.0%), 100% (95% CI 66.4-100%), and 90.9% (95% CI 58.7-99.8%), respectively. Dot-ELISA has the potential to be developed as a cheaper, simpler, and more practical method for detection of anti- Toxocara antibodies on accidental hosts. This is a preliminary study conducted on experimental animals before optimization and standardization for human serum samples.

Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

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Zoheiry Mona K

2011-09-01

Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

SERO-DIAGNOSIS OF TUBERCULOSIS WITH A60 ANTIGEN ENZYME-LINKED-IMMUNOSORBENT-ASSAY - FAILURE IN HIV-INFECTED INDIVIDUALS IN GHANA

NARCIS (Netherlands)

VANDERWERF, TS; DAS, PK; VANSOOLINGEN, D; YONG, S; VANDERMARK, TW

In order to assess the diagnostic usefulness of the A60 (ANDA Biologicals, Strassbourg, France) sero-diagnostic enzyme-linked immunosorbent assay (ELISA) kit for tuberculosis in Africa, sera of 53 pulmonary smear-positive tuberculosis (TB) patients, 30 apparently healthy control subjects and 6 AIDS

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

Science.gov (United States)

BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

2015-01-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

Screen Printed Carbon Electrode Based Electrochemical Immunosensor for the Detection of Dengue NS1 Antigen

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Om Parkash

2014-11-01

Full Text Available An electrochemical immunosensor modified with the streptavidin/biotin system on screen printed carbon electrodes (SPCEs for the detection of the dengue NS1 antigen was developed in this study. Monoclonal anti-NS1 capture antibody was immobilized on streptavidin-modified SPCEs to increase the sensitivity of the assay. Subsequently, a direct sandwich enzyme linked immunosorbent assay (ELISA format was developed and optimized. An anti-NS1 detection antibody conjugated with horseradish peroxidase enzyme (HRP and 3,3,5,5'-tetramethybezidine dihydrochloride (TMB/H2O2 was used as an enzyme mediator. Electrochemical detection was conducted using the chronoamperometric technique, and electrochemical responses were generated at −200 mV reduction potential. The calibration curve of the immunosensor showed a linear response between 0.5 µg/mL and 2 µg/mL and a detection limit of 0.03 µg/mL. Incorporation of a streptavidin/biotin system resulted in a well-oriented antibody immobilization of the capture antibody and consequently enhanced the sensitivity of the assay. In conclusion, this immunosensor is a promising technology for the rapid and convenient detection of acute dengue infection in real serum samples.

Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

OpenAIRE

Jiménez, T; Díaz, A M; Zlotnik, H

1990-01-01

Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Myco...

An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

DEFF Research Database (Denmark)

Hansen, Soren; Schmidt, Vivi; Steffensen, Maria Abildgaard

2008-01-01

characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which......Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other...... innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were...

CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens.

Science.gov (United States)

Chen, Peter; Mesci, Aruz; Carlyle, James R

2011-01-01

Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen

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Anita Verma

2018-02-01

Full Text Available Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA, the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

Evaluation of two Taenia solium cysticercal antigenic preparations (vesicular fluid and a glycoprotein fraction with affinity for lentil lectin for the immunodiagnosis of neurocysticercosis by enzyme-linked immunosorbent assay (ELISA

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Lisandra Akemi Suzuki

2011-06-01

Full Text Available OBJECTIVE: To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. METHOD: Fifty-six cerebrospinal fluid (CSF samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA. RESULTS: The VF ELISA showed 100% sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9% and 97.1%, with the CSF samples and 95.5% and 100% with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. CONCLUSION: Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.

Determination of PCBs in fish using enzyme-linked immunosorbent assay (ELISA)

Science.gov (United States)

Lasrado, J.A.; Santerre, C.R.; Zajicek, J.L.; Stahl, J.R.; Tillitt, D.E.; Deardorff, D.

2003-01-01

Polychlorinated biphenyls (PCBs) were determined in fish tissue using an enzyme-linked immunosorbent assay (ELISA). Standard curves for Aroclor 1248, 1254, and 1260 in catfish tissue were developed with ranges from 0.05 to 0.5 ppm and 0.5 to 5.0 ppm. Wild fish were initially analyzed using gas chromatography/electron-capture detection (GC/ECD) and those having residues within the standard curve ranges were analyzed with ELISA. Results obtained using ELISA and GC/ECD were not significantly different (p < 0.05) from 0.05 to 0.5 ppm. From 0.5 to 5.0 ppm, the standard curve for Aroclor 1254 was the best predictor of total PCB in wild fish samples.

An enzyme-linked immuno-filtration assay used to compare infant and maternal antibody profiles in toxoplasmosis.

Science.gov (United States)

Pinon, J M; Thoannes, H; Gruson, N

1985-02-28

Enzyme-linked immuno-filtration assay is carried out on a micropore membrane. This doubly analytical technique permits simultaneous study of antibody specificity by immunoprecipitation and characterisation of antibody isotypes by immuno-filtration with enzyme-labelled antibodies. Recognition of the same T. gondii antigenic constituent by IgG, IgA, IgM or IgE antibodies produces couplets (IgG-IgM; IgG-IgA) or triplets (IgG-IgM-IgA; IgG-IgM-IgE) which identify the functional fractions of the toxoplasmosis antigen. In acquired toxoplasmosis, the persistence of IgM antibody long after infestation puts in question the implication of recent infestation normally linked to detection of this isotype. For sera of comparable titres, comparison of immunological profiles by the method described demonstrates disparities in the composition of the specific antibody content as expressed in international units. Use of the same method to detect IgM antibodies or distinguish between transmitted maternal IgG and IgG antibodies synthesised by the foetus or neonate makes a diagnosis of congenital toxoplasmosis possible in 85% of cases during the first few days of life. With the method described the diagnosis may be made on average 5 months earlier than with classical techniques. In the course of surveillance for latent congenital toxoplasmosis, the appearance of IgM or IgE antibodies raises the possibility of complications (hydrocephalus, chorioretinitis). After cessation of treatment, a rise in IgG antibodies indicating persistence of infection is detected earlier by the present than by classical methods.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

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Dong Huahuang

2012-08-01

Full Text Available Abstract Background HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1 which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA assays combined with polymerase chain reaction (PCR and gel electrophoresis to quantify HIV-1 p24 antigen. Method A pair of anti-p24 monoclonal antibodies (mAbs were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. Conclusions When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected

Enzyme-Linked Immunosorbent Assay To Differentiate the Antibody Responses of Animals Infected with Brucella Species from Those of Animals Infected with Yersinia enterocolitica O9

OpenAIRE

Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

2003-01-01

Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

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Spiropoulou Christina F

2010-06-01

Full Text Available Abstract Background Outbreaks of Hendra (HeV and Nipah (NiV viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry. Results Through screening of hybridomas derived from mice immunized with γ-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N protein and the other, the phosphoprotein (P of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig. Conclusion The antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

Science.gov (United States)

Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

2017-02-01

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately

A competitive enzyme linked immunosorbent assay for the ...

African Journals Online (AJOL)

A competitive enzyme linked immunosorbent assay for the determination of diminazene residues in animal tissues. ... After six washes with buffer, enzyme activity was determined by adding tetramethyl-benzidine and hydrogen peroxide as substrate. The assay detection limits for diminazene were 2.4 ng/g in muscle, 2.5 ...

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

Science.gov (United States)

Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

2014-12-01

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

Science.gov (United States)

Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

2005-01-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

DEFF Research Database (Denmark)

Jensen, A T; Gaafar, A; Ismail, A

1996-01-01

An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...... samples from healthy Sudanese individuals living in an area endemic for malaria but free of leish-maniasis were negative in all the assays. Significantly higher levels of antibodies were found in the patients who had suffered from the disease for more than eight weeks than in patients with a shorter...

Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles

Science.gov (United States)

Guo, Longhua; Xu, Shaohua; Ma, Xiaoming; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

2016-09-01

Colorimetric enzyme-linked immunosorbent assay utilizing 3‧-3-5‧-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3‧-3-5‧-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.

Zinc oxide nanoparticle-enhanced ultrasensitive chemiluminescence immunoassay for the carcinoma embryonic antigen

International Nuclear Information System (INIS)

Pal, Souvik; Bhand, Sunil

2015-01-01

An ultrasensitive enzyme-linked immunosorbent assay (ELISA) is reported for the determination of carcinoma embryonic antigen (CEA) in human serum. It was realized using a microplate reader using a 384-well plate. Monoclonal antibody (Ab) against CEA (1° Ab) acting as the capture probe was immobilized on zinc oxide nanoparticles (ZnO-NPs) in the form of self-assembled monolayers (SAMs). CEA captured by 1° Ab was quantified using a sandwich ELISA wherein a polyclonal second antibody against CEA (2° Ab) was used for detection and quantified using an HRP-labeled secondary antibody (3° Ab). The ZnO-NPs-CEA capture probe was deposited on the bottom of the wells in order to enhance capture of CEA. A 3-fold enhancement in the chemiluminescence (CL) signal of luminol is found (compared to a conventional ELISA). CEA can be quantified by this method in concentrations as low as 1 pg · mL −1 . The upper limit of detection is 20 ng · mL −1 . The use of ZnO-NPs also imparts improved thermal stability. When stored at 4 °C in phosphate-buffered saline of pH 7.4, the probe displays stability of up to 30 days. (author)

Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Andresen, Lars Ole; Klausen, Joan; Barfod, Kristen

2002-01-01

and from herds declared free of infection with Ap. The Ap serotype 12 blocking ELISA showed a herd sensitivity of 0.77 (95% confidence interval, 0.62-0.88) and a herd specificity of 1.00 (0.95-1.00) with a cut-off value at 40% relative absorbance or 60% inhibition. The assay may be used advantageously......The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen...... in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotypc 12 infection...

Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

Directory of Open Access Journals (Sweden)

William H Roldán

2009-05-01

Full Text Available To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis. Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%, whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

Polymerase chain reaction versus enzyme-linked immunosorbent ...

African Journals Online (AJOL)

Polymerase chain reaction versus enzyme-linked immunosorbent assay in detection of Chlamydia trachomatis infection among gynaecological patients in southwestern Nigeria. ... Socio-demographic bio-data and gynaecological history were obtained with questionnaire; data was analyzed using SPSS version 20.0.

Comparison of IgM Capture Enzyme- Linked Immunosorbent Assay (ELISA) using Inhouse method and commercially available MRL kit for serological confirmation of dengue infection

International Nuclear Information System (INIS)

Hapugoda, D.M.; De Silva R, Nilanthi; Abeywickreme, W.; Gunasena, Sunethra; Prithimala, L.D.; Jayawardene, S.L.G.J.; Kumari, Thamara

2003-01-01

Laboratory diagnosis of dengue infection is important for the management of the patients. In this study igM capture ELISA using an inhouse method and commercially available kit (MRL diagnostics,USA) was compared to detect diagnostic capability of Inhouse IgM ELISA for provision of diagnostic facilities to the public at an affordable cost. Eighty acute and convalescent serum samples were collected from serologically confirmed dengue patients. Serological confirmation of patients were performed by Haemagglutination Inhibition (HI) assay, gold standard assay for dengue on paired serum samples. All collected acute and convalescent sera were tested by IgM ELISA using the inhouse method and MRL kit. Antigen and conjugate for the inhouse IgM method were prepared in the laboratory. A cocktail of four dengue antigens containing 25 Antigen ELISA units of each type was prepared and used as the assay antigen. Conjugate was prepared using a serum sample with high dengue Anti flavi IgG antibody titre conjugated with Horseradish peroxidase. A prospective study of both IgM ELISA assays were performed using 113 acute sera collected from dengue suspected cases. Overall results showed that 46% and 52% acute sera collected from dengue confirmed patients were positive by inhouse ELISA assay and MRL kits respectively. In the prospective study done using acute sera collected from dengue suspected patients showed that 44% and 52% were positive by inhouse ELISA assay and MRL kits. There was no significant difference in positivity between these two assays. (P=0.18). Inhouse IgM ELISA can be used for provision of laboratory diagnosis of dengue virus infection more than 5 days. The assay is 10 times less costly than using MRL kits as assay antigen and conjugate can be prepared easily in the laboratory

Development of enzyme linked immunosorbent assay (ELISA) for the detection of root-knot nematode Meloidogyne incognita.

Science.gov (United States)

Kapur-Ghai, J; Kaur, M; Goel, P

2014-09-01

Root-knot nematodes (Meloidogyne incognita) are obligate, sedentary plant endoparasites that are extremely polyphagous in nature and cause severe economic losses in agriculture. Hence, it is essential to control the parasite at an early stage. For any control strategy to be effective, an early and accurate diagnosis is of paramount importance. Immunoassays have the inherent advantages of sensitivity and specificity; have the potential to identify and quantify these plant-parasitic nematodes. Hence, in the present studies, enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of M.incognita antigens. First an indirect ELISA was developed for detection and titration of anti-M.incognita antibodies. Results indicated as high as 320 K titre of the antisera. Finally competitive inhibition ELISA was developed employing these anti-M.incognita antibodies for detection of M.incognita antigens. Sensitivity of ELISA was 10 fg. Competitive inhibition ELISA developed in the present studies has the potential of being used as an easy, rapid, specific and sensitive diagnostic tool for the detection of M.incognita infection.

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus

International Nuclear Information System (INIS)

Salfeld, J.; Pfaff, E.; Noah, M.; Schaller, H.

1989-01-01

The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis virus nucleocapsid

A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide

DEFF Research Database (Denmark)

Barington, T; Sparholt, S; Juul, L

1992-01-01

A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary...... antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality...... and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection...

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

Science.gov (United States)

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

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

Science.gov (United States)

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (assay variability assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Antigen-decorated shell cross-linked nanoparticles: synthesis, characterization, and antibody interactions.

Science.gov (United States)

Joralemon, Maisie J; Smith, Norah L; Holowka, David; Baird, Barbara; Wooley, Karen L

2005-01-01

Antigen-decorated shell cross-linked knedel-like nanoparticles (SCKs) were synthesized and studied as multivalent nanoscale surfaces from which antibody-binding units were presented in a manner that was designed to approach virus particle surfaces. The SCK nanostructures were fabricated with control over the number of antigenic groups, from mixed micellization of amphiphilic diblock copolymer building blocks that contained either an antigen (2,4-dinitrophenyl) or an ethylpropionate group at the hydrophilic alpha-chain terminus. Amphiphilic diblock copolymers were synthesized by atom transfer radical polymerization of tert-butyl acrylate and methyl acrylate sequentially from either a 2,4-dinitrophenyl-functionalized initiator or ethyl 2-bromopropionate, followed by selective removal of the tert-butyl groups to afford 2,4-dinitrophenyl-poly(acrylic acid)60-b-poly(methyl acrylate)60 (DNP-PAA(60)-b-PMA60) and poly(acrylic acid)70-b-poly(methyl acrylate) (PAA70-b-PMA70). Micelles were assembled via addition of water to THF solutions of the polymers in 0:1, 1:1, and 1:0 molar ratios of DNP-PAA60-b-PMA60 to PAA70-b-PMA70, followed by dialysis against water. The acrylic acid groups of the micelle coronas were partially cross-linked (nominally 50%) with 2,2'-(ethylenedioxy)bis(ethylamine), in the presence of 1-(3'-dimethylaminopropyl)-3-ethylcarbodiimide methiodide. Following extensive dialysis against water, the 0%, 50%, and 100% dinitrophenylated shell cross-linked nanoparticles (DNP-SCKs) were characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared and UV-vis spectroscopies, and analytical ultracentrifugation (AU). The surface accessibility and bioavailability of the DNP units upon the DNP-SCKs were investigated by performing quenching titrations of fluorescein-labeled IgE antibody in solution and degranulation of Ig

Optimized enzyme-linked immunosorbent assay for detecting cytomegalovirus infections during clinical trials of recombinant vaccines.

Science.gov (United States)

Pagnon, Anke; Piras, Fabienne; Gimenez-Fourage, Sophie; Dubayle, Joseline; Arnaud-Barbe, Nadège; Hessler, Catherine; Caillet, Catherine

2017-11-01

In clinical trials of cytomegalovirus (CMV) glycoprotein B (gB) vaccines, CMV infection is detected by first depleting serum of anti-gB antibodies and then measuring anti-CMV antibodies with a commercially available enzyme-linked immunosorbent assay (ELISA) kit, with confirmation of positive findings by immunoblot. Identification of CMV immunoantigens for the development of an ELISA that detects specifically CMV infection in clinical samples from individuals immunized with gB vaccines. Sensitivity and specificity of ELISAs using antigenic regions of CMV proteins UL83/pp65, UL99/pp28, UL44/pp52, UL80a/pp38, UL57, and UL32/pp150 were measured. An IgG ELISA using a UL32/pp150 [862-1048] capture peptide was the most specific (93.7%) and sensitive (96.4%) for detecting CMV-specific antibodies in sera. The ELISA successfully detected CMV-specific antibodies in 22 of 22 sera of subjects who had been vaccinated with a gB vaccine but who had later been infected with CMV. The ELISA was linear over a wide range of CMV concentrations (57-16,814 ELISA units/mL) and was reproducible as indicated by a 5% intra-day and 7% inter-day coefficients of variation. The signal was specifically competed by UL32/pp150 [862-1048] peptide but not by CMV-gB or herpes simplex virus 2 glycoprotein D. Lipid and hemoglobin matrix did not interfere with the assay. The UL32/pp150 [862-1048] IgG ELISA can be used for the sensitive and specific detection of CMV infection in gB-vaccinated individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

Estimation of transfused red cell survival using an enzyme-linked antiglobulin test

International Nuclear Information System (INIS)

Kickler, T.S.; Smith, B.; Bell, W.; Drew, H.; Baldwin, M.; Ness, P.M.

1985-01-01

An enzyme-linked antiglobulin test (ELAT) method was developed to estimate survival of transfused red cells. This procedure is based on a principle analogous to that of the Ashby technique were antigenically distinct red cells are transfused and their survival studied. The authors compared the ELAT survival to the 51 Chromium method ( 51 Cr) in four patients. Three patients with hypoproliferative anemias showed T 1/2 by ELAT of 17.5, 18, and 17 days versus 18.5, 20, and 19 days by the 51 Cr method. A fourth patient with traumatic cardiac hemolysis had two studies performed. In this case, the ELAT showed a T 1/2 of 10 and 8.1 days while 51 Cr T 1/2 values were 11 and 10.5 days. The ELAT method for measuring red cell survival yielded data which agreed closely with the results of the 51 Cr method. Although 51 Cr is the accepted method for red cell survival, the ELAT method can be used to estimate transfused red cell survival

Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

International Nuclear Information System (INIS)

Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

2016-01-01

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders

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

Science.gov (United States)

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

Determination of histamine in Iranian cheese using enzyme-linked ...

African Journals Online (AJOL)

john

enzyme-linked immunosorbent assay (ELISA) method. Mojtaba ... Histamine is a simple chemical substance created during processing of the amine acid histidine. Histamine is also an .... Institute of environment Health and Forensic. Sciences ...

Penggunaan Crude Antigen Cysticercus cellulosae Isolat Bali Untuk Optimalisasi Uji ELISA

Directory of Open Access Journals (Sweden)

Inti Sari Pati R U Sianturi

2017-01-01

Full Text Available Sistiserkosis merupakan penyakit parasitik yang disebabkan oleh larva stadium metacestoda dari cacing pita yang disebut Cysticercus.  Cysticercus yang ditemukan pada babi adalah Cysticercus cellulosae yang merupakan larva dari cacing pita Taenia solium. Penelitian ini dilakukan dengan tujuan mengevaluasi antigen crude Cysticercus cellulosae untuk mendeteksi sistiserkosis pada babi. Cysticercus celllulosae yang digunakan adalah isolat lokal yang diperoleh dari babi terinfeksi Taenia solium asal Karangasem-Bali. Penelitian dilakukan untuk optimalisasi ELISA (Enzyme Linked Immunosorbent Assay terhadap antigen, serum, dan konjugat dengan cara mencari konsentrasi optimal dari antigen, pengenceran optimal serum, dan pengenceran optimal konjugat. Hasil penelitian didapatkan bahwa crude antigen Cysticercus cellulosae isolat Bali bersifat antigenik dan dapat digunakan untuk mendeteksi sistiserkosis pada babi dengan konsentrasi optimal antigen 0,3125 µg/ml, pengenceran optimal serum 1:50, dan pengenceran konjugat 1:2000.

Seasonal variations in antibody response to a Plasmodium ...

African Journals Online (AJOL)

An Enzyme Linked Immunosorbent Assay (ELISA), employing a recombinant peptide capture antigen (R32tet32) was used to detect antibodies against the circumsporozoite protein (CSP) of the malaria parasite, Plasmodium falciparum in 169 ...

An enzyme-linked immunoabsorbent assay for estimating red cell survival of transfused red cells-validation using CR-51 labeling

International Nuclear Information System (INIS)

Drew, H.; Kickler, T.; Smith, B.; LaFrance, N.

1984-01-01

The survival time of transfused red cells antigenically distinct from the recipient's red cells was determined using an indirect enzyme linked antiglobulin test. These results were then compared to those determined by Cr-51 labeling. Three patients with hypoproliferative anemias and one patient (2 studies) with traumatic hemolytic anemia caused by a prosthetic heart valve were studied. Survival times were performed by transfusing a 5cc aliquot of Cr-51 labeled cells along with the remaining unit. One hour post transfusion, a blood sample was drawn and used as the 100% value. Subsequent samples drawn over a 2-3 week period were then compared to the initial sample to determine percent survival for both methods. The ELISA method for measuring red cell survival in antigenically distinct cells is in close agreement with the Cr-51 method. Although CR-51 labeling is the accepted method for red cell survival determination the ELISA method can be used when radioisotopes are unavailable or contraindicated or when the decision to estimate red cell survival is made after transfusion

Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay

OpenAIRE

Gulati, Baldev R.; Munir, Shirin; Patnayak, Devi P.; Goyal, Sagar M.; Kapur, Vivek

2001-01-01

The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detecti...

The use of enzyme-linked immunosorbent assay systems for the serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. Groen (Jan); H.F. Egberink (Herman); G.H.A. Borst (Gerrit); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractComplex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-,

Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

Science.gov (United States)

Li, Zhi; Zhang, Yanlong; Wang, Huiguo; Jin, Jinhua; Li, Wenzhe

2013-01-01

A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease. PMID:24124274

Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum

Science.gov (United States)

Moreno, Elizabeth Castro; Gonçalves, Andréa Vieira; Chaves, Anderson Vieira; Melo, Maria Norma; Lambertucci, José Roberto; Andrade, Antero Silva Ribeiro; Negrão-Corrêa, Deborah; Antunes, Carlos Mauricio de Figueiredo; Carneiro, Mariângela

2009-01-01

Background One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period. Methodology Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve. Findings The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10). Conclusions Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

Science.gov (United States)

Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

2009-05-15

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, pautomated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

Nitrocellulose membrane-based enzyme-linked immunoassay for dengue serotype-1 IgM detection

International Nuclear Information System (INIS)

Leon, S.; Guevara, C.; Chunga, A.

1999-01-01

To evaluate the sensitivity and specifity of a nitrocellulose membrane-based immunoassay for dengue IgM, with respect to capture enzyme immunoassay, for the diagnosis of dengue virus infection. 101 serum samples were processed and divided into 2 groups: 53 from dengue serotype 1 (DEN1) infected patients, and 48 from healthy subjects. Both groups were tested with a nitrocellulose membrane-based IgM capture enzyme immunoassay (NMB-EIA) and also with an ELISA as referential pattern. NMB-EIA testing detected IgM anti-DEN1 in 94,34% of samples from infected patients, and in 14,58% of control samples, whereas ELISA fails to report false positive or false negative results: NMB-EIA appears to be a good alternative for dengue infection diagnosis. (authors)

Indirect Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The indirect competitive ELISA (indirect cELISA) pits plate-immobilized antigen against antigens in solution for binding to antigen-specific antibody. The antigens in solution are in the test sample and are first incubated with antigen-specific antibody. These antibody-antigen complexes are then added to microtiter plates whose wells have been coated with purified antigen. The wells are washed to remove unbound antigen-antibody complexes and free antigen. A reporter-labeled secondary antibody is then added followed by the addition of substrate. Substrate hydrolysis yields a signal that is inversely proportional to antigen concentration within the sample. This is because when antigen concentration is high in the test sample, most of the antibody is bound before adding the solution to the plate. Most of the antibody remains in solution (as complexes) and is thus washed away before the addition of the reporter-labeled secondary antibody and substrate. Thus, the higher the antigen concentration in the test sample, the weaker the resultant signal in the detection step. The indirect cELISA is often used for competitive detection and quantification of antibodies against viral diseases in biological samples. © 2017 Cold Spring Harbor Laboratory Press.

Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

Energy Technology Data Exchange (ETDEWEB)

Ivanov, A P; Rezapkin, G V; Dzagurova, T K; Tkachenko, E A [Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow

1984-05-01

Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

Development and Application of an Indirect Enzyme-Linked Immunosorbent Assay Using Recombinant Mag1 for Serodiagnosis of Toxoplasma gondii In Dogs.

Science.gov (United States)

Zhuo, Xunhui; Sun, Hongchao; Zhang, Zhi; Luo, Jiaqing; Shan, Ying; Du, Aifang

2017-06-01

Serologic tests are widely accepted and applied as means to detect anti- Toxoplasma gondii immunoglobulin G antibodies. In this study, recombinant matrix antigen (rMAG1) was induced by isopropyl-β-d-thiogalactoside and purified by nickel-nitrilotriacetic acid purification system. We then developed and optimized an indirect enzyme-linked immunosorbent assay (ELISA) through checkerboard assays using serial dilutions of antigens and sera to assess the potential use of rMAG1 in serologic detection of T. gondii infection in dogs. Serum samples from 93 domestic dogs were analyzed by western blot and rMAG1-ELISA. The results were compared with those obtained from an ELISA with the soluble Toxoplasma lysate antigens (TLA). We found that although yielding an excellent agreement (96.7%) with western blot data (κ = 0.9659), rMAG1-ELISA produced higher sensitivity (93.9% vs. 87.8%) and specificity (98.3% vs. 96.7%) than TLA-ELISA. In addition, receiver operating characteristic analysis also revealed that rMAG1-ELISA is in more agreement with western blot (area under the curve [AUC] = 0.985) relative to TLA-ELISA (AUC = 0.955). These results indicated that the rMAG1-ELISA established in this study provides a promising and reliable tool for serologic detection of T. gondii infection in dogs.

Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

International Nuclear Information System (INIS)

Safarik, I. Ivo; Safarikova, Mirka

2001-01-01

New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

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Edith S Málaga-Machaca

2017-11-01

Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

Magnetic cross-linked enzyme aggregates (CLEAs): a novel concept towards carrier free immobilization of lignocellulolytic enzymes.

Science.gov (United States)

Bhattacharya, Abhishek; Pletschke, Brett I

2014-01-01

The enzymatic conversion of lignocellulosic biomass into biofuels has been identified as an excellent strategy to generate clean energy. However, the current process is cost-intensive as an effective immobilization approach to reuse the enzyme(s) has been a major challenge. The present study introduces the concept and application of novel magnetic cross-linked enzyme aggregates (mag-CLEAs). Both mag-CLEAs and calcium-mag-CLEAs (Ca-mag-CLEAs) exhibited a 1.35 fold higher xylanase activity compared to the free enzyme and retained more than 80.0% and 90.0% activity, respectively, after 136h of incubation at 50°C, compared to 50% activity retained by CLEAs. A 7.4 and 9.0 fold higher sugar release from lime-pretreated and NH4OH pre-treated sugar bagasse, respectively, was achieved with Ca-mag-CLEAs compared to the free enzymes. The present study promotes the successful application of mag-CLEAs and Ca-mag-CLEAs as carrier free immobilized enzymes for the effective hydrolysis of lignocellulolytic biomass and associated biofuel feedstocks. Copyright © 2014 Elsevier Inc. All rights reserved.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis.

Science.gov (United States)

Bu, Ri-E; Wang, Jin-Liang; Wu, Jin-Hua; Xilin, Gao-Wa; Chen, Jin-Long; Wang, Hua

2017-03-01

The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

The Effect of Different Methods of Fermentation on the Detection of Milk Protein Residues in Retail Cheese by Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Ivens, Katherine O; Baumert, Joseph L; Hutkins, Robert L; Taylor, Steve L

2017-11-01

Milk and milk products are among the most important allergenic food ingredients, both in the United States and throughout the world; cheeses are among the most important of these milk products. Milk contains several major antigenic proteins, each with differing susceptibilities to proteolytic enzymes. The extent of proteolysis in cheese varies as a result of conditions during manufacture and ripening. Proteolysis has the potential to degrade antigenic and allergenic epitopes that are important for residue detection and elicitation of allergic reactions. Commercial enzyme-linked immunosorbent assays (ELISAs) are not currently validated for use in detecting residues in hydrolyzed or fermented food products. Eighteen retail cheeses produced using 5 different styles of fermentation were investigated for detectable milk protein residues with 4 commercial ELISA kits. Mozzarella, Swiss, Blue, Limburger, and Brie cheeses were assessed. The Neogen Veratox® Casein and Neogen Veratox® Total Milk kits were capable of detecting milk residues in most cheeses evaluated, including blue-veined cheeses that exhibit extensive proteolysis. The other 2 ELISA kits evaluated, r-Biopharm® Fast Casein and ELISA Systems™ Casein, can detect milk residues in cheeses other than blue-veined varieties. ELISA results cannot be quantitatively compared among kits. The quantitative reliability of ELISA results in detection of cheese residues is questionable, but some methods are sufficiently robust to use as a semi-quantitative indication of proper allergen control for the validation of cleaning programs in industry settings. Many commercially available enzyme-linked immunosorbent assays (ELISAs) are not validated for detection of allergenic residues in fermented or hydrolyzed products. This research seeks to determine if commercial milk ELISAs can detect milk residues in varieties of cheese that have undergone different styles of fermentation and different degrees of proteolysis. Only certain

Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

Directory of Open Access Journals (Sweden)

Raba Julio

2011-10-01

Full Text Available Abstract Background Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA for quantification of B. cinerea in apple (Red Delicious, table grape (pink Moscatel, and pear (William's tissues. Results The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. Conclusions The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

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.

Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

NARCIS (Netherlands)

Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

2000-01-01

The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

Pelacakan Secara Imunohistokimiawi Antigen Virus pada Ayam yang Diinfeksi dengan Virus Penyakit Tetelo (IMMUNOHISTOCHEMICAL DETECTION OF VIRAL ANTIGEN IN TISSUE OF CHICKENS EXPERIMENTALLY INFECTED WITH NEWCASTLE DISEASE VIRUS

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Anak Agung Ayu Mirah Adi

2013-07-01

Full Text Available In order to study the distribution of Newcastle disease virus (NDV following infection, chickenswere experimentally infected with visceretropic velogenic NDV isolate. Monoclonal antibodies (mAbsagainst the NDV LaSota vaccine strain were then produced to detect viral antigen in the infectedorgans. The mAbs were firstly tested for their specificity by enzyme linked immunosorbent assay(ELISA using NDV and normal allantoic fluids as antigens. Eight mAbs specific against NDVwere isolated and two mAbs were used for immunodetection of NDV antigen in chicken’s tissues.By immunohistochemistry labeled streptavidin-biotin (LSAB staining NDV–antigen was detectedin paraffin embedded tissues of NDV-infected chickens. NDV antigen was not detected in noninfected chickens. In the infected chickens, high intensity of NDV antigen was detected in thelymphoid tissues, lung and intestine. The NDV antigen with a lesser intensity was detected in thebrain, trachea, liver and myocardium. This study shows that although viscerotropic velogenicNDV isolate can infect almost all organs, the main target of infection are lung, intestine andlymphoids tissues

Low-Energy Solvents For Carbon Dioxide Capture Enabled By A Combination Of Enzymes And Vacuum Regeneration

Energy Technology Data Exchange (ETDEWEB)

Salmon, Sonja [Novozymes North America, Inc., Franklinton, NC (United States); House, Alan [Novozymes North America, Inc., Franklinton, NC (United States); Liu, Kun [Univ. of Kentucky, Lexington, KY (United States); Frimpong, Reynolds [Univ. of Kentucky, Lexington, KY (United States); Liu, Kunlei [Univ. of Kentucky, Lexington, KY (United States); Freeman, Charles [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Whyatt, Greg [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Slater, Jonathan [Doosan Babcock, Renfew (United Kingdom); Fitzgerald, David [Doosan Babcock, Renfew (United Kingdom)

2015-08-31

An integrated bench-scale system combining the attributes of the bio-renewable enzyme carbonic anhydrase (CA) with low-enthalpy CO2 absorption solvents and vacuum regeneration was designed, built and operated for 500 hours using simulated flue gas. The objective was to develop a CO2 capture process with improved efficiency and sustainability when compared to NETL Case 10 monoethanolamine (MEA) scrubbing technology. The use of CA accelerates inter-conversion between dissolved CO2 and bicarbonate ion to enhance CO2 absorption, and the use of low enthalpy CO2 absorption solvents makes it possible to regenerate the solvent at lower temperatures relative to the reference MEA-based solvent. The vacuum regeneration-based integrated bench-scale system operated successfully for an accumulated 500 hours using aqueous 23.5 wt% K2CO3-based solvent containing 2.5 g/L enzyme to deliver an average 84% CO2 capture when operated with a 20% enzyme replenishment rate per ~7 hour steady-state run period. The total inlet gas flow was 30 standard liters per minute with 15% CO2 and 85% N2. The absorber temperature was 40°C and the stripper operated under 35 kPa pressure with an approximate 77°C stripper bottom temperature. Tests with a 30°C absorber temperature delivered >90% capture. On- and off-line operational measurements provided a full process data set, with recirculating enzyme, that allowed for enzyme replenishment and absorption/desorption kinetic parameter calculations. Dissolved enzyme replenishment and conventional process controls were demonstrated as straightforward approaches to maintain system performance. Preliminary evaluation of a novel flow-through ultrasonically enhanced regeneration system was also conducted, yet resulted in CO2 release within the range of temperature-dependent release, and further work would be needed to validate the benefits of ultrasonic enhanced stripping. A full technology assessment was completed in which four techno-economic cases for

Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

Science.gov (United States)

Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

2015-08-10

The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

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

OpenAIRE

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 th...

Immobilization of cross-linked tannase enzyme on multiwalled carbon nanotubes and its catalytic behavior.

Science.gov (United States)

Ong, Chong-Boon; Annuar, Mohamad S M

2018-02-07

Immobilization of cross-linked tannase on pristine multiwalled carbon nanotubes (MWCNT) was successfully performed. Cross-linking of tannase molecules was made through glutaraldehyde. The immobilized tannase exhibited significantly improved pH, thermal, and recycling stability. The optimal pH for both free and immobilized tannase was observed at pH 5.0 with optimal operating temperature at 30°C. Moreover, immobilized enzyme retained greater biocatalytic activities upon 10 repeated uses compared to free enzyme in solution. Immobilization of tannase was accomplished by strong hydrophobic interaction most likely between hydrophobic amino acid moieties of the glutaraldehyde-cross-linked tannase to the MWCNT.

Enzyme-linked immunosorbent assay and polymerase chain reaction performance using Mexican and Guatemalan discrete typing unit I strains of Trypanosoma cruzi.

Science.gov (United States)

Ballinas-Verdugo, Martha; Reyes, Pedro Antonio; Mejia-Dominguez, Ana; López, Ruth; Matta, Vivian; Monteón, Victor M

2011-12-01

Thirteen Trypanosoma cruzi isolates from different geographic regions of Mexico and Guatemala belonging to discrete typing unit (DTU) I and a reference CL-Brener (DTU VI) strain were used to perform enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). A panel of 57 Mexican serum samples of patients with chronic chagasic cardiopathy and asymptomatic infected subjects (blood bank donors) were used in this study. DNA from the above 14 T. cruzi strains were extracted and analyzed by PCR using different sets of primers designed from minicircle and satellite T. cruzi DNA. The chronic chagasic cardiopathy serum samples were easily recognized with ELISA regardless of the source of antigenic extract used, even with the CL-Brener TcVI, but positive serum samples from blood bank donors in some cases were not recognized by some Mexican antigenic extracts. On the other hand, PCR showed an excellent performance despite the set of primers used, since all Mexican and Guatemalan T. cruzi strains were correctly amplified. In general terms, Mexican, Guatemalan, and CL-Brener T. cruzi strains are equally good sources of antigen when using the ELISA test to detect Mexican serum samples. However, there are some strains with poor performance. The DTU I strains are easily detected using either kinetoplast or satellite DNA target designed from DTU VI strains.

Genipin Cross-Linked Glucose Oxidase and Catalase Multi-enzyme for Gluconic Acid Synthesis.

Science.gov (United States)

Cui, Caixia; Chen, Haibin; Chen, Biqiang; Tan, Tianwei

2017-02-01

In this work, glucose oxidase (GOD) and catalase (CAT) were used simultaneously to produce gluconic acid from glucose. In order to reduce the distance between the two enzymes, and therefore improve efficiency, GOD and CAT were cross-linked together using genipin. Improvements in gluconic acid production were due to quick removal of harmful intermediate hydrogen peroxide by CAT. GOD activity was significantly affected by the proportion of CAT in the system, with GOD activity in the cross-linked multi-enzyme (CLME) being 10 times higher than that in an un-cross-linked GOD/CAT mixture. The glucose conversion rate after 15 h using 15 % glucose was also 10 % higher using the CLME than was measured using a GOD/CAT mixture.

Validity and reliability of enzyme immunoassays using Leishmania major or L. infantum antigens for the diagnosis of canine visceral leishmaniasis in Brazil.

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Mauro Maciel de Arruda

Full Text Available BACKGROUND: American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. METHODS: A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. RESULTS: The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. INTERPRETATION: ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.

Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

Science.gov (United States)

Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

2018-05-01

We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

Science.gov (United States)

Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

2018-03-01

We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

Purification and characterization of a major human Pneumocystis carinii surface antigen

DEFF Research Database (Denmark)

Lundgren, B; Lipschik, G Y; Kovacs, J A

1991-01-01

. To evaluate humoral immune responses to the human P. carinii protein, an enzyme-linked immunosorbent assay using purified protein was developed. Some, but not all, patients who subsequently developed P. carinii pneumonia demonstrated a serum antibody response to the surface antigen. Nearly all subjects...... without a history of P. carinii pneumonia had no detectable antibodies. Purified P. carinii proteins will greatly facilitate the investigation of host-P. carinii interactions....

Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

Energy Technology Data Exchange (ETDEWEB)

Birch, C J; Lehmann, N I; Hawker, A J; Marshall, J A; Gust, I D [Fairfield Hospital for Communicable Diseases, Victoria (Australia). Virology Dept.

1979-07-01

Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.

Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

International Nuclear Information System (INIS)

Birch, C.J.; Lehmann, N.I.; Hawker, A.J.; Marshall, J.A.; Gust, I.D.

1979-01-01

Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy. (author)

The Biological Deep Sea Hydrothermal Vent as a Model to Study Carbon Dioxide Capturing Enzymes

Directory of Open Access Journals (Sweden)

Premila D. Thongbam

2011-04-01

Full Text Available Deep sea hydrothermal vents are located along the mid-ocean ridge system, near volcanically active areas, where tectonic plates are moving away from each other. Sea water penetrates the fissures of the volcanic bed and is heated by magma. This heated sea water rises to the surface dissolving large amounts of minerals which provide a source of energy and nutrients to chemoautotrophic organisms. Although this environment is characterized by extreme conditions (high temperature, high pressure, chemical toxicity, acidic pH and absence of photosynthesis a diversity of microorganisms and many animal species are specially adapted to this hostile environment. These organisms have developed a very efficient metabolism for the assimilation of inorganic CO2 from the external environment. In order to develop technology for the capture of carbon dioxide to reduce greenhouse gases in the atmosphere, enzymes involved in CO2 fixation and assimilation might be very useful. This review describes some current research concerning CO2 fixation and assimilation in the deep sea environment and possible biotechnological application of enzymes for carbon dioxide capture.

Comparison of agal gel precipitation test (AGPT) and enzyme linked ...

African Journals Online (AJOL)

The use of agar gel precipitation test (AGPT) and enzyme linked immunosorbent assay (ELISA) in assaying for the presence of infectious bursal disease (IBD) virus antibody in village chickens in Oyo State, Nigeria, was compared. Out of 400 sera subjected to ELISA, 360 (90%) samples where positive for IBD virus ...

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

DEFF Research Database (Denmark)

Rasmussen, M; Dahl, M; Buus, S

2014-01-01

. 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....../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...

A New Enzyme-linked Sorbent Assay (ELSA) to Quantify Syncytiotrophoblast Extracellular Vesicles in Biological Fluids.

Science.gov (United States)

Göhner, Claudia; Weber, Maja; Tannetta, Dionne S; Groten, Tanja; Plösch, Torsten; Faas, Marijke M; Scherjon, Sicco A; Schleußner, Ekkehard; Markert, Udo R; Fitzgerald, Justine S

2015-06-01

The pregnancy-associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are available and accepted, though imperfect. For this purpose, we aimed to provide an enzyme-linked sorbent assay (ELSA) to quantify STBEV in fluid samples based on their microvesicle characteristics and placental origin. Ex vivo placenta perfusion provided standards and samples for the STBEV quantification. STBEV were captured by binding of extracellular phosphatidylserine to immobilized annexin V. The membranous human placental alkaline phosphatase on the STBEV surface catalyzed a colorimetric detection reaction. The described ELSA is a rapid and simple method to quantify STBEV in diverse liquid samples, such as blood or perfusion suspension. The reliability of the ELSA was proven by comparison with nanoparticle tracking analysis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

DEFF Research Database (Denmark)

Deufel, T; Grove, A; Kofod, Hans

1985-01-01

Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

Science.gov (United States)

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

Directory of Open Access Journals (Sweden)

Sam Horrell

2018-05-01

Full Text Available High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT, providing an unparallelled level of structural information during catalysis for redox enzymes.

Enzyme-linked immunosorbent assays for Z-DNA.

OpenAIRE

Thomas, M J; Strobl, J S

1988-01-01

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e....

Schistosome circulating anodic antigen in serum of individuals infected with Schistosoma japonicum from the Philippines before and after chemotherapy with praziquantel

NARCIS (Netherlands)

van 't Wout, A. B.; de Jonge, N.; Tiu, W. U.; Garcia, E. E.; Mitchell, G. F.; Deelder, A. M.

1992-01-01

The presence of the schistosome circulating anodic antigen (CAA) in serum of patients infected with Schistosoma japonicum from The Philippines has been investigated using an enzyme-linked immunosorbent assay (ELISA). Serum samples were tested from 48 patients who excreted S. japonicum eggs, 9

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

Directory of Open Access Journals (Sweden)

Gardenia Braz Figueiredo Carvalho

2011-11-01

Full Text Available The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.

Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA

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Paula Ciaurriz

2017-01-01

Full Text Available The enzyme-linked immunosorbent assay (ELISA technique is based on the specific recognition ability of the molecular structure of an antigen (epitope by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs as a vehicle for secondary antibodies and peroxidase (HRP. The design of experiments technique (DOE and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof. As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.

Development of enzyme-linked immunosorbent assay for detecting Ornithobacterium rhinotracheale (ORT infection in chicken

Directory of Open Access Journals (Sweden)

Adin Priadi

2006-10-01

Full Text Available Ornithobacterium rhinotracheale (ORT has been recognized in chicken in Indonesia and incriminated as a possible additional causative agent in respiratory disease complex. An enzyme-linked immunosorbent assay (ELISA has been developed for the seroepidemiological study of ORT infection in chickens. Ten weeks old chickens are injected with 0.5 ml of killed O. rhinotracheale emulsified in Freund's complete adjuvant at a concentration of 109 CFU/ml. Hyperimmune sera and non-reactive control sera were used to standardized the ELISA for ORT infection. Optimum condition for the ORT ELISA was antigen dilution 1/800, serum dilution 1/100 and 1/4000 conjugate dilution. Optical density cut-off point was determined by using 31 serum samples from 2 broiler farms. Cut-off for negative serum was 0.27 (mean + 3 standard deviation. With these optima, 187 chicken sera from broiler, layer and broiler breeder farms were collected and screened. Seroconvertions were detected from broiler and layer farms in Magelang district, Central Java (Bojong I, Paremono, Bojong II, Keblukan and a broiler breeder farm in West Java. The seraconvertion were 0, 10, 94, 88 and 100 percents respectively. These figures show that the prevalence of O. rhinotracheale infection in chicken in layer and breeder farms were very high.

Improving dengue viral antigens detection in dengue patient serum specimens using a low pH glycine buffer treatment.

Science.gov (United States)

Shen, Wen-Fan; Galula, Jedhan Ucat; Chang, Gwong-Jen J; Wu, Han-Chung; King, Chwan-Chuen; Chao, Day-Yu

2017-04-01

Early diagnosis of dengue virus (DENV) infection to monitor the potential progression to hemorrhagic fever can influence the timely management of dengue-associated severe illness. Nonstructural protein 1 (NS1) antigen detection in acute serum specimens has been widely accepted as an early diagnostic assay for dengue infection; however, lower sensitivity of the NS1 antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) in secondary dengue viral infection has been reported. In this study, we developed two forms of Ag-ELISA capable of detecting E-Ag containing virion and virus-like particles, and secreted NS1 (sNS1) antigens, respectively. The temporal kinetics of viral RNA, sNS1, and E-Ag were evaluated based on the in vitro infection experiment. Meanwhile, a panel of 62 DENV-2 infected patients' sera was tested. The sensitivity was 3.042 ng/mL and 3.840 ng/mL for sNS1 and E, respectively. The temporal kinetics of the appearance of viral RNA, E, NS1, and infectious virus in virus-infected tissue culture media suggested that viral RNAs and NS1 antigens could be detected earlier than E-Ag and infectious virus. Furthermore, a panel of 62 sera from patients infected by DENV Serotype 2 was tested. Treating clinical specimens with the dissociation buffer increased the detectable level of E from 13% to 92% and NS1 antigens from 40% to 85%. Inclusion of a low-pH glycine buffer treatment step in the commercially available Ag-ELISA is crucial for clinical diagnosis and E-containing viral particles could be a valuable target for acute DENV diagnosis, similar to NS1 detection. Copyright © 2015. Published by Elsevier B.V.

Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24)

International Nuclear Information System (INIS)

Neurath, A.R.; Strick, N.; Lee, Y.S.; Nilsen, T.; Baker, L.; Sproul, P.; Rubinstein, P.; Taylor, P.; Stevens, C.E.; Gold, J.W.M.

1985-01-01

Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and 125 I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA) - or enzyme-linked immunoassay (ELISA) - inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed. (Auth.)

Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

Science.gov (United States)

Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

2012-04-01

Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Evidence for carcinoembryonic antigen using radioimmunoassay and enzyme immunoassay

International Nuclear Information System (INIS)

Kungda Gao, L.

1980-01-01

A commercially available radioimmunoassay for the determination of carcinoembryonic antigen (CEA) was initially compared with an enzyme immunoassay (EIA). Considerable differences were found between the individual value. For three patients suffering from carcinomas of the digestive tract a better indication of the disease was given in the RIA than in the EIA. A further 110 patients with various illnesses were examined for serum CEA-levels using RIA. A method for measuring CEA in feces by RIA was developed. The normal range lies below 300 ng/ml. This assay could be of significance for the early recognition of colo-rectal carcinoma. In part II of this dissertation CEA was isolated from colo-rectal carcinomas using three different gel filtration media. It was only possible to obtain almost pure CEA (24 μg CEA per μg protein) by one of the methods. Six guinea pigs were immunized with the isolated CEA and all developed antibodies. The isolated CEA was labelled with 125 I and an own RIA saturation sequence and double antibody separation was developed. One of the antisera was able to distinguish without overlap 7 healthy patients from 7 suffering from colo-rectal carcinomas in non-extracted serum. (orig./MG) [de

Enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin.

Science.gov (United States)

Gao, Shunxiang; Zheng, Xin; Hu, Bo; Sun, Mingjuan; Wu, Jihong; Jiao, Binghua; Wang, Lianghua

2017-03-15

In this study, we coupled biolayer interferometry (BLI) with competitive binding assay through an enzyme-linked aptamer and developed a real-time, ultra-sensitive, rapid quantitative method for detection of the marine biotoxin palytoxin. Horseradish peroxidase-labeled aptamers were used as biorecognition receptors to competitively bind with palytoxin, which was immobilized on the biosensor surface. The palytoxin: horseradish peroxidase-aptamer complex was then submerged in a 3,3'-diaminobenzidine solution, which resulted in formation of a precipitated polymeric product directly on the biosensor surface and a large change in the optical thickness of the biosensor layer. This change could obviously shift the interference pattern and generate a response profile on the BLI biosensor. The biosensor showed a broad linear range for palytoxin (200-700pg/mL) with a low detection limit (0.04pg/mL). Moreover, the biosensor was applied to the detection of palytoxin in spiked extracts and showed a high degree of selectivity for palytoxin, good reproducibility, and stability. This enzyme-linked, aptamer-based, competitive BLI biosensor offers a promising method for rapid and sensitive detection of palytoxin and other analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

[Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium].

Science.gov (United States)

Alvarado, Primavera; Ostos, Ana; Franquiz, Nohelys; Roschman-González, Antonio; Zambrano, Edgar A; Mendoza, Mireya

2015-06-01

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Preparation of anti-Sudan red monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of Sudan red in chilli jam and chilli oil.

Science.gov (United States)

Xu, Jing; Zhang, Yuanyang; Yi, Jian; Meng, Meng; Wan, Yuping; Feng, Caiwei; Wang, Shanliang; Lu, Xiao; Xi, Rimo

2010-10-01

Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC(50) value of 1.7 μg L(-1) in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 μg L(-1) and 19.6 μg L(-1), respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%-110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

Design of indirect solid-phase immunosorbent methods for detecting arenavirus antigens and antibodies

Energy Technology Data Exchange (ETDEWEB)

Ivanov, A P; Rezapkin, G V; Dzagurova, T K; Tkachenko, E A

1984-05-01

Specifications have been elaborated for formulating indirect solid-phase enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (SPRIA) methods that employ anti-human and anti-mice G class immunoglobulin (IgG), conjugated with horseradish peroxidase and /sup 125/I for detecting the arenaviruses Junin, Machupo, Tacaribe, Amalpari, Tamiami, Lassa, and LCM (lymphocytic choriomeningitis). These methods make it possible to identify with a high degree of sensitivity arenavirus antigens and antibodies in various kinds of material.

Antigen based detection of cystic echinococcosis in buffaloes using ELISA and Dot-EIA.

Science.gov (United States)

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 %.

Dictyostelium discoideum: mutants in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides

International Nuclear Information System (INIS)

Freeze, H.; Willies, L.; Hamilton, S.

1986-01-01

The lysosomal enzymes of Dictyostelium discoideum share highly immunogenic oligosaccharides which contain multiple Man-6-SO 4 residues. Two mutant strains which lack the shared antigenic determinant were analyzed in an attempt to identify the primary defect in each. [ 3 H]Man labelled N-linked oligosaccharides of secreted glycoproteins were released by Endo/PNGaseF digestion and analyzed. Both of the mutant strains produced smaller, less sulfated oligosaccharides compared to the wild-type, yet both still contained considerable amounts of Man-6-SO 4 . The size of the precursor lipid-linked oligosaccharide from the wild-type is consistent with a Glc 3 Man 9 GlcNAc 2 structure, while those from both of the mutants have an oligosaccharide the size of Man 5 GlcNAc 2 . The authors conclude that both of the mutants are defective in the biosynthesis of the precursor oligosaccharide. Both oligosaccharides from the mutants contain a tri-mannosyl core and are not glucosylated. Two of the five Man residues are released by a 1,2 specific α mannosidase. Based on the size and mannosidase digestions the authors conclude that 4/5 of the Man residues on the α1,6 branch of the β-linked Man residues are missing. Thus, these residues must be required to define the shared antigenic determinant

Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

Science.gov (United States)

Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

2001-10-01

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

Correlation between the e-antigen, Pre-S2 antigen and DNA of hepatitis B virus

International Nuclear Information System (INIS)

Cai Changhui; Liang Jinsheng

2006-01-01

Objective: To study the relationship between the hepatitis B e-antigen (HBeAg), Pre-S1 antigen (Pre-S1), Pre-S2 antigen (Pre-S2) and DNA of hepatitis B virus (HBV). Methods: The blood samples of 268 cases of viral B hepatitis were collected. The HBV DNA of all samples were tested by fluorescent-quantitating PCR method, and HBeAg were assayed by time-resolved fluoro-immunoassay method, and their Pre-S1 and Pre-S2 were assayed by enzyme linked immunosorbentassay method. Results: The positive rates of HBeAg, Pre-S1 and Pre-S2 in HBV DNA positive group were 48.2%, 76.4% and 100% respectively, and 1.6%, 36.3% and 32.3% respectively in HBV DNA negative group. There was significantly difference between the HBeAg, Pre-S1 and Pre-S2 positive rates of the two groups (Chi-square test, P<0.01). Conclusions: There was positive relationship between the HBeAg, Pre-S1, Pre-S2 and DNA which all were indicators of HBV reproduction. Comparing to HBV DNA, Pre-S2 was the most, Pre-S1 the second, and HBeAg the third sensitive indicator for evaluating HBV reproduction. Pre-S1 and Pre-S2 could be used as the supplementary indicator for the reproduction of HBV. (authors)

Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24)

Energy Technology Data Exchange (ETDEWEB)

Neurath, A R; Strick, N; Lee, Y S; Nilsen, T; Baker, L; Sproul, P; Rubinstein, P; Taylor, P; Stevens, C E; Gold, J W.M.

1985-10-01

Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and /sup 125/I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA) - or enzyme-linked immunoassay (ELISA) - inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed. 17 refs.; 2 figs.; 1 table.

B-cell responses to pregnancy-restricted and -unrestricted Plasmodium falciparum erythrocyte membrane protein 1 antigens in Ghanaian women naturally exposed to malaria parasites

DEFF Research Database (Denmark)

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F

2014-01-01

-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two...... commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods...

Definition of a virulence-related antigen of Neisseria gonorrhoeae with monoclonal antibodies and lectins.

Science.gov (United States)

Demarco de Hormaeche, R; Bundell, C; Chong, H; Taylor, D W; Wildy, P

1986-03-01

Variants of one strain of Neisseria gonorrhoeae, grown in vivo or in vitro, that have been previously shown to differ in infectivity, serum resistance, and capsule production were compared with use of monoclonal antibodies and lectins. Monoclonal antibodies to virulent gonococci recognized an antigenic site of the lipopolysaccharide (LPS) produced in large amounts by gonococci grown in vivo but present only in a small proportion of in vitro-grown gonococci. This antigen (C-LPS) was found in all 85 different gonococcal isolates studied but not among nonpathogenic neisseriae. It was shared by group B and C meningococci but not by groups A and D. Enzyme-linked immunosorbent assay and Western blot analysis showed that N-acetylglucosamine and N-acetylgalactosamine form part of the epitope. The C-LPS antigen was shown by immunofluorescence to be present on the surface of the gonococci and also free as slime. This antigen appears to confer resistance to killing by normal sera.

HUMORAL IMMUNE-RESPONSE TO 2,4-DINITROPHENYL KEYHOLE LIMPET HEMOCYANIN IN ANTIGEN-FREE, GERM-FREE AND CONVENTIONAL BALB/C MICE

NARCIS (Netherlands)

BOS, NA; PLOPLIS, VA

The B cell immune response to 2,4-dinitrophenyl (DNP) keyhole limpet hemocyanin was compared in antigen-free, germ-free and conventional BALB/c mice. The numbers of total and of DNP-specific IgM-, IgG- and IgA-secreting cells in the spleen were determined by enzyme-linked immunosorbent plaque assays

Cystatin capture elisa immunodiagnosis of human fasciolosis at Chupaca-Junin Province

International Nuclear Information System (INIS)

Cornejo, W.; Alva, P.; Sevilla, C.; Huiza, A.; Universidad Nacional Mayor de San Marcos, Lima

2003-01-01

To determine the prevalence of human fasciolosis in an endemic area by means of an Enzyme-Linked Immunosorbent Assay (ELISA) using cystatin as a capture agent for the detection of specific antibodies to fasciola hepatica cysteine proteinases. An ELISA plate was sensitized with cystatin, incubated with excretory-secretory products of adult flukes, and followed by standard ELISA procedures. Clinical applicability of the cystatin capture ELISA for the immunodiagnosis of fasciolosis was tested with 200 serum samples of children and adults from an endemic area in Chupaca province, Junin department. Serum samples from the endemic area tested by cystatin capture ELISA showed 27/200 (13,5%) of positive cases. Fasciolosis remains a major health problem at Chupaca province, Junin department

Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases

International Nuclear Information System (INIS)

Falo, L.D. Jr.; Haber, S.I.; Herrmann, S.; Benacerraf, B.; Rock, K.L.

1987-01-01

To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) the authors analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A 2 and phospholipase C. In addition it is observed with three distinct antigens - ovalbumin, bovine insulin, and poly(LGlu 56 LLys 35 LPhe 9 )[(GluLysPhe)/sub n/]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to control in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or processing independent antigens. In parallel studies 125 I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125 I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. These studies demonstrate that phospholipase effectively removes processed cell surface antigen

Antibody-based enzyme-linked lectin assay (ABELLA) for the sialylated recombinant human erythropoietin present in culture supernatant.

Science.gov (United States)

Kim, Hyoung Jin; Lee, Seung Jae; Kim, Hong-Jin

2008-11-04

The terminal sialic acid of human erythropoietin (hEPO) is essential for in vivo activity. The current resorcinol and HPLC methods for analyzing alpha2,3-linked sialic acid require more than a microgram of purified rhEPO, and purification takes a great deal of time and labor. In this study, we assessed the use of an antibody-based enzyme-linked lectin assay (ABELLA) for analyzing non-purified recombinant hEPO (rhEPO). The major problem of this method was the high background due to terminal sialylation of components of the assay (antibody and bovine serum albumin) other than rhEPO. To solve this problem, we used a monoclonal antibody (Mab 287) to capture the rhEPO, and oxidized the bovine serum albumin used for blocking with meta-periodate. The sialic acid content of non-purified rhEPO measured by ABELLA was similar to that obtained by the resorcinol method on purified rhEPO. ABELLA has advantages such as adaptability and need for minimal amounts of rhEPO (40 ng/ml). Our observations suggest that ABELLA should reduce the time and labor needed to improve culture conditions so as to increase protein sialylation, and also facilitate the study of sialylation mechanisms.

Enhancement of Human Antigen-Specific Memory T-Cell Responses by Interleukin-7 May Improve Accuracy in Diagnosing Tuberculosis▿ †

Science.gov (United States)

Feske, Marsha; Nudelman, Rodolfo J.; Medina, Miguel; Lew, Justin; Singh, Manisha; Couturier, Jacob; Graviss, Edward A.; Lewis, Dorothy E.

2008-01-01

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2 = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB. PMID:18753334

Parasite enzymes as a tool to investigate immune responses

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Italo M. Cesari

1992-01-01

Full Text Available Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzume antigens might be also intersting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg*+, pH 9.5, type I phosphodiesterase (PDE, pH 9.5, cysteine proteinase (CP, dithiothreitol, pH 5.5 and N-acetyl-ß-D-glucosaminidase (NAG, pH 5.5. The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes.

Enzyme-linked immunosorbent Assay for detecting of antibody to canine distemper virus

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Sudarisman

2006-03-01

Full Text Available Serum neutralisation test (SNT has been established for evaluating canine distemper vaccination, but until now SNT was rarely used due to the need for continuous tissue culture facilities and requires 3 days to perform. For detecting antibody to canine distemper virus, an enzyme-linked immunosorbent assay (ELISA is relatively simple and rapid seroassay. ELISA for canine immunoglobulin (Ig G antibodies to canine distemper virus (CDV was developed by using Onderstepoort strain of canine distemper virus as coating antigen. Rabbit anti canine IgG labelled with horse radish peroxidase was used as the conjugate, while phenylenediamine dihydrochloride (OPD was used as the substrate. The ELISA results were then compared with the results of the SNT, using the sera of 312 random-source dogs from West Java. The two test-results had a high degree of correlation. Very few discrepancies occurred and most of these were at the lower limits of each test. When the sera were tested at 1 : 100 dilutions, there was a 95.5% agreement between the ELISA and SNT. Their sensitivity and spesificity were 83.9 and 98.4%. Titrated SNT and ELISA also were performed on sera from 7 dogs whose lifetime medical histories were known. The antibodies were inclining up after two months of post vaccination, where the titre was not in zero/lower position at the day of vaccination. However, antibody zero or low position were found at 28 days post vaccination. All of the results indicated that ELISA can be used for evaluating antibody to canine distemper virus response, replacing the SNT.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts.

Science.gov (United States)

Dalton, K P; Podadera, A; Granda, V; Nicieza, I; Del Llano, D; González, R; de Los Toyos, J R; García Ocaña, M; Vázquez, F; Martín Alonso, J M; Prieto, J M; Parra, F; Casais, R

2018-01-01

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×10 8 molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Reduced Serum IgG Responses to Pneumococcal Antigens in Otitis-Prone Children May Be Due to Poor Memory B-Cell Generation

Science.gov (United States)

Sharma, Sharad K.; Casey, Janet R.

2012-01-01

A low level of serum antibody to antigens expressed by Streptococcus pneumoniae has been proposed to explain the susceptibility of children to recurrent episodes of acute otitis media (hereafter, “otitis-prone children”). By use of enzyme-linked immunospot assays, the percentages of memory B cells to pneumococcal protein antigens PhtD, LytB, PcpA, PhtE, and Ply were compared between otitis-prone and non–otitis-prone children at the time of acute otitis media or nasopharyngeal colonization with S. pneumoniae. We found significantly lower percentages of memory B cells to 3 pneumococcal protein antigens (PhtD, PhtE, and Ply) and reduced antigen-specific immunoglobulin G concentrations in otitis-prone children, compared with non–otitis-prone children. PMID:22383675

The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15.

Science.gov (United States)

Ahmed, Zubair M; Goodyear, Richard; Riazuddin, Saima; Lagziel, Ayala; Legan, P Kevin; Behra, Martine; Burgess, Shawn M; Lilley, Kathryn S; Wilcox, Edward R; Riazuddin, Sheikh; Griffith, Andrew J; Frolenkov, Gregory I; Belyantseva, Inna A; Richardson, Guy P; Friedman, Thomas B

2006-06-28

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation.

Shedding of leukemia-associated P24 antigen by lymphoblastoid cell lines.

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Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

1987-12-01

We report the development of a unique enzyme-linked immunosorbent assay (ELISA) which makes possible the detection of leukemia-associated P24 antigen, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4 simultaneously. Using the RCA1/SJ-9A4-ELISA, P24 antigen, as few as 50 X 10(3) cells from a common acute lymphoblastic leukemia (C-ALL) cell line could be detected. The presence of D-galactose gave complete and specific inhibition of P24 antigen binding to RCA1. Matched concentrations of D-glucose and D-sucrose had no effect on binding. The release of the P24 antigen into the culture medium by a C-ALL cell line maintained at 37 degrees C could be detected; however, no P24 antigen was present in the culture medium when the cells were maintained at 4 degrees C. Sequential analysis of the culture medium for soluble P24 antigen revealed that release of the P24 antigen associated with cell growth. Molecular sieve chromatography of concentrated culture medium indicated that shed P24 antigen was eluted in the macromolecule fraction. P24 antigen was detected in the cerebrospinal fluid (CSF) of four patients with P24 positive ALL at the time of relapse of the central nervous system (CNS) and was undetectable while in complete remission. The CSF from three patients with P24 negative ALL and three patients with aseptic meningitis had no detectable activity.

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

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Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease.

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Gonzales Santana, Bibiana; Dalton, John P; Vasquez Camargo, Fabio; Parkinson, Michael; Ndao, Momar

2013-01-01

Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.

Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato

1988-01-01

We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-γ. In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G 2 /M phases of the cell cycle than in G 0 /G 1 . The expression of YH206 antigen was enhanced in the S and G 2 /M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens. (author)

AN EVALUATION STUDY OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA USING RECOMBINANT PROTEIN GRA1 FOR DETECTION OF IGG ANTIBODIES AGAINTS TOXOPLASMA GONDII INFECTIONS

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Nina Difla Muflikhah

2017-08-01

Full Text Available Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, an intracellular protozoan parasite that live inside the cells of the reticulo endothelial and parenchymal cells of human and animals (mammals and birds. Some cases of toxoplasmosis usually have no symptoms, but in any cases caused severe symptoms, such as hydrocephalus, microcephalus, intracranial calcification, retinal damage, brain abscess, mental retardation, lymphadenopathy, and others. Its severe symptoms usually showed a long time after first exposure, except symptoms showed by congenital transmission caused by infected mother. Early diagnosis is important to prevent the illness but methods for toxoplasmosis screening are still too expensive for developing country. Enzyme-linked immunosorbent assay (ELISA allow the testing of a large number samples within short time frame and based on antibody or antigen detection. This study aimed to know the sensitivity and specificity of recombinat protein GRA1 as antigen using ELISA methods. We tested the sensitivity and spesificity of GRA1 protein as antigen in ELISA methods to diagnose toxoplasmosis and compared with ELISA Kit Commercial. Reliable laboratory testing is important to detect Toxoplasma gondii infection, and focused to improving the low cost and easy-to-use diagnostic instrument. Seventy sera collected and tested using both indirect ELISA, commercial ELISA kit and GRA1 protein coated as antigen. Fourty eight and fifty one samples showed positive IgG antibody result of ELISA-GRA1 and ELISA kit. Negative sample tested by ELISA-GRA1 was 22 samples and 19 sample tested by ELISA Kit. The sensitivity and specificity of GRA1-based on ELISA were 100% and 86.36%, positive prediction value (ppv was 94.11%. These data indicate that the recombinant protein GRA1 is a highly immunogenic protein in human toxoplasmosis and become a promising marker for the screening of toxoplasmosis.

The use of a synthetic antigen for the serological diagnosis of human trichinellosis

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Bruschi F.

2001-06-01

Full Text Available Hosts infected with Trichinella produce antibodies specific for an epitope common to the TSL-1 family antigens. This epitope contained uncommon terminal 3, 6-dideoxy-D-arabinohexose (so called tyvelose residues. The disaccharide moiety was synthesized and an immunodiagnostic assay was developed, which was specific and sensitive in swine trichinellosis. We aimed to verify the specificity and sensitivity of this immunodiagnostic test in human trichinellosis. 15 sera from normal subjects, 12 from patients with other parasitic diseases and 50 from trichinellosis patients were tested. Indirect enzyme linked immunosorbent assay (ELISA for specific IgG and an amplified ELISA for specific IgE were performed using β-tyvelose-GalNAc-bovine serum albumin (BSA disaccharide conjugate or T. spiralis muscle larvae excretory/secretory (E/S products, as antigens. Neither control sera nor other parasitic infection sera resulted positive both for IgG and IgE when synthetic or E/S antigens were used. In trichinellosis patient sera, specific IgG were present in 100 % of cases, irrespective of the antigen used, but whereas specific IgE were detected in 78 % using E/S antigens, a 100% positivity rate was obtained, using the β-tyvelose- BSA conjugate.

Tumor immunolocalization using 124I-iodine-labeled JAA-F11 antibody to Thomsen-Friedenreich alpha-linked antigen

International Nuclear Information System (INIS)

Chaturvedi, Richa; Heimburg, Jamie; Yan, Jun; Koury, Stephen; Sajjad, Munawwar; Abdel-Nabi, Hani H.; Rittenhouse-Olson, Kate

2008-01-01

Clinical immunolocalization has been attempted by others with an anti-Thomsen-Friedenreich antigen (TF-Ag) mAb that bound both alpha- and beta-linked TF-Ag. In this report, 124 I-labeled mAb JAA-F11 specific for alpha-linked TF-Ag showed higher tumor specificity in in vivo micro-positron emission tomography (micro-PET) of the mouse mammary adenocarcinoma line, 4T1, showing no preferential uptake by the kidney. Labeled product remained localized in the tumor for at least 20 days. Glycan array analysis showed structural specificity of the antibody

Improved porous silicon (P-Si) microarray based PSA (prostate specific antigen) immunoassay by optimized surface density of the capture antibody

Science.gov (United States)

Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

2014-01-01

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL−1, 80pgmL−1, and 800fgmL−1 when arraying the PSA antibody, H117 at the concentration 15µgmL−1, 35µgmL−1 and 154µgmL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL−1 to 500ngmL−1. The microarray showed a LOD of 800fgmL−1 and a dynamic range of 800 fgmL−1 to 80ngmL−1 in serum spiked samples. PMID:24016590

Bluetongue virus: comparative evaluation of enzyme-linked immunosorbent assay, immunodiffusion, and serum neutralization for detection of viral antibodies.

OpenAIRE

Poli, G; Stott, J; Liu, Y S; Manning, J S

1982-01-01

Comparative studies on the detection of bovine serum immunoglobulin G antibodies to bluetongue virus with an enzyme-linked immunosorbent assay, an immunodiffusion method, and a serum neutralization assay demonstrated complete concordance between the enzyme-linked immunosorbent assay and the serum neutralization assay results. However, the immunodiffusion method failed to detect bluetongue virus antibody in a substantial number of sera found to possess bluetongue virus immunoglobulin G with th...

Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

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Fleur S. van de Bovenkamp

2018-04-01

Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

Trichinella britovi human infection in Spain : antibody response to surface, excretory/secretory and somatic antigens

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Rodríguez-Osorio M.

2003-06-01

Full Text Available A third outbreak of Trichinella britovi with 140 people involved, occurred in Granada Spain (December 1998. The source of infection was sausage made from uninspected wild boar meat. Fifty-two patients agreed to participated in this study. An elevated eosinophil level (> 5 % was detected in 59.6 % of patients, and persisted in most of these cases for two months. A moderate IgG response was observed. At the onset of symptoms, Western blot (WB test detected more positive cases than Enzyme linked immunosorbent assay (ELISA and indirect immunofluorescence (IIF. Six months from infection, ELISA revealed fewer positive cases than the other two tests. It would appear that the response to somatic antigens starts earlier than those to cuticular and excretory/secretory (ES antigens and that the response to ES antigens is the first to decrease.

Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

Science.gov (United States)

Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

Functional Enzyme-Based Approach for Linking Microbial Community Functions with Biogeochemical Process Kinetics

Energy Technology Data Exchange (ETDEWEB)

Li, Minjing [School; Qian, Wei-jun [Pacific Northwest National Laboratory, Richland, Washington 99354, United States; Gao, Yuqian [Pacific Northwest National Laboratory, Richland, Washington 99354, United States; Shi, Liang [School; Liu, Chongxuan [Pacific Northwest National Laboratory, Richland, Washington 99354, United States; School

2017-09-28

The kinetics of biogeochemical processes in natural and engineered environmental systems are typically described using Monod-type or modified Monod-type models. These models rely on biomass as surrogates for functional enzymes in microbial community that catalyze biogeochemical reactions. A major challenge to apply such models is the difficulty to quantitatively measure functional biomass for constraining and validating the models. On the other hand, omics-based approaches have been increasingly used to characterize microbial community structure, functions, and metabolites. Here we proposed an enzyme-based model that can incorporate omics-data to link microbial community functions with biogeochemical process kinetics. The model treats enzymes as time-variable catalysts for biogeochemical reactions and applies biogeochemical reaction network to incorporate intermediate metabolites. The sequences of genes and proteins from metagenomes, as well as those from the UniProt database, were used for targeted enzyme quantification and to provide insights into the dynamic linkage among functional genes, enzymes, and metabolites that are necessary to be incorporated in the model. The application of the model was demonstrated using denitrification as an example by comparing model-simulated with measured functional enzymes, genes, denitrification substrates and intermediates

Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions.

Science.gov (United States)

Nguyen, Le Truc; Yang, Kun-Lin

2017-05-01

Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5±0.5μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3±0.3μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic, antigenic and pathogenic characterization of four infectious bursal disease virus isolates from China suggests continued evolution of very virulent viruses.

Science.gov (United States)

Li, Kai; Courtillon, Céline; Guionie, Olivier; Allée, Chantal; Amelot, Michel; Qi, Xiaole; Gao, Yulong; Wang, Xiaomei; Eterradossi, Nicolas

2015-03-01

Infectious bursal disease virus (IBDV) causes an economically significant disease of young chickens worldwide. The emergence of very virulent IBDV (vvIBDV) strains has brought more challenges for effective prevention and control of this disease. The aim of the present study was to characterize four IBDV isolates from various regions of China between late 1990s and recent years and to compare them with previously isolated European IBDV strains. In this study, one Chinese vvIBDV strain isolated in 1999 and three strains isolated between 2005 and 2011 were analyzed at the genetic, antigenic and pathogenic levels. Strain SH99 was closely related and clustered in the same genetic lineage as the typical vvIBDV based on the genomic sequences of segments A and B. However, the three more recent Chinese vvIBDV (HLJ0504, HeB10 and HuN11) showed several genetic changes in both segments and clustered in a distinct lineage from the typical vvIBDV and the previously known Chinese vvIBDV. Based on the binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, all Chinese vvIBDVs exhibited similar antigenicity with the European typical vvIBDV strains. Nonetheless, the pathogenicity caused by the recent Chinese vvIBDV was higher than that induced by the European typical vvIBDV. This study calls for a sustained surveillance of IBD situation in China in order to support a better prevention and control of the disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens.

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Anderson Messias Rodrigues

Full Text Available Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans.We explored the presence and diversity of serum antibodies (IgG specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49, healthy cats (n = 19, and cats with other diseases (n = 20.Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94-1; P<0.0001 versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis.A convergent IgG-response in various hosts (mice, cats, and humans has important implications for our

Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar

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Gortázar Christian

2008-11-01

Full Text Available Abstract Background Bovine tuberculosis (bTB remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures. Results An enzyme-linked immunosorbent assay (ELISA to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off. Conclusion Although some negative group animals showed an ELISA positive reaction (

Increased serum anti-mycobacterial antibody titers in rheumatoid arthritis patients: Is there any specific antigenic target?

International Nuclear Information System (INIS)

Cetin, Emel S.; Aksoy, Ali M

2007-01-01

Objective was to investigate the presence of immunoreactivity against mycobacterial antigens in the sera of patients with rheumatoid arthritis (Ra) and to detect the target of the immune reaction. This study was carried out on 60 patients with RA, and 25 patients with no joint diseases in the laboratory of Clinical Microbiology Department of Ankara University Medical Faculty, Ankara, Turkey between July 2003 to January 2004. Secreted and cellular antigens of Mycobacterium tuberculosis (M. tuberculosis) H37Rv and Mycobacterium bovis (M. bovis) were isolated and purified by high performance liquid chromatography to antigenic fractions. The immunoreactivity of patient and control sera against these antigens were determined by enzyme-linked immunosorbent assay (ELISA). Immunoreactivity against mycobacterial antigens in RA patients were significantly higher than controls. Significant difference between patients and controls has been determined with M. bovis Bacillus Calmette Guerin (BCG) culture fluid and sonicate antigens, but not with M. tuberculosis H37Rv. This suggests that the antigen triggering immune response in patients with RA may belong to or mainly expressed on M. bovis BCG. The ELISA results showed significant difference between RA patients and controls with all antigenic fractions. Presence of increased immunoreactivity against mycobacterial antigens in the sera of patients with RA was detected. When statistical analysis was considered, we cannot put forward any antigenic fraction alone as the one responsible for the increased reactivity. (author)

Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp.

OpenAIRE

Oaks, E V; Hale, T L; Formal, S B

1986-01-01

The serum antibody response to proteins encoded by the virulence-associated plasmid of Shigella flexneri was determined in monkeys challenged with virulent S. flexneri serotype 2a. With water-extractable antigen in an enzyme-linked immunosorbent assay, a significant increase in antibody titer against proteins from a plasmid-carrying, virulent strain of S. flexneri serotype 5 could be demonstrated in convalescent sera. There were minimal antibody titers against proteins from an avirulent (plas...

Immunoglobulin M antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi in chronic chagasic patients.

Science.gov (United States)

Vasconcelos, Romero H T; Azevedo, Elisa A N; Cavalcanti, Maria G A M; Silva, Edimilson D; Ferreira, Antonio G P; Morais, Clarice N L; Gomes, Yara M

2011-05-01

Previous works of our research group have demonstrated aspects of the humoral immune response of chronic Chagas disease using the cytoplasmatic repetitive antigen (CRA) and the flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The aim of this work was to analyze the presence of specific immunoglobulin M (IgM) antibodies in chronic chagasic patients using these recombinant antigens of T. cruzi. The positivity of IgM in chronic chagasic patients against CRA and FRA antigens was determined by indirect enzyme-linked immunosorbent assay. We reported no statistical significant differences between the levels of IgM for both recombinant antigens and the different chronic clinical forms of Chagas disease. However, a small proportion of chronic chagasic patients analyzed in this study was positive for this antibody isotype. The findings of this study indicate that the IgM antibodies cannot be used to elucidate the differences in the profile of humoral immune response among chronic chagasic patients with different clinical forms using the CRA and FRA recombinant antigens of T. cruzi. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

New technology for regiospecific covalent coupling of polysaccharide antigens in ELISA for serological detection

DEFF Research Database (Denmark)

Jauho, E.S.; Boas, Ulrik; Wiuff, Camilla

2000-01-01

In this study we demonstrate a new UV irradiation technique for covalent coupling of bacterial polysaccharides derived from lipopolysaccharides to microtiter plates and the use of such plates in an enzyme linked immunosorbent assay (ELISA). Lipopolysaccharides were cleaved by mild acid hydrolysis...... describe the use of this technique for the immobilization of Lipopolysaccharide derived polysaccharides from Salmonella Typhimurium and Salmonella Choleraesuis lipopolysaccharides, representing the O-antigens 1, 4, 5, 6, 7, and 12, The functional polysaccharide surface gave similar ELISA results to plates...

Crystal Structure of TDP-Fucosamine Acetyl Transferase (WECD) from Escherichia Coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

International Nuclear Information System (INIS)

Hung, M.; Rangarajan, E.; Munger, C.; Nadeau, G.; Sulea, T.; Matte, A.

2006-01-01

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence →(3)-α-D-Fuc4NAc-(1→4)-β-D-ManNAcA-(1→4)-α-D-GlcNAc-(1→). While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-D-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstroms resolution and bound to acetyl-CoA at a 1.66-Angstroms resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

rK39 enzyme-linked immunosorbent assay for diagnosis of Leishmania donovani infection

NARCIS (Netherlands)

Zijlstra, E. E.; Daifalla, N. S.; Kager, P. A.; Khalil, E. A.; El-Hassan, A. M.; Reed, S. G.; Ghalib, H. W.

1998-01-01

The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months

Immobilization of enzymes on radiation-modified gelatine gel by using a chemical cross-linking agent

International Nuclear Information System (INIS)

Bachmann, S.; Gebicka, L.; Galant, S.

1981-01-01

Investigations into the effect of ionizing radiation on the gelatine gels have shown that water-insoluble gel can be formed under suitable irradiation conditions. To establish the optimal conditions for the processing of the insoluble gel, the yield of cross-linking has been determined for gelatine solutions and its gels irradiated with various doses in the absence and in the presence of oxygen. Glucose isomerase (GI) was used as a test enzyme for immobilization on the gelatine gel. This enzyme which catalyses the isomerization of glucose to fructose has been used on the commercial-scale production of high fructose syrups. The support matrix chosen for the enzyme immobilization has been obtained by irradiating 4% wt/vol. de-aerated gelatine gel at a dose of 1.5 x 10 4 kGy at 15 0 C. Actinoplanes missouriensis cells containing GI were mixed with gelatine gel particles and cross-linked with glutaraldehyde. It was found that the immobilized GI can be successfully applied in the continuous isomerization of glucose to fructose. (author)

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

Science.gov (United States)

Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

2014-07-01

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Determinants of wheat antigen and fungal alpha-amylase exposure in bakeries.

Science.gov (United States)

Burstyn, I; Teschke, K; Bartlett, K; Kennedy, S M

1998-05-01

The study's objectives were to measure flour antigen exposure in bakeries and define the determinants of exposure. Ninety-six bakery workers, employed in seven different bakeries, participated in the study. Two side-by-side full-shift inhalable dust samples were obtained from each study participant on a single occasion. The flour antigen exposure was measured as wheat antigen and fungal alpha-amylase content of the water-soluble fraction of inhalable dust, assayed via enzyme-linked immunosorbent assays. During the entire sampling period bakers were observed and information on 14 different tasks was recorded at 15-minute intervals. Other production characteristics were also recorded for each sampling day and used in statistical modeling to identify significant predictors of exposure. The mean alpha-amylase antigen exposure was 22.0 ng/m3 (ranging from below the limit of detection of 0.1 ng/m3 to 307.1 ng/m3) and the mean wheat antigen exposure was 109 micrograms/m3 (ranging from below the limit of detection of 1 microgram/m3 to 1018 micrograms/m3). Regression models that explained 74% of variability in wheat antigen and alpha-amylase antigen exposures were constructed. The models indicated that tasks such as weighing, pouring, and operating dough-brakers increased flour antigen exposure, while packing and decorating resulted in lower exposures. Croissant, puff-pastry, and bread/bun production lines were associated with increased exposure, while cake production and substitution of dusting with the use of divider oil were associated with decreased exposure. Exposure levels can be reduced by the automation of forming tasks, alteration of tasks requiring pouring of flour, and changes to the types of products manufactured.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

Science.gov (United States)

Cui, Xiping; Vasylieva, Natalia; Wu, Panpan; Barnych, Bogdan; Yang, Jun; Shen, Ding; He, Qiyi; Gee, Shirley J; Zhao, Suqing; Hammock, Bruce D

2017-10-17

Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC 50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

Immunodetection of Fasciola gigantica circulating antigen in sera of infected individuals for laboratory diagnosis of human fascioliasis.

Science.gov (United States)

Attallah, Abdelfattah M; Bughdadi, Faisal A; El-Shazly, Atef M; Ismail, Hisham

2013-10-01

Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961, P 93%), and a significant correlation (r = 0.715, P fascioliasis.

Monoclonal antibody against a serotype antigen of Porphyromonas (Bacteroides) endodontalis and characteristics of the antigen.

Science.gov (United States)

Hanazawa, S; Sagiya, T; Amano, S; Nishikawa, H; Kitano, S

1990-01-01

Recent studies have demonstrated the presence of three serotypes (O1K1, O1K2, and O1K-) of Porphyromonas (Bacteroides) endodontalis. In the present study, a hybridoma cell line producing monoclonal antibody (BEE11) specific for serotype O1K1 of P. endodontalis was established. The specificity of the antibody was evaluated by enzyme-linked immunosorbent assay and immunoslot blot analysis. BEE11 antibody reacted with strains ATCC 35406, HG 400, and HG 421 of the bacterium. However, it did not react with HG 422 or HG 948. Also, the antibody did not react with any of the black-pigmented Bacteroides strains tested. Although the antibody reacted with total cell envelope and capsule materials, it did not do so with lipopolysaccharide. The antibody reacted with antigen material having a molecular mass of 110 kilodaltons (kDa), as judged from fractionation by Superose 12 prep gel chromatography. When the peak fraction from the Superose 12 column was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the reactivity was detected as a single band at an apparent molecular mass of about 52 kDa. The antigen material purified partially by high-performance liquid chromatography was sensitive to trypsin, V8 protease, and heating to 80 degrees C but not to neuraminidase. Therefore, the present study shows that BEE11 antibody recognizes a serotype antigen of P. endodontalis which may be a dimer consisting of monomers having molecular masses of approximately 52 kDa and sensitivity to proteases and heat. Images PMID:2370106

Enhanced acquired antibodies to a chimeric Plasmodium falciparum antigen; UB05-09 is associated with protective immunity against malaria.

Science.gov (United States)

Dinga, J N; Gamua, S D; Titanji, V P K

2017-08-01

It has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of UB05 and UB09 antigens (UB05-09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross-sectional study, recombinant UB05-09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria-endemic zone, using the enzyme-linked immunosorbent assay (ELISA). Anti-UB05-09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05-09-specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B-cell epitope prediction tools confirmed the presence of B-cell epitopes in the study antigens. UB05-09 chimera is a marker of protective immunity against malaria that needs to be studied further. © 2017 John Wiley & Sons Ltd.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

Directory of Open Access Journals (Sweden)

A. M. Marassá

2008-01-01

Full Text Available The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells.

Science.gov (United States)

Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

1990-07-01

The accurate identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of acute lymphoblastic leukemia (ALL). We demonstrated that soluble CD9 antigen was shed into CSF obtained from children with ALL, using enzyme-linked immunosorbent assay (ELISA), which used the activity of CD9 antigen to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4, simultaneously. Using RCA1/SJ-9A4 ELISA, CD9 antigen was detectable in CSF but not in plasma from 12 cases of CD9+ ALL in central nervous system (CNS) relapse. However, CD9 antigen was not released into CSF from 11 cases of CD9- ALL with CNS involvement, 136 cases of CD9+ ALL in complete remission (CR), 29 cases of CD9- ALL in CR, or 21 cases of aseptic meningitis. Interestingly, the levels of CD9 antigen were elevated in CSF from 7 of 10 CD9+ ALL patients without cytologically proven CNS involvement at diagnosis, with subsequent return to undetectable levels after initial induction chemotherapy was begun. In addition, sequential analysis of CSF from a 5-year-old boy with CD9+ ALL in CNS relapse showed that levels of CD9 antigen correlated well with the number of leukemic cells in CSF. Serial quantitative analysis of CD9 antigen in CSF could be useful to detect the proliferation of residual leukemic cells before the clinical manifestation.

The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

Science.gov (United States)

Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael

2013-01-01

Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this

Magnetically responsive enzyme powders

Energy Technology Data Exchange (ETDEWEB)

Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

2015-04-15

Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

Science.gov (United States)

Hasanzadeh, Leila; Ghaznavi-Rad, Ehsanollah; Soufian, Safieh; Farjadi, Vahideh; Abtahi, Hamid

2013-01-01

Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis . PMID:23997913

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

Directory of Open Access Journals (Sweden)

Leila Hasanzadeh

2013-07-01

Full Text Available Objective(s: Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.   Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3 pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism.

Science.gov (United States)

Cheng, W-F; Chang, M-C; Sun, W-Z; Lee, C-N; Lin, H-W; Su, Y-N; Hsieh, C-Y; Chen, C-A

2008-07-01

A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.

Antigenic evidence of bluetongue virus from small ruminant population of two different geographical regions of Odisha, India

Directory of Open Access Journals (Sweden)

Shaswati Subhadarsini Pany

2016-03-01

Full Text Available Aim: The aim of the present study was to carry out antigenic detection of bluetongue virus (BTV among the small ruminant population of two different geographical regions of Odisha (coastal and central using recombinant VP7 (r-VP-7 based sandwich enzyme-linked immunosorbent assay (s-ELISA. Materials and Methods: Blood samples (n=274 were collected from two different geographical pockets of Odisha, which covered mostly the coastal and central regions. Of the total samples under study 185 were from goat and 89 were from sheep. The blood samples were tested for the presence of BTV antigen by r-VP7 based s-ELISA. Results: r-VP-7 s-ELISA detected BTV antigen in 52.43% and 44.94% of the goat and sheep population under study, respectively. This study highlights the antigenic persistence of BTV in the state for the 1st time. Conclusion: This high antigenic presence in both sheep and goat population suggests an alarming BTV infection in field conditions which warrants more systematic study directed toward isolation and characterization studies as well as the implementation of control strategy for BT in Odisha.

An electric detection of immunoglobulin G in the enzyme-linked immunosorbent assay using an indium oxide nanoparticle ion-sensitive field-effect transistor

International Nuclear Information System (INIS)

Lee, Dongjin; Cui, Tianhong

2012-01-01

Semiconducting nanoparticle ion-sensitive field-effect transistors (ISFETs) are used to detect immunoglobulin G (IgG) in the conventional enzyme-linked immunosorbent assay (ELISA). Indium oxide and silica nanoparticles were layer-by-layer self-assembled with the oppositely charged polyelectrolyte as the electrochemical transducer and antibody immobilization site, respectively. The assay was conducted on a novel platform of indium oxide nanoparticle ISFETs, where the electric signals are generated in response to the concentration of target IgG using the labeled detecting antibody. The sandwiched ELISA structure catalyzed the conversion of the acidic substrate into neutral substance with the aid of horseradish peroxidase. The pH change in the substrate solution was detected by nanoparticle ISFETs. Normal rabbit IgG was used as a model antigen whose detection limit of 0.04 ng ml −1 was found. The facile electric detection in the conventional assay through the semiconducting nanoparticle ISFET has potential applications as a point-of-care detection or a sensing element in a lab-on-a-chip system

Evaluation of ascitic soluble human leukocyte antigen-G for distinguishing malignant ascites from benign ascites.

Science.gov (United States)

Sun, Juan; Chang, Yan-Xiang; Niu, Chun-Yan

2017-11-01

The overexpression of soluble human leukocyte antigen-G is associated with malignant tumours. The purpose of our study was to detect soluble human leukocyte antigen-G concentrations in ascites and to evaluate the value of ascitic soluble human leukocyte antigen-G for the diagnosis of malignant ascites. Enzyme-linked immunosorbent assay was used to detect soluble human leukocyte antigen-G levels in 64 patients with malignant ascites and 30 patients with benign ascites. Receiver operating characteristic curves were used to evaluate the diagnostic efficacy of ascitic soluble human leukocyte antigen-G for the detection of malignant ascites. Ascitic soluble human leukocyte antigen-G levels were significantly higher in the malignant ascites group than in the benign ascites group (20.718 ± 3.215 versus 12.467 ± 3.678 µg/L, t = 7.425, p human leukocyte antigen-G was 0.957 (95% confidence interval, 0.872-0.992). At a cut-off value of 19.60 µg/L, the sensitivity and specificity of ascitic soluble human leukocyte antigen-G were 87.5% (95% confidence interval, 71.0%-96.5%) and 100% (95% confidence interval, 88.4%-100%), respectively. With respect to area under the receiver operating characteristic curve, sensitivity and specificity, ascitic carcinoembryonic antigen (0.810, 68.75% and 83.33%, respectively) and carbohydrate antigen 19-9 (0.710, 65.63% and 70%, respectively) significantly differed (all p human leukocyte antigen-G was 75%, which was higher than the corresponding rates for ascitic carcinoembryonic antigen (31.25%) and carbohydrate antigen 19-9 (6.25%; both p human leukocyte antigen-G exhibited good performance for diagnosing malignant ascites, and particularly those that were cytology-negative and biopsy-positive.

Multicountry Prospective Clinical Evaluation of Two Enzyme-Linked Immunosorbent Assays and Two Rapid Diagnostic Tests for Diagnosing Dengue Fever

Science.gov (United States)

Dauner, Allison L.; Valks, Andrea; Forshey, Brett M.; Long, Kanya C.; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C.; Halsey, Eric S.; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G.; Fernandez, Stefan; An, Ung Sam; Kochel, Tadeusz J.; Jasper, Louis E.; Wu, Shuenn-Jue L.

2015-01-01

We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks. PMID:25588659

Application of an improved enzyme-linked immunosorbent assay method for serological diagnosis of canine leishmaniasis

NARCIS (Netherlands)

Santarém, Nuno; Silvestre, Ricardo; Cardoso, Luís; Schallig, Henk; Reed, Steven G.; Cordeiro-da-Silva, Anabela

2010-01-01

Accurate diagnosis of canine leishmaniasis (CanL) is essential toward a more efficient control of this zoonosis, but it remains problematic due to the high incidence of asymptomatic infections. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based

Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells. Immunological study using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Hareyama, Masato

1988-12-01

We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-..gamma... In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G/sub 2//M phases of the cell cycle than in G/sub 0//G/sub 1/. The expression of YH206 antigen was enhanced in the S and G/sub 2//M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens.

Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

Science.gov (United States)

Attallah, Abdelfattah M.; Bughdadi, Faisal A.; El-Shazly, Atef M.

2013-01-01

Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961, P 93%), and a significant correlation (r = 0.715, P fascioliasis. PMID:23945158

Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

Science.gov (United States)

Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

2015-10-21

A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

Transmission of hepatitis-B virus through salivary blood group antigens in saliva

International Nuclear Information System (INIS)

Meo, S.A.; Abdo, A.A.; Baksh, N.D.; Sanie, F.M.

2010-01-01

To determine an association between transmission of hepatitis B virus and secretor and non-secretor status of salivary blood group antigens. Study Design: Cross-sectional, analytical study. Place and Duration of Study: The Department of Physiology and Division of Hepatology, College of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Kingdom of Saudi Arabia, from 2007 to 2009. Methodology: Eighty eight known patients, who were positive for Hepatitis B Surface Antigen [HBsAg] were recruited. Saliva was collected for investigating the secretor and non-secretor status by using blood typing kit number Kemtec Educational Science USA. Hepatitis B Surface antigen test was performed on Enzyme Linked Immunosorbent Assay technique. Polymerase chain reaction [PCR] on saliva was also carried out in High Performance Thermal Cycler-Palm- Cycler [Corbett Life Science, Sydney, Australia] and enzymatic amplification of extracted viral DNA was performed using primers covering the promoter of the core region of HBV. Results: Out of the 88 subjects, 61 belong to blood group O, 20 to A and 7 subjects to blood group B. Fifty subjects were secretors [salivary blood group antigens positive] and 38 subjects were non-secretors [salivary blood group antigens negative]. Among core gene positive 25 (69.4%) were secretors and 11 (30.6%) were non-secretors. However, in core gene negative 25 (48.1%) were secretors and 27 (51.9%) were non-secretors. Conclusion: The result shows an association [p=0.047] between secretor and non-secretors status of the salivary blood group antigens with core gene positive and core gene negative. (author)

A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

Science.gov (United States)

Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

2012-01-01

ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

Science.gov (United States)

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

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

Science.gov (United States)

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.

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens.

Science.gov (United States)

Rodrigues, Anderson Messias; Fernandes, Geisa Ferreira; Araujo, Leticia Mendes; Della Terra, Paula Portella; dos Santos, Priscila Oliveira; Pereira, Sandro Antonio; Schubach, Tânia Maria Pacheco; Burger, Eva; Lopes-Bezerra, Leila Maria; de Camargo, Zoilo Pires

2015-01-01

Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans. We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20). Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94-1; Psporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis. A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of lipopolysaccharide-derived antigens covalently attached to polystyrene microwells

DEFF Research Database (Denmark)

Wiuff, C.; Jauho, E. S.; Stryhn, H.

2000-01-01

to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from......Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled...

EVALUATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR BIOLOGICAL MONITORING OF 3-PHENOXYBENZOIC ACID IN URINE

Science.gov (United States)

Abstract describes the development of an enzyme-linked immunosorbent assay (ELISA) method for monitoring 2,4-dichlorophenoxyacetic acid (2,4-D exposures). The ELISA is compared with a gas chromatograhy/mass spectrometry procedure. ELISA method development steps and comparative ...

Improvement of an enzyme immunosorbent assay for detecting antibodies against Dioctophyma renale.

Science.gov (United States)

Pedrassani, Daniela; do Nascimento, Adjair Antonio; André, Marcos Rogério; Machado, Rosangela Zacarias

2015-09-15

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-Dioctophyma renale antibodies in the sera of dogs using, detection of parasite eggs in urine sediment as a reference test. ELISA uses a soluble antigenic preparation of esophagus of D. renale and the optimal dilutions of the antigen, serum and conjugate were determined by means of checker board titration, using positive (n=13) and negative (n=27) reference serum. The specificity and sensitivity of the ELISA were 93.8% and 92.3% respectively and the kappa index was good (0.76). These results suggest that ELISA described may prove to be an effective serological test for detecting dogs infected and exposed to this parasite mainly dogs that are not eliminating parasite eggs through their urine. Copyright © 2015 Elsevier B.V. All rights reserved.

Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

Science.gov (United States)

Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

2003-01-01

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

Identification of the antigenic region of Neospora caninum dense granule protein 7 using ELISA.

Science.gov (United States)

Abdelbaky, Hanan H; Fereig, Ragab M; Nishikawa, Yoshifumi

2018-06-26

Dense granule protein 7 (NcGRA7) is a potent diagnostic antigen of Neospora caninum. Some studies have reported on the difficult expression, low yield, and variable degree of solubility of recombinant NcGRA7. We aimed to unravel the possible causes for these issues and tested NcGRA7 antigenicity in enzyme linked immunosorbent assays (ELISAs). The NcGRA7 coding sequence (217 amino acids) was split into five amino acid regions: NcGRA7m (27-217), NcGRA7m3 (27-160), NcGRA7m4 (27-135), NcGRA7m5 (161-190), and NcGRA7m6 (188-217). Three fragments, NcGRA7m, NcGRA7m3 and NcGRA7m4, exhibited high antigenic properties when tested against experimentally-infected mice and dog sera by ELISA. High levels of IgG2 antibodies against NcGRA7m were observed in field dog sera. In experimentally and naturally-infected cattle, the N. caninum-specific sera only reacted with NcGRA7m, indicating that this antigenic region differs among the three animal species. This study presents valuable information about the antigenic properties and topology of NcGRA7, and highlights its suitability for diagnostic purposes. Copyright © 2018. Published by Elsevier B.V.

Enzyme-linked immunospot: an alternative method for the detection of interferon gamma in Johne's disease

DEFF Research Database (Denmark)

Begg, Douglas J.; de Silva, Kumudika; Bosward, Katrina

2009-01-01

To date, the sensitivity of the interferon gamma (IFN-) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN- detection in the early stages of infection, an alternate assay needs to be ...

Enzyme catalyzed oxidative cross-linking of feruloylated pectic polysaccharides from sugar beet

DEFF Research Database (Denmark)

Abang Zaidel, Dayang Norulfairuz

beet pulp as a potential starting material for production of pectin derived products which could help maintain the competitiveness of the sugar beet based industry. The overall objective of this study has been focusing on understanding the kinetics of enzyme catalyzed oxidative crosslinking......-linked by HRP catalysis in the presence of hydrogen peroxide (H2O2) to form ferulic acid dehydrodimers (diFAs). The composition of the substrate was analyzed by HPAEC, HPLC and MALDI-TOF, confirming the structural make up of the arabinan-oligosaccharide (Arabinose: 2.9- 3.4 mmol?g-1 DM; FA: 2.5-7.0 mg?g-1 DM......, identically composed, oil-in-water emulsion systems to study the effect of different methods of emulsion preparation on the emulsion stability in the presence of SBP and the kinetics of enzyme catalyzed oxidative gelation of SBP. The result shows that the different methods of emulsion preparation affect...

Optimization of AFP-radioimmunoassay using Antibody Capture Technique

International Nuclear Information System (INIS)

Moustafa, K.A.

2003-01-01

Alpha-fetoprotein (AFP) is a substance produced by the unborn baby. When the neural tube is not properly formed large amounts of AFP pass into the amniotic fluid and reach the mother's blood. By measuring AFP in the mother's blood and amniotic fluid, it is possible to tell whether or not there is a chance that the unborn baby has a neural tube defect. AFP also used as a tumor marker for hepatocellular carcinoma. There are many different techniques for measuring AFP in blood, but the most accurate one is the immunoassay technique. The immunoassays can be classified on the basis of methodology into three classes; (1) the antibody capture assays, (2) the antigen capture assay, (3)the two-antibody sandwich assays. In this present study, the antibody capture assay in which the antigen is attached to a solid support, and labeled antibody is allowed to bind, will be optimized

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

Science.gov (United States)

Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

2012-01-01

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and

Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

Science.gov (United States)

Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

1988-07-01

The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested.

Tumor antigens as proteogenomic biomarkers in invasive ductal carcinomas

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Campos, Benito; Winther, Ole

2014-01-01

directly linked to the hallmarks of cancer. The results found by proteogenomic analysis of the 32 tumor antigens studied here, capture largely the same pathway irregularities as those elucidated from large-scale screening of genomics analyses, where several thousands of genes are often found......Background: The majority of genetic biomarkers for human cancers are defined by statistical screening of high-throughput genomics data. While a large number of genetic biomarkers have been proposed for diagnostic and prognostic applications, only a small number have been applied in the clinic....... Similarly, the use of proteomics methods for the discovery of cancer biomarkers is increasing. The emerging field of proteogenomics seeks to enrich the value of genomics and proteomics approaches by studying the intersection of genomics and proteomics data. This task is challenging due to the complex nature...

Cross-sensitization between xeno- and allo-antigens on subsequent allogeneic and xenogeneic pancreatic islet transplantation in a murine model.

Science.gov (United States)

Kim, Hyun-Je; Byun, Nari; Kwon, Ohsang; Park, Chung-Gyu

2016-11-18

The number of patients in need of organ transplantation is continuously on the rise. However, because of organ donor shortage, xenotransplantation has been highlighted as an alternative. Among the various porcine organs and tissues, porcine islets are considered to be the best-matching implantable candidates for clinical application based on recent progress in nonhuman primate pre-clinical studies. Nevertheless, before initiation of clinical trials, it should be confirmed whether the requisite xeno-antigen sensitization would have a deleterious effect on subsequent allo-transplantation or vice versa. Therefore, in the present study, the survival rate of islets grafted in naïve recipients was compared with that in cross-sensitized recipients. Enzyme-linked immunosorbent spot, fluorescence-activated cell sorting, and immunohistochemistry were conducted to assess the cellular and humoral immune responses. The survival days of Balb/c mouse islets transplanted into B6 mice that had been previously sensitized with porcine cells (i.e., xeno-sensitized) showed no significant difference from that of naïve B6 mice. Moreover, the survival days of porcine islets transplanted into allo-antigen (Balb/c)-sensitized B6 recipients was not significantly different from that in naïve B6 mice. Furthermore, our data provide the first demonstration that the cellular xenogeneic immune response (against porcine antigen) measured by an enzyme-linked immunosorbent spot assay is not cross-reactive to the allogeneic immune responses in a murine islet transplantation model. These results suggest that clinical application of islet xenotransplantation is not likely to have a deleterious effect on subsequent allogeneic islet transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

Science.gov (United States)

Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

2013-01-01

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

Directory of Open Access Journals (Sweden)

Claudio R Madruga

2006-11-01

Full Text Available There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Development of a monoclonal antibody-based sandwich-type enzyme-linked immunosorbent assay (ELISA) for detection of abrin in food samples.

Science.gov (United States)

Zhou, Yu; Tian, Xiang-Li; Li, Yan-Song; Pan, Feng-Guang; Zhang, Yuan-Yuan; Zhang, Jun-Hui; Wang, Xin-Rui; Ren, Hong-Lin; Lu, Shi-Ying; Li, Zhao-Hui; Liu, Zeng-Shan; Chen, Qi-Jun; Liu, Jing-Qiu

2012-12-15

Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1-100 μg L(-1) with a detection limit of 0.5 μg L(-1) for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5-98.6%, 95.8-98.4% and 94.8-9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of antigenic domains in the non-structural protein of Muscovy duck parvovirus.

Science.gov (United States)

Yu, Tian-Fei; Li, Ming; Yan, Bing; Shao, Shu-Li; Fan, Xing-Dong; Wang, Jia; Wang, Dan-Na

2016-08-01

Muscovy duck parvovirus (MDPV) infection is widespread in many Muscovy-duck-farming countries, leading to a huge economic loss. By means of overlapping peptides expressed in Escherichia coli in combination with Western blot, antigenic domains on the non-structural protein (NSP) of MDPV were identified for the first time. On the Western blot, the fragments NS(481-510), NS (501-530), NS (521-550), NS (541-570), NS (561-590), NS (581-610) and NS (601-627) were positive (the numbers in parentheses indicate the location of amino acids), and other fragments were negative. These seven fragments were also reactive in an indirect enzyme-linked immunosorbent assay (i-ELISA). We therefore conclude that a linear antigenic domain of the NSP is located at its C-terminal end (amino acid residues 481-627). These results may facilitate future investigations into the function of NSP of MDPV and the development of immunoassays for the diagnosis of MDPV infection.

Detection of Rabies antigen in brains of suspected Rabid dogs ...

African Journals Online (AJOL)

Objective: To detect the presence of rabies antigen in brains of suspected rabid dogs. Materials and Methods: Ninety six (96) brain specimens from suspected rabid dogs were examined for the presence of rabies antigen using Seller's staining technique and enzyme immunoassay. Results: The two techniques were both ...

A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues.

Directory of Open Access Journals (Sweden)

Monique da Rocha Queiroz Lima

Full Text Available UNLABELLED: / BACKGROUND: Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4. Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23 that occurred in 2002 in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: A total of 74 tissue specimens were available: liver (n = 23, lung (n = 14, kidney (n = 04, brain (n = 10, heart (n = 02, skin (n = 01, spleen (n = 15, thymus (n = 03 and lymph nodes (n = 02. We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics, Platelia NS1 (BioRad Laboratories and the rapid test NS1 Ag Strip (BioRad Laboratories. The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23, 60.8% (14/23 and 91.3% (21/23, respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74 of the specimens analyzed and the Platelia NS1 in 45.9% (34/74. The highest sensitivity (78.3%; 58/74 was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p<0.05. The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23, lung (71.4%; 10/14, kidney (100%; 4/4, brain (80%; 8/10, spleen (66.6%, 10/15 and thymus (100%, 3/3 when compared to the other two ELISA assays. CONCLUSIONS/SIGNIFICANCE: This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an

Significance of Serology by Multi-Antigen ELISA for Tissue Helminthiases in Korea

Science.gov (United States)

2017-01-01

It is clinically important to differentiate tissue-invading helminthiasis. The purpose of this study was to assess the specific immunoglobulin G (IgG) antibody positive rates for clonorchiasis, paragonimiasis, cysticercosis, and sparganosis 4 helminthiases from 1996 to 2006 using multi-antigen enzyme-linked immunosorbent assay (ELISA) in Korea. Results of 6,017 samples, which were referred to our institute for serodiagnosis, were analyzed. The subjects with positive serum IgG antibodies were 1,502 (25.0%) for any of the 4 helminthiases. The overall positive numbers for clonorchiasis, paragonimiasis, cysticercosis, and sparganosis were 728 (12.1%), 166 (2.8%), 729 (12.1%), and 263 (4.4%), respectively. The positive serologic reaction to multi-antigens was determined in 309 (20.6%) of the 1,502 total seropositive subjects. Those with multi-antigen positivity were regarded as positive for the antigen of strongest reaction but cross-reaction to others with weak positive reaction. Annual seropositive rates for those 4 tissue helminthiases ranged from 12.1% to 35.7%. The highest rate was observed in age from 60 to 69 years old and prevalence of men (27.4%; 1,030/3,763) was significantly higher than of women (19.1%; 332/1,741). Hospital records of 165 ELISA positive patients were reviewed to confirm correlation with their clinical diagnosis. Paragonimiasis was highly correlated as 81.8% (9/11), cysticercosis 29.9% (20/67), clonorchiasis 29.0% (20/69), and sparganosis 11.1% (2/18). In conclusion, the multi-antigen ELISA using 4 helminth antigens is useful to differentiate suspected tissue-invading helminthiases, especially ELISA diagnosis of paragonimiasis is reliable. The seropositivity is still high among suspected patients in Korea. PMID:28581268

Enzyme II/sup Mtl/ of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

International Nuclear Information System (INIS)

Pas, H.H.; Robillard, G.T.

1988-01-01

The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [ 14 C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing

Increased levels of IgA antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi differentiate digestive forms of Chagas disease.

Science.gov (United States)

Vasconcelos, Romero H T; Amaral, Fábio N; Cavalcanti, Maria G A M; Silva, Edimilson D; Ferreira, Antonio G P; Morais, Clarice N L; Gomes, Yara M

2010-10-01

In the chronic phase of Chagas disease, individuals infected by Trypanosoma cruzi may be asymptomatic or may present cardiac and/or digestive complications. Our aim here was to analyze the relationship between the presence of specific immunoglobulin A antibodies and the different chronic clinical forms of Chagas disease using two recombinant antigens of Trypanosoma cruzi, cytoplasmatic repetitive antigen and flagellar repetitive antigen. The association of this immunoglobulin isotype with the digestive and cardio-digestive forms of the disease determined by indirect enzyme-linked immunosorbent assay, strongly suggests that IgA antibodies against these recombinant antigens of T. cruzi can be used as an immunological marker of the digestive alterations caused by Chagas disease. The tests performed in this study show that it is possible to differentiate digestive forms of Chagas disease. The knowledge provided by these results may help physicians to manage early alterations in the digestive tract of patients with the indeterminate or cardiac forms of Chagas disease. Prospective studies, however, with follow-up of the patients that presenting with high levels of immunoglobulin A against cytoplasmatic repetitive antigen and flagellar repetitive antigen recombinant antigens, need to be conducted to confirm this hypothesis. 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Validation of an improved enzyme-linked immunosorbent assay for the diagnosis of trypanosomal antibodies in Ghanaian cattle

International Nuclear Information System (INIS)

Doku, C.K.; Seidu, I.B.M.

2000-01-01

The validation of an enzyme-linked immunosorbent assay (Ab-ELISA) for the detection of antibodies to pathogenic trypanosomes in cattle is described. Two hundred known negative sera obtained from the tsetse-free zone of Dori (Burkina Faso) were analyzed using microtitre plates pre-coated with crude antigen lysates of Trypanosoma congolense and T. vivax. A pre-test optimization was carried out and a percent positivity (PP) of 50% was chosen (specificity: >82%) for assaying field sera. A total of 440 serum samples collected from cattle in areas of known and unknown disease prevalence were assayed. For all animals the packed red cell volume (PCV) was determined and the buffy coat technique (BCT) and blood smears were examined to detect trypanosomes at the species level. A comparison of the BCT and Ab-ELISA results showed there was a much higher prevalence of antibodies to both species than the parasite prevalence as shown by the BCT (10 fold). The rate of agreement between BCT-positive and Ab-ELISA-positive samples for both species was low (<10%). No conclusion could be drawn from this finding because of the low number of known BCT positive cases that were identified. There was a better, albeit highly variable, agreement between BCT-negative and Ab-ELISA-negative samples (30-70%). Proposals for further improvement of the Ab-ELISA and prospects for the use of the assay in the monitoring of trypanosomosis control in Ghana are discussed. (author)

Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

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Widiastuti R

2013-06-01

Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis

Science.gov (United States)

Zahabiun, Farzaneh; Sadjjadi, Seyed Mahmoud; Yunus, Muhammad Hafiznur; Rahumatullah, Anizah; Moghaddam, Mohammad Hosein Falaki; Saidin, Syazwan; Noordin, Rahmah

2015-01-01

Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory–secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis. PMID:26033026

Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

Science.gov (United States)

Higgins, Melanie A; Whitworth, Garrett E; El Warry, Nahida; Randriantsoa, Mialy; Samain, Eric; Burke, Robert D; Vocadlo, David J; Boraston, Alisdair B

2009-09-18

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

Synergistic immune responses induced by endogenous retrovirus and herpesvirus antigens result in increased production of inflammatory cytokines in multiple sclerosis patients

DEFF Research Database (Denmark)

Brudek, T; Christensen, T; Hansen, H J

2008-01-01

Human endogenous retroviruses (HERV) and herpesviruses are increasingly associated with the pathogenesis of the neurological inflammatory disease multiple sclerosis (MS). Herpesviruses are capable of HERV activation and simultaneous presence of HERV and herpesvirus antigens have a synergistic...... effect on cell-mediated immune responses, which tend to be higher in MS patients in comparison with healthy individuals. Here, we investigate whether these synergistic immune responses are reflected in changes in the production of proinflammatory cytokines. Using enzyme-linked immunosorbent assays...

Development of Two Antibody Detection Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Human Chronic Fascioliasis

Science.gov (United States)

Cabán-Hernández, Kimberly; Gaudier, José F.; Ruiz-Jiménez, Caleb

2014-01-01

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories. PMID:24353000

Development of two antibody detection enzyme-linked immunosorbent assays for serodiagnosis of human chronic fascioliasis.

Science.gov (United States)

Cabán-Hernández, Kimberly; Gaudier, José F; Ruiz-Jiménez, Caleb; Espino, Ana M

2014-03-01

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories.

Development of an enzyme-linked immunosorbent assay method to detect mustard protein in mustard seed oil

NARCIS (Netherlands)

Koppelman, S.J.; Vlooswijk, R.; Bottger, G.; Duijn, G. van; Schaft, P. van der; Dekker, J.; Bemgen, H. van

2007-01-01

An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the

Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

Science.gov (United States)

Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

2008-02-15

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Acrolein inhibits NADH-linked mitochondrial enzyme activity: implications for Alzheimer's disease.

Science.gov (United States)

Pocernich, Chava B; Butterfield, D Allan

2003-01-01

In Alzheimer's disease (AD) brain increased lipid peroxidation and decreased energy utilization are found. Mitochondria membranes contain a significant amount of arachidonic and linoleic acids, precursors of lipid peroxidation products, 4-hydroxynonenal (HNE) and 2-propen-1-al (acrolein), that are extremely reactive. Both alkenals are increased in AD brain. In this study, we examined the effects of nanomolar levels of acrolein on the activities of pyruvate dehydrogenase (PDH) and Alpha-ketoglutarate dehydrogenase (KGDH), both reduced nicotinamide adenine dinucleotide (NADH)-linked mitochondrial enzymes. Acrolein decreased PDH and KGDH activities significantly in a dose-dependent manner. Using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS), acrolein was found to bind lipoic acid, a component in both the PDH and KGDH complexes, most likely explaining the loss of enzyme activity. Acrolein also interacted with oxidized nicotinamide adenine dinucleotide (NAD(+)) in such a way as to decrease the production of NADH. Acrolein, which is increased in AD brain, may be partially responsible for the dysfunction of mitochondria and loss of energy found in AD brain by inhibition of PDH and KGDH activities, potentially contributing to the neurodegeneration in this disorder.

Association Between PAI-1 Activity Levels and t-PA Antigen with Glycemic Status in Prediabetic Population

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Andi Fachruddin Benyamin

2016-11-01

Full Text Available Aim: to evaluate an association between fibrinolysis defect and glycemic status in prediabetic population by assessing the levels of t-PA antigen and PAI-1 activity. Methods: it was an observational study with cross-sectional approach. There were 72 subjects aged 30-50 years who had met the inclusion criteria. The diagnosis of diabetes mellitus (DM and glycemic index were determined based on the American Diabetes Association (ADA criteria. The PAI-1 and t-PA antigen levels were measured quantitatively using enzyme-linked immunosorbent assay (ELISA. Analysis between the levels of t-PA antigen and PAI-1 activity was performed using ANOVA. Results: the t-PA antigen level was significantly higher in subjects with impaired glucose tolerance (IGT and impaired fasting blood glucose (IFBG as well as subject with impaired fasting blood glucose (IFBG than those with normal glucose tolerance (NGT (p=0.047. The PAI-1 activity was significantly higher in subjects with IGT, IFBG and subjects with IFBG than NGT (p=0.024. There was a significant association between glycemic status in prediabetic subjects and PAI-1 activity (p=0.04. Conclusion: the level of t-PA antigen and PAI-1 activity were significantly higher in prediabetic subjects than those with NGT; and there was a significant association between glycemic status in prediabetic subjects and PAI-1 activity.

Added value of antigen ELISA in the diagnosis of neurocysticercosis in resource poor settings.

Directory of Open Access Journals (Sweden)

Sarah Gabriël

Full Text Available BACKGROUND: Neurocysticercosis (NCC is the most common cause of acquired epilepsy in Taenia solium endemic areas, primarily situated in low-income countries. Diagnosis is largely based upon the "Del Brutto diagnostic criteria" using the definitive/probable/no NCC diagnosis approach. Neuroimaging and specific T. solium cysticercosis antibody detection results are at the mainstay of this diagnosis, while antigen detection in serum has never been included. This study aimed at evaluating the addition of antigen detection as a major diagnostic criterion, especially in areas where neuroimaging is absent. METHODS: The B158/B60 monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA for the detection of circulating cysticercus antigen was carried out retrospectively on serum samples collected during a hospital-based study from 83 people with epilepsy (PWE in an endemic area. RESULTS: The addition of antigen results as a major criterion allowed the correct diagnosis of definitive NCC in 10 out of 17 patients as opposed to 0/17 without antigen results in the absence of neuroimaging. A sensitivity of 100% and a specificity of 84% were determined for the diagnosis of active NCC using antigen ELISA. While the use of a higher cutoff improves the specificity of the test to 96%, it decreases its sensitivity to 83%. CONCLUSIONS: In areas where neuroimaging is absent, NCC diagnosis according to the existing criteria is problematic. Taking into account its limitations for diagnosis of inactive NCC, antigen detection can be of added value for diagnosing NCC in PWE by supporting diagnostic and treatment decisions. Therefore, we recommend a revision of the "Del Brutto diagnostic criteria" for use in resource poor areas and suggest the inclusion of serum antigen detection as a major criterion.

Quantification of patient specific assay interference in different formats of enzyme linked immunoassays for therapeutic monoclonal antibodies

NARCIS (Netherlands)

Grebenchtchikov, N.J.; Geurts-Moespot, A.; Heijmen, L.; Laarhoven, H.W.M. van; Herpen, C.M.L. van; Thijs, A.M.J.; Span, P.N.; Sweep, F.C.

2014-01-01

BackgroundThe use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are

Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans

NARCIS (Netherlands)

van Doorn, H. Rogier; Koelewijn, Rob; Hofwegen, Henk; Gilis, Henk; Wetsteyn, Jose C. F. M.; Wismans, Pieter J.; Sarfati, Claudine; Vervoort, Tony; van Gool, Tom

2007-01-01

A homemade enzyme-linked immunosorbent assay (ELISA) (Academic Medical Center ELISA [AMC-ELISA]) and a dipstick assay for the detection of anti-Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA [IVD Research, Inc.

Low energy, low cost, efficient CO{sub 2} capture

Energy Technology Data Exchange (ETDEWEB)

Michael C. Trachtenberg; Lihong Bao; David A. Smith; Remy Dumortier [Carbozyme, Inc., Monmouth Junction, NJ (United States)

2006-07-01

This paper discusses the development and some characteristics of a new, enzyme-based, contained liquid membrane contactor to capture CO{sub 2}. The enzyme carbonic anhydrase catalyzes the removal of CO{sub 2} while the membrane contactor increases the surface area to allow the reduction of the size of the system. The modular system design is easily scaled to any required size reducing the investment costs. The system captures CO{sub 2} at a low energy and low cost promising to be a cost effective technology for CO{sub 2} capture. 5 refs., 7 figs.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant truncated Cap protein for the diagnosis of porcine circovirus-like virus P1.

Science.gov (United States)

Wen, Li-bin; Wen, Shi-fu; He, Kong-wang

2016-01-19

Porcine circovirus-like virus P1 is a newly discovered virus. To date, there has been no specific serological assay for use in the diagnosis of P1 infection. Because P1 has high homology to porcine circovirus type 2 (PCV2) at the nucleotide level, the C-terminal portion of the capsid protein (amino acids 73-114), a discriminative antigen, was expressed in a prokaryotic expression system. The recombinant product (rctCap), composed of three identical repeated domains, was shown to be strongly immunoreactive to P1-specific serum. This assay was validated by comparison with an indirect immunofluorescence assay (IFA). The diagnostic sensitivity and specificity of the rctCap enzyme-linked immunosorbent assay (ELISA) developed in this study are 93.6% and 98.3%, respectively, compared with the results from IFAs on 450 sera samples from pigs. The indirect ELISA that we developed with rctCap, the recombinant capsid fragment containing the 217-342 nt repeat domain, was sensitive, specific, and suitable for the large-scale detection of P1 infections in swine.

Quantification of patient-specific assay interference in different formats of enzyme-linked immunoassays for therapeutic monoclonal antibodies

NARCIS (Netherlands)

Grebenchtchikov, Nicolai; Geurts-Moespot, Anneke J.; Heijmen, Linda; van Laarhoven, Hanneke W. M.; van Herpen, Carla M. L.; Thijs, Annemarie M. J.; Span, Paul N.; Sweep, Fred C. G. J.

2014-01-01

The use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are subject to a

Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

Directory of Open Access Journals (Sweden)

Fred Luciano Neves Santos

Full Text Available The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6, demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

Science.gov (United States)

Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

2015-09-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Rethinking fundamentals of enzyme action.

Science.gov (United States)

Northrop, D B

1999-01-01

Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

Reduced response to Epstein–Barr virus antigens by T-cells in systemic lupus erythematosus patients

Science.gov (United States)

Draborg, Anette Holck; Jacobsen, Søren; Westergaard, Marie; Mortensen, Shila; Larsen, Janni Lisander; Houen, Gunnar; Duus, Karen

2014-01-01

Objective Epstein–Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. Methods T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. Results SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. Conclusions These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed. PMID:25396062

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

Science.gov (United States)

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

Development of real-time motion capture system for 3D on-line games linked with virtual character

Science.gov (United States)

Kim, Jong Hyeong; Ryu, Young Kee; Cho, Hyung Suck

2004-10-01

Motion tracking method is being issued as essential part of the entertainment, medical, sports, education and industry with the development of 3-D virtual reality. Virtual human character in the digital animation and game application has been controlled by interfacing devices; mouse, joysticks, midi-slider, and so on. Those devices could not enable virtual human character to move smoothly and naturally. Furthermore, high-end human motion capture systems in commercial market are expensive and complicated. In this paper, we proposed a practical and fast motion capturing system consisting of optic sensors, and linked the data with 3-D game character with real time. The prototype experiment setup is successfully applied to a boxing game which requires very fast movement of human character.

Multiantigen Print Immunoassay for Comparison of Diagnostic Antigens for Taenia solium Cysticercosis and Taeniasis▿

Science.gov (United States)

Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N.; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E.; Garcia, Hector H.; Gilman, Robert H.; Tsang, Victor C. W.; Wilkins, Patricia P.

2010-01-01

One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases. PMID:19906893

Multiantigen print immunoassay for comparison of diagnostic antigens for Taenia solium cysticercosis and taeniasis.

Science.gov (United States)

Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E; Garcia, Hector H; Gilman, Robert H; Tsang, Victor C W; Wilkins, Patricia P

2010-01-01

One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases.

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Science.gov (United States)

Galli, Vanessa; Simionatto, Simone; Marchioro, Silvana Beutinger; Klabunde, Gustavo Henrique Ferrero; Conceição, Fabricio Rochedo

2013-01-01

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae. PMID:23803903

Phytochrome quantitation in crude extracts of Avena by enzyme-linked immunosorbent assay with monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Shimazaki, Y; Cordonnier, M M; Pratt, L H

1983-01-01

An enzyme-linked immunosorbent assay (ELISA), which uses both rabbit polyclonal and mouse monoclonal antibodies to phytochrome, has been adapted for quantitation of phytochrome in crude plant extracts. The assay has a detection limit of about 100 pg phytochrome and can be completed within 10 h. Quantitation of phytochrome in crude extracts of etiolated oat seedlings by ELISA gave values that agreed well with those obtained by spectrophotometric assay. When etiolated oat seedlings were irradiated continuously for 24 h, the amount of phytochrome detected by ELISA and by spectrophotometric assay decreased by more than 1000-fold and about 100-fold, respectively. This discrepancy indicates that phytochrome in light-treated plants may be antigenically distinct from that found in fully etiolated plants. When these light-grown oat seedlings were kept in darkness for 48 h, phytochrome content detected by ELISA increased by 50-fold in crude extracts of green oat shoots, but only about 12-fold in extracts of herbicide-treated oat shoots. Phytochrome reaccumulation in green oat shoots was initially more rapid in the more mature cells of the primary leaf tip than near the basal part of the shoot. The inhibitory effect of Norflurazon on phytochrome accumulation was much more evident near the leaf tip than the shoot base. A 5-min red irradiation of oat seedlings at the end of a 48-h dark period resulted in a subsequent, massive decrease in phytochrome content in crude extracts from both green and Norflurazon-bleached oat shoots. These observations eliminate the possibility that substantial accumulation of chromophore-free phytochrome was being detected and indicate that Norflurazon has a substantial effect on phytochrome accumulation during a prolonged dark period. 25 references, 9 figures, 3 tables.

A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments.

Directory of Open Access Journals (Sweden)

Hannah L Albritton

Full Text Available Chlamydia trachomatis (CT is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs from two of the most common genital serovars (D and E were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

DEFF Research Database (Denmark)

Dziegiel, M; Borre, Mette; Petersen, E

1992-01-01

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...

Evaluation of Chicken IgY Generated Against Canine Parvovirus Viral-Like Particles and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Assay for Canine Parvovirus Detection.

Science.gov (United States)

He, Jinxin; Wang, Yuan; Sun, Shiqi; Zhang, Xiaoying

2015-11-01

Immunoglobulin Y (IgY) antibodies were generated against canine parvovirus virus-like particles (CPV-VLPs) antigen using chickens. Anti-CPV-VLPs-IgY was extracted from hen egg yolk and used for developing enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for the detection of CPV in dog feces. The cutoff negative values for anti-CPV-VLPs-IgY were determined using negative fecal samples (already confirmed by polymerase chain reaction [PCR]). In both ELISA and ICA, there was no cross-reaction with other diarrheal pathogens. Thirty-four fecal samples were collected from dogs with diarrhea, of which 26.47% were confirmed as CPV-positive samples by PCR, while 29.41% and 32.35% of the samples were found to be positive by ELISA and ICA, respectively. The developed ELISA and ICA exhibited 97.06% and 94.12% conformity with PCR. Higher sensitivity and specificity were observed for IgY-based ELISA and ICA. Thus, they could be suitable for routine use in the diagnosis of CPV in dogs.

Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

Science.gov (United States)

Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

2011-02-01

Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

Science.gov (United States)

A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

Science.gov (United States)

Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

2017-10-01

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

Hematology, plasma biochemistry, and tissue enzyme activities of invasive red lionfish captured off North Carolina, USA.

Science.gov (United States)

Anderson, E T; Stoskopf, M K; Morris, J A; Clarke, E O; Harms, C A

2010-12-01

The red lionfish Pterois volitans is important not only in the aquarium trade but also as an invasive species in the western Atlantic. Introduced to waters off the southeastern coast of the United States, red lionfish have rapidly spread along much of the East Coast and throughout Bermuda, the Bahamas, and much of the Caribbean. Hematology and plasma biochemistry were evaluated in red lionfish captured from the offshore waters of North Carolina to establish baseline parameters for individual and population health assessment. Blood smears were evaluated for total and differential white blood cell counts, and routine clinical biochemical profiles were performed on plasma samples. To improve the interpretive value of routine plasma biochemistry profiles, tissue enzyme activities (alkaline phosphatase [ALP], alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyl transferase [GGT], lactate dehydrogenase [LD], and creatine kinase [CK]) were analyzed from liver, kidney, skeletal muscle, gastrointestinal tract, and heart tissues from five fish. The hematological and plasma biochemical values were similar to those of other marine teleosts except that the estimated white blood cell counts were much lower than those routinely found in many species. The tissue enzyme activity findings suggest that plasma LD, CK, and AST offer clinical relevance in the assessment of red lionfish.

Comparative study of the enzyme linked immunosorbent assay (ELISA) and two radioimmunoassays (RIA'S) for in-sulin

Energy Technology Data Exchange (ETDEWEB)

Klimes, I; Jurcovicova, J; Palkovic, M [Slovenska Akademia Vied, Bratislava (Czechoslovakia). Ustav Experimentalnej Endokrinologie

1978-06-30

The results of the quality control tests for enzyme linked immunosorbent assay (ELISA) were compared with the results of two different radioimmunoassays (RIA'S) for insulin. Using the manufacturer's procedure for the ELISA kit we found that the analytical variables such as assay sensitivity, recovery study and the 50% binding intercept were in good agreement with those obtained with the RIA method.

Trypanosomosis surveillance on Zanzibar island, using the trypanosomal antigen detection ELISA (enzyme-linked immunosorbent assay) technique

Energy Technology Data Exchange (ETDEWEB)

Mbwambo, H A [Animal Disease Research Inst. (ADRI), Dar-es-Salaam (Tanzania, United Republic of)

1997-02-01

The effectiveness of trypanosomosis control programs depends greatly on prior knowledge of basic data of the epidemiological situation of the disease, which in turns depends, among others, on the use of techniques that give a fairly quick and accurate diagnosis. An antigen-detection (Ag) ELISA was first introduced into Tanzania and validated at the Animal Disease Research Institute (ADRI) through the FAO/IAEA Research Contract (RC) No. 5030/NL. Incorporation of the Ag-ELISA technique into a FAO animal disease control project (1986-1993) on Unguja island, in 1992, revealed useful information of high trypanosomosis prevalence in an area previously declared free of the disease using just stained blood smears and buffy coat examinations. This triggered further efforts into more intensive surveys of the tsetse and trypanosomosis situation on Unguja island. The present study is a continuation of previous work in an effort to confirm the practical application of Ag-ELISA in trypanosomosis control operations. Results obtained from a known tsetse and trypanosomosis-free area, on Pemba island, showed a high specificity of the test for Trypanosoma congolense, T. vivax and T. brucei. A preliminary cut-off value of 10% (Percent Positivity = PP) was used. When the PP of 10 was used on sera of trypanosomosis-endemic areas (Mangapwani, Ndijani, Dunga and Kikungwi) on Unguja island, the results reflected the `real` trypanosomis situation in the affected area. This was most strongly felt in the Mangapwani area, where tsetse and trypanosomosis were considered under control by 1994 (no tsetse flies were caught and no samples were encountered positive by the buffy coat technique). However, it should be stressed that the buffy coat technique and the Ag-ELISA complement each other and should be used in conjunction. (author). 8 refs, 1 fig., 5 tabs.

Trypanosomosis surveillance on Zanzibar island, using the trypanosomal antigen detection ELISA (enzyme-linked immunosorbent assay) technique

International Nuclear Information System (INIS)

Mbwambo, H.A.

1997-01-01

The effectiveness of trypanosomosis control programs depends greatly on prior knowledge of basic data of the epidemiological situation of the disease, which in turns depends, among others, on the use of techniques that give a fairly quick and accurate diagnosis. An antigen-detection (Ag) ELISA was first introduced into Tanzania and validated at the Animal Disease Research Institute (ADRI) through the FAO/IAEA Research Contract (RC) No. 5030/NL. Incorporation of the Ag-ELISA technique into a FAO animal disease control project (1986-1993) on Unguja island, in 1992, revealed useful information of high trypanosomosis prevalence in an area previously declared free of the disease using just stained blood smears and buffy coat examinations. This triggered further efforts into more intensive surveys of the tsetse and trypanosomosis situation on Unguja island. The present study is a continuation of previous work in an effort to confirm the practical application of Ag-ELISA in trypanosomosis control operations. Results obtained from a known tsetse and trypanosomosis-free area, on Pemba island, showed a high specificity of the test for Trypanosoma congolense, T. vivax and T. brucei. A preliminary cut-off value of 10% (Percent Positivity = PP) was used. When the PP of 10 was used on sera of trypanosomosis-endemic areas (Mangapwani, Ndijani, Dunga and Kikungwi) on Unguja island, the results reflected the 'real' trypanosomis situation in the affected area. This was most strongly felt in the Mangapwani area, where tsetse and trypanosomosis were considered under control by 1994 (no tsetse flies were caught and no samples were encountered positive by the buffy coat technique). However, it should be stressed that the buffy coat technique and the Ag-ELISA complement each other and should be used in conjunction. (author). 8 refs, 1 fig., 5 tabs

Epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from Japanese encephalitis virus infections in equine sera.

Science.gov (United States)

Kitai, Yoko; Shoda, Mizue; Kondo, Takashi; Konishi, Eiji

2007-08-01

West Nile virus (WNV) is now widely distributed worldwide, except in most areas of Asia where Japanese encephalitis virus (JEV) is distributed. Considering the movement and migration of reservoir birds, there is concern that WNV may be introduced in Asian countries. Although manuals and guidelines for serological tests have been created in Japan in preparedness for the introduction of WNV, differential diagnosis between WNV and JEV may be complicated by antigenic cross-reactivities between these flaviviruses. Here, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS1) of WNV and established an epitope-blocking enzyme-linked immunosorbent assay that can differentiate WNV from JEV infections in horse sera. Under conditions well suited for our assay system, samples collected from 95 horses in Japan (regarded as negative for WNV antibodies), including those collected from horses naturally infected with JEV, showed a mean inhibition value of 8.2% and a standard deviation (SD) of 6.5%. However, inhibition values obtained with serum used as a positive control (obtained after 28 days from a horse experimentally infected with WNV) in nine separate experiments showed a mean of 54.4% and an SD of 7.1%. We tentatively determined 27.6% (mean + 3 x SD obtained with 95 negative samples) as the cutoff value to differentiate positive from negative samples. Under this criterion, two horses experimentally infected with WNV were diagnosed as positive at 12 and 14 days, respectively, after infection.

Cellular immune response from Chagasic patients to CRA or FRA recombinant antigens of Trypanosoma cruzi.

Science.gov (United States)

Lorena, Virginia M B; Verçosa, Alinne F A; Machado, Raquel C A; Moitinho-Silva, Lucas; Cavalcanti, Maria G A; Silva, Edimilson D; Ferreira, Antonio G P; Correa-Oliveira, Rodrigo; Pereira, Valéria R A; Gomes, Yara M

2008-01-01

We propose to analyze the relation between the cellular immune response of Chagas' disease patients after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant antigens cytoplasmatic repetitive antigen (CRA) or flagellar repetitive antigen (FRA) of T. cruzi and the chronic clinical forms of disease. Cells were stimulated using phytohemagglutinin, CRA, FRA, or a soluble antigen of Epimastigota (Ag-Epi) for 24 hr, 72 hr, or 6 days. The proliferation of cells was evaluated after 6 days of culture by quantification of incorporated 3H-thymidine. Cytokines were measured in the supernatants obtained after 24 hr (tumor necrosis factor [TNF]-alpha and interleukin [IL]-4), 72 hr (IL-10), and 6 days (interferon [IFN]-gamma) using enzyme-linked immunosorbent assay (ELISA). Cells of the Chagas patients stimulated with the recombinant antigens exhibited higher proliferation responses compared with that of non-Chagas (NC) individuals. However, when proliferation was compared between patients with the cardiac form (CF) or indeterminate form (IF), it was not possible to establish a difference in the response. So far as the cytokines secreted in the culture supernatants after stimulation in vitro with T. cruzi antigens were concerned, the results showed that CRA, as well as Epi-Ag, were able to stimulate the production of TNF-alpha and IFN-gamma in Chagas patients as compared with NC individuals. However, the cytokine levels after stimulation with the T. cruzi antigens were not different between the patients with CF and IF. CRA was capable of inducing a T helper type 1 (Th1) immune response, with elevated production of TNF-alpha and IFN-gamma in Chagas patients that are carriers of CF and IF clinical forms. (Copyright ) 2008 Wiley-Liss, Inc.

Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products.

Science.gov (United States)

Zhang, Hongyan; Wang, Shuo

2009-10-31

The current status of enzyme-linked immunosorbent assays (ELISAs) for sulfonamides in edible animal products is reviewed. The attention was focused on the design and synthesis of haptens, conjugation to carrier protein, production of antibody, application of homologous and heterologous systems, as well as the molecular modeling of the haptens and sulfonamides. Researches have shown that sulfonamides seem to be particularly resistant to attempts to produce broad specificity antibodies. By summarizing the available research on sulfonamide ELISAs, it is hoped that it can be considered as a basis for further investigation aimed at developing the most efficient approaches for detection.

Changes of the antigenic and allergenic properties of a hen's egg albumin in a cake with gamma-irradiated egg white

International Nuclear Information System (INIS)

Lee, J.-W.; Seo, J.-H.; Kim, J.-H.; Lee, S.-Y.; Kim, K.-S.; Byun, M.-W.

2005-01-01

Changes of the antigenicity and allergenicity of a hen's egg albumin (ovalbumin, OVA) in white layer cakes containing egg white gamma-irradiated with 10 or 20 kGy were monitored by an enzyme-linked immunosorbent assay (ELISA), individually formatted with mouse anti-OVA IgG (mouse IgG) and with egg allergic patients' IgE. Mouse IgG recognized OVA in the cakes with irradiated egg white better than that in the control with a non-irradiated one. Whereas, the detected concentrations of intact OVA in the control significantly decreased in the treatments, when determined by IgE-based ELISA. The results appeared to indicate that the antigenicity of the OVA increased, but that the allergenicity was decreased by irradiation and processing. Egg white irradiated for reducing the egg allergy could be used for producing a safer cake from the egg allergy

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

DEFF Research Database (Denmark)

Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

2004-01-01

's thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture......B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto...

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

DEFF Research Database (Denmark)

Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

2004-01-01

B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto......'s thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture...

A nanoparticle label/immunochromatographic electrochemical biosensor for rapid and sensitive detection of prostate-specific antigen

Energy Technology Data Exchange (ETDEWEB)

Lin, Ying-Ying; Wang, Jun; Liu, Guodong; Wu, Hong; Wai, Chien M.; Lin, Yuehe

2008-06-15

We present a nanoparticle (NP) label/immunochromatographic electrochemical biosensor (IEB) for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. This IEB integrates the immunochromatographic strip with the electrochemical detector for transducing quantitative signals. The NP label, made of CdSe@ZnS, serves as a signal-amplifier vehicle. A sandwich immunoreaction was performed on the immunochromatographic strip. The captured NP labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable-screen-printed electrode, which is embedded underneath the membrane of the test zone. Experimental parameters (e.g., immunoreaction time, the amount of anti-PSA-NP conjugations applied) and electrochemical detection conditions (e.g., preconcentration potential and time) were optimized using this biosensor for PSA detection. The analytical performance of this biosensor was evaluated with serum PSA samples according to the “figure-of-merits” (e.g., dynamic range, reproducibility, and detection limit). The results were validated with enzyme-linked immunosorbent assay (ELISA) and show high consistency. It is found that this biosensor is very sensitive with the detection limit of 0.02 ng/mL PSA and is quite reproducible. This method is rapid, clinically accurate, and less expensive than other diagnosis tools for PSA; therefore, this IEB coupled with a portable electrochemical analyzer shows great promise for simple, sensitive, quantitative point-of-care testing of disease-related protein biomarkers.

Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

Directory of Open Access Journals (Sweden)

Cheng-Liang Xie

2014-01-01

Full Text Available Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50 of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR. The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

Vaccination with an adenoviral vector encoding the tumor antigen directly linked to invariant chain induces potent CD4(+) T-cell-independent CD8(+) T-cell-mediated tumor control

DEFF Research Database (Denmark)

Sorensen, Maria R; Holst, Peter J; Pircher, Hanspeter

2009-01-01

of the vaccine antigen to invariant chain (Ii). To evaluate this strategy we used a mouse model, in which an immunodominant epitope (GP33) of the LCMV glycoprotein (GP) represents the tumor-associated neoantigen. Prophylactic vaccination of C57BL/6 mice with a replication-deficient human adenovirus 5 vector...... encoding GP linked to Ii (Ad-Ii-GP) resulted in complete protection against GP33-expressing B16.F10 tumors. Therapeutic vaccination with Ad-Ii-GP delayed tumor growth by more than 2 wk compared with sham vaccination. Notably, therapeutic vaccination with the linked vaccine was significantly better than...... the tumor degradation. Finally, Ad-Ii-GP but not Ad-GP vaccination can break the immunological non-reactivity in GP transgenic mice indicating that our vaccine strategy will prove efficient also against endogenous tumor antigens....

How-to-do-it: Immunological Assays for the Classroom 1. Enzyme Linked Immunosorbent Assay (ELISA): A Laboratory Tool for Demonstration of Antibody-Antigen Interaction.

Science.gov (United States)

Russo, A. J.; And Others

1984-01-01

Background information, list of required materials, and procedures are provided for an immunological assay which has been modified for use as a classroom/laboratory demonstration of antigen-antibody reaction. The assay is designed for a two and one-half hour laboratory period but may be modified for one hour laboratories. (JN)

Enzyme-linked immunosorbent assay and radioimmunoassay of serum pepsinogen A

Energy Technology Data Exchange (ETDEWEB)

Pals, G; Meuwissen, S G.M.; Frants, R R; Kostense, P J; Eriksson, A W; Raesaenen, V

1987-02-01

The determination of serum pepsinogen A (=pepsinogen I) levels is of clinical importance in the study of duodenal ulcer, atrophic gastritis and gastric cancer. In the present study two different quantitative immunological techniques for serum pepsinogen A were compared: a radioimmunoassay (RIA) (Helsinki) and an enzyme-linked immunosorbent assay (ELISA) (Amsterdam). Serum samples of 177 subjects with various gastric diseases were tested in a double blind study. The correlation was excellent (r=0.954 in the range 0-760 ..mu..g/l and r=0.971 in the range 0-100 ..mu..g/l). The functional relationship between ELISA (x) and RIA (y), determined by weighted model II regression, was y=1.12x-0.54. Initially the use of goat anti-PGA in the ELISA resulted in falsely high values in about 10% of the individuals. This was caused by circulating antibodies cross-reacting with goat IgG. This artefact was eliminated by pre-incubation of all samples with non-immune goat serum.

Enzyme-linked immunosorbent assay and radioimmunoassay of serum pepsinogen A

International Nuclear Information System (INIS)

Pals, Gerard; Meuwissen, S.G.M.; Frants, R.R.; Kostense, P.J.; Eriksson, A.W.; Raesaenen, Vesa

1987-01-01

The determination of serum pepsinogen A (=pepsinogen I) levels is of clinical importance in the study of duodenal ulcer, atrophic gastritis and gastric cancer. In the present study two different quantitative immunological techniques for serum pepsinogen A were compared: a radioimmunoassay (RIA) (Helsinki) and an enzyme-linked immunosorbent assay (ELISA) (Amsterdam). Serum samples of 177 subjects with various gastric diseases were tested in a double blind study. The correlation was excellent (r=0.954 in the range 0-760 μg/l and r=0.971 in the range 0-100 μg/l). The functional relationship between ELISA (x) and RIA (y), determined by weighted model II regression, was y=1.12x-0.54. Initially the use of goat anti-PGA in the ELISA resulted in falsely high values in about 10% of the individuals. This was caused by circulating antibodies cross-reacting with goat IgG. This artefact was eliminated by pre-incubation of all samples with non-immune goat serum. (author)

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

Science.gov (United States)

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

The use of subcutaneous fat tissue for amyloid typing by enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Olsen, K E; Sletten, K; Westermark, Per

1999-01-01

for typing the most common systemic amyloidoses of AL, AA, and transthyretin types by enzyme-linked immunosorbent assay (ELISA), using abdominal wall subcutaneous fat biopsy specimens. The method was tested on 21 abdominal fat biopsy specimens that were sent to the laboratory. Of these, 15 contained amyloid......The amyloidoses are biochemically heterogeneous diseases with pathophysiologic deposits of various proteins. The clinical course, prognosis, and therapy are different for each type of amyloidosis and, therefore, a type-specific diagnosis is demanded as early as possible. We describe a method...

Simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles.

Science.gov (United States)

Markwalter, Christine F; Ricks, Keersten M; Bitting, Anna L; Mudenda, Lwiindi; Wright, David W

2016-12-01

We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5-8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Expression and immunological characterisation of Eimeria tenella glycosylphosphatidylinositol-anchored surface antigen-5

Science.gov (United States)

Ho, Sue-Kim; Nathan, Sheila; Wan, Kiew-Lian

2016-11-01

Eimeria tenella is the most pathogenic of the Eimeria species that infect chickens and causes huge economic losses to the poultry industry. The glycosylphosphatidylinositol-anchored surface antigen-5 (SAG5) found on the surface of the parasite has been shown to activate the chicken's immune system. In this study, recombinant SAG5 was expressed, purified and used to investigate the immune-inducing characteristics of the molecule. Chickens were immunized with purified recombinant SAG5 and sera were subjected to Enzyme-linked Immunosorbant Assay (ELISA). Results indicated that specific antibodies against rSAG5 were produced, with IgG detected at a higher level compared to IgA and IgM. Information on the immunological responses elicited by SAG5 provides essential knowledge that will contribute towards the effort to develop more effective strategies against coccidiosis.

Development of an enzyme-linked immunosorbent assay-based method for measuring galactosyltransferase activity using a synthetic glycopolymer acceptor substrate.

Science.gov (United States)

Oubihi, M; Kitajima, K; Kobayashi, K; Adachi, T; Aoki, N; Matsuda, T

1998-03-15

A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring beta 1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a beta-linked N-acetylglucosamine (GlcNAc beta) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, beta-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by using Ricinus communis agglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAc beta), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.

INDUCTION OF AUTOANTIBODIES TO HUMAN ENZYMES FOLLOWING VIRAL-INFECTION - A BIOLOGICALLY RELEVANT HYPOTHESIS

NARCIS (Netherlands)

WEIJERS, RNM; LAWSON, C; LEUNISSEN, J

Macro enzymes, i. e. complexes of normal (iso-)enzymes with an immunoglobulin, may be due to immunological cross-reactions evoked by specific viral antigenic determinants that are homologous to regions in the target enzymes. A search of the National Biomedical Research Foundation protein databank

Management of bacterial kidney disease in Chinook Salmon hatcheries based on broodstock testing by enzyme-linked immunosorbent assay: A multiyear study

Science.gov (United States)

Munson, A. Douglas; Elliott, Diane G.; Johnson, Keith

2010-01-01

From the mid-1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin-medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme-linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen levels, or (2) culling of eggs from females with high ELISA OD values. The ELISA-based culling program had the most profound positive effects on the study populations. Mortality of juvenile fish during rearing was significantly lower at each hatchery for brood years derived from culling compared with brood years for which culling was not practiced. The prevalence of R. salmoninarum in juvenile fish, as evidenced by detection of the bacterium in kidney smears by the direct fluorescent antibody test, also decreased significantly at each hatchery. In addition, the proportions of returning adult females with kidney ELISA OD values of 0.25 or more decreased 56–85% for fish reared in brood years during which culling was practiced, whereas the proportions of ELISA-negative adults increased 55–58%. This management strategy may allow IDFG Chinook salmon hatcheries to reduce or eliminate prophylactic erythromycin-medicated feed treatments. We recommend using ELISA

Central nervous system blastomycosis diagnosed using the MVista® Blastomyces quantitative antigen enzyme immunoassay test on cerebrospinal fluid: A case report and review of the literature.

Science.gov (United States)

Walkty, Andrew; Keynan, Yoav; Karlowsky, James; Dhaliwal, Perry; Embil, John

2018-02-01

Blastomyces dermatitidis is a thermally dimorphic fungus that is capable of causing pulmonary and extra-pulmonary disease, including infections of the central nervous system (CNS). Diagnosis of CNS blastomycosis with non-invasive testing can be difficult, and a surgical biopsy may ultimately be required for microbiological and/or histopathological confirmation. A case of B. dermatitidis meningitis is presented where the diagnosis was made by testing cerebrospinal fluid (CSF) using the MVista® Blastomyces Quantitative Antigen Enzyme Immunoassay test. The utility of performing this test on CSF for diagnosis of CNS mass lesions/abscesses caused by B. dermatitidis in the absence of associated meningitis remains unclear. Cross reaction of the Blastomyces antigen test with other dimorphic fungi is a concern, necessitating that positive test results are interpreted in the context of the patient's exposure and travel history. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

NARCIS (Netherlands)

Damen, Carola W. N.; de Groot, Els R.; Heij, Marianne; Boss, David S.; Schellens, Jan H. M.; Rosing, Hilde; Beijnen, Jos H.; Aarden, Lucien A.

2009-01-01

Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to

Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

Directory of Open Access Journals (Sweden)

Rafaella Fortini Queiroz Grenfell

2012-08-01

Full Text Available INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62. In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively, whereas ELISA using egg antigens (ELISA-SEA detected samples after 140 days (p=0.03. CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

Linking Hydrolysis Performance to Trichoderma reesei Cellulolytic Enzyme Profile

DEFF Research Database (Denmark)

Lehmann, Linda Olkjær; Petersen, Nanna; I. Jørgensen, Christian

2016-01-01

Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, e.g. by spiking with single enzymes and monitoring hydrolysis performance. In this study a multivariate...... approach, partial least squares regression, was used to see if it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by Trichoderma reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed...

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

Science.gov (United States)

Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma.

Science.gov (United States)

Thuring, Camilla; Follin, Elna; Geironson, Linda; Freyhult, Eva; Junghans, Victoria; Harndahl, Mikkel; Buus, Søren; Paulsson, Kajsa M

2015-09-15

Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with β2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols.

Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins.

Science.gov (United States)

Venkatachalam, M; Teuber, S S; Roux, K H; Sathe, S K

2002-06-05

Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart.

AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D)

Science.gov (United States)

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk pl...

Continuum capture in the three-body problem

International Nuclear Information System (INIS)

Sellin, I.A.

1980-01-01

The three-body problem, especially the problem of electron capture to the continuum in heavy particle collisions is reviewed. Major topics covered include: second born-induced asymmetry in electron capture to the continuum; historical context, links to other tests of atomic scattering theory; experiments characterizing the velocity distribution of ECC electrons; other atomic physics tests of high velocity Born expansions; atom capture; capture by positrons; and pion capture to the continuum

Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay.

Science.gov (United States)

Niu, Qingli; Liu, Zhijie; Yang, Jifei; Yu, Peifa; Pan, Yuping; Zhai, Bintao; Luo, Jianxun; Guan, Guiquan; Yin, Hong

2016-12-01

Ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

Directory of Open Access Journals (Sweden)

Rodríguez Islay

2002-01-01

Full Text Available A recombinant-antigen enzyme immunoassay (EIA, BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38, secondary (n = 10, early latent (n = 28 and congenital syphilis (n = 2, patients with leptospirosis ( n= 8, infectious mononucleosis (n = 7, hepatitis (n = 9, diabetes mellitus (n = 11, rheumatoid arthritis (n = 13, leprosy (n = 11, tuberculosis (n = 9, HIV/Aids ( n= 12, systemic lupus erythematosus (n = 4, rheumatic fever (n = 3, old-persons (n = 9, pregnant women (n = 29 and blood donors (n = 164. The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

Protamine-based nanoparticles as new antigen delivery systems.

Science.gov (United States)

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Two competitive enzyme immunoassays for the detection of IgG class antibodies to hepatitis a antigen

Directory of Open Access Journals (Sweden)

Claudia Lamarca Vitral

1991-06-01

Full Text Available Two competitive enzyme immunoassays (EIA techniques were developed: in the first (COMP-1, test sera were added together with HAV antigen on anti-HAV IgG-coated wells followed by an anti-HA VHRP conjugate; in the second (COMP-2, test sera and anti-HA VHRP conjugate competed for HAV epitopes previously adsorbed to anti-HA V IgG-coated wells. Both procedures used tetramethylbenzidine (TMB as a substrate. Both competitive tests were shown to be reproducible and suitable for routine diagnosis and research purposes.Foram desenvolvidos dois ensaios imunoenzimáticos (EIA competitivos: no primeiro (COMP-1 colocou-se numa placa sensibilizada com anti-HAVIgG as amostras teste juntamente como antígeno HA Vea seguir o conjugado anti-HA VHRP; no segundo (COMP-2, as amostras teste e o conjugado anti-HAV HRP competem pelos epitopos do antígeno HAV previamente absorvido na placa sensibilizada do anti-HAV IgG. O substrato utilizado foi tetrametilbenzidina (TMB. Ambas as técnicas mostraram ser produtíveis e aplicáveis para fins de diagnóstico e pesquisa.

Use of a trypanosomal antigen ELISA to monitor tsetse and trypanosomosis control programmes in Kenya

Energy Technology Data Exchange (ETDEWEB)

Olaho-Mukani, W; Munga, L K; Nyanga` O, J N.M.; Ouma, J O; Masika, P; Okech, G; Ndungu, J M [Kenya Trypanosomiasis Research Inst., Kikuyu (Kenya)

1997-02-01

The capture Antigen-ELISA was used to monitor serum samples originating from three study areas in Kenya. At the Galana ranch the test was used to assess re-invasion of an area previously cleared of Glossina pallidipes. In Busia district the Ag-ELISA is being used to monitor the progress of a tsetse and trypanosomosis control programme. At Taita and Tara ranches the capture Antigen ELISA and the buffy coat technique (BCT) were used to evaluate the efficacy of a commercial Cypermethrin dip for the control of Glossina pallidipes. (author). 3 refs, 4 figs, 2 tabs.

Specific capture of target bacteria onto sensor surfaces for infectious disease diagnosis

International Nuclear Information System (INIS)

Kim, Jong-Hoon; Inoue, Shinnosuke; Chung, Jae-Hyun; Cangelosi, Gerard A; Lee, Kyong-Hoon

2014-01-01

A long-sought goal for infectious disease care is a rapid and accurate diagnostic tool that is compatible with the needs of low-resource settings. To identify target biomarkers of infectious diseases, immunoassays utilizing the binding affinity between antigen and antibody have been widely used. In immunoassays, the interaction between antigen and antibody on sensor surfaces should be precisely controlled for specific identification of targets. This paper studies the specific capturing mechanisms of target bacteria onto sensor surfaces through investigation of combined effects of capillary action and binding affinity. As a model system, cells of both Escherichia coli and the Bacillus Calmette-Guérin strain of Mycobacterium bovis were used to study specific and nonspecific capturing mechanisms onto a microtip sensor. The capillary action was observed to arrange the concentrated cells onto the two-dimensional sensor surface. Due to the capillary-induced organization of target cells on the antibody-functionalized sensor surface, the number of the captured target cells was three times greater than that of the non-targeted cells. The capturing and detection capabilities varied with the width of a microtip. The specific capturing mechanism can be used to enhance the sensitivity and specificity of an immunoassay. (paper)

Development of horseradish peroxidase-based cross-linked enzyme aggregates and their environmental exploitation for bioremediation purposes.

Science.gov (United States)

Bilal, Muhammad; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2017-03-01

In the present study, horseradish peroxidase (HRP), in-house isolated crude cocktail enzyme, from Armoracia rusticana was cross-linked using a new type of cross-linking agent, i.e., ethylene glycol-bis [succinic acid N-hydroxysuccinimide, (EG-NHS)], which is mild in nature as compared to the glutaraldehyde (GA). The HRP-immobilized cross-linked enzyme aggregates (HRP-CLEAs) were developed using a wider range of EG-NHS and notably no adverse effect was observed. In a comparative evaluation, in the case of EG-NHS, a high-level stability in the residual activity was recorded, whereas a sharp decrease was observed in the case of glutaraldehyde. Following initial cross-linker evaluation, the HRP-CLEAs were tested to investigate their bio-catalytic efficacy for bioremediation purposes using a newly developed packed bed reactor system (PBRS). A maximal of 94.26% degradation of textile-based methyl orange dye was recorded within the shortest time frame, following 91.73% degradation of basic red 9, 84.35% degradation of indigo, 81.47% degradation of Rhodamin B, and 73.6% degradation of Rhodamine 6G, respectively, under the same working environment. Notably, the HRP-CLEAs retained almost 60% of its original activity after methyl orange dye degradation in seven consecutive cycles using PBRS. Furthermore, after HRP-CLEAs-mediated treatment in the PBRS, a significant toxicity reduction in the dye samples was recorded as compared to their pristine counterparts. In conclusion, the results suggest that the newly developed HRP-CLEAs have a great potential for industrial exploitation, to tackle numerous industrial dye-based emergent pollutants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay

Directory of Open Access Journals (Sweden)

Hossam Murad

2017-05-01

Full Text Available BackgroundMonitoring blood levels of human growth hormone (hGH in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin®. Besides, misusing of rhGH by sportsmen is banned by the World Anti-Doping Agency and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies. They were successfully generated against numerous antigens including hormones.MethodsA fully nanobody-based sandwich ELISA method was developed in this work for direct measurement of GH in biological samples.ResultsTwo major characteristics of nanobody were exploited for this goal: the robust and stable structure of the nanobody (NbGH04 used to capture hGH from tested samples, and the great ability of tailoring, enabling the display of the anti-GH detector nanobody (NbGH07 on the tip of M13-phage. Such huge, stable, and easy-to-prepare phage-Nb was used in ELISA to provide an amplified signal. Previously, NbGH04 was retrieved on immobilized hGH by phage display from a wide “immune” cDNA library prepared from a hGH-immunized camel. Here, and in order to assure epitope heterogeneity, NbGH07 was isolated from the same library using NbGH04-captured hGH as bait. Interaction of both nanobodies with hGH was characterized and compared with different anti-GH nanobodies and antibodies. The sensitivity (~0.5 ng/ml and stability of the nanobody-base sandwich ELISA were assessed using rhGH before testing in the quantification of hGH in blood sera and cell culture supernatants.ConclusionIn regard to all advantages of nanobodies; stability, solubility, production affordability in Escherichia coli, and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.

Chemoselective ligation and antigen vectorization.

Science.gov (United States)

Gras-Masse, H

2001-01-01

The interest in cocktail-lipopeptide vaccines has now been confirmed by phase I clinical trials: highly diversified B-, T-helper or cytotoxic T-cell epitopes can be combined with a lipophilic vector for the induction of B- and T-cell responses of predetermined specificity. With the goal of producing an improved vaccine that should ideally induce a multispecific response in non-selected populations, increasing the diversity of the immunizing mixture represents one of the most obvious strategies.The selective delivery of antigens to professional antigen-presenting cells represents another promising approach for the improvement of vaccine efficacy. In this context, the mannose-receptor represents an attractive entry point for the targeting to dendritic cells of antigens linked to clustered glycosides or glycomimetics. In all cases, highly complex but fully characterized molecules must be produced. To develop a modular and flexible strategy which could be generally applicable to a large set of peptide antigens, we elected to explore the potentialities of chemoselective ligation methods. The hydrazone bond was found particularly reliable and fully compatible with sulphide ligation. Hydrazone/thioether orthogonal ligation systems could be developed to account for the nature of the antigens and the solubility of the vector systems. Copyright 2001 The International Association for Biologicals.

Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins

International Nuclear Information System (INIS)

Huang Yong; Shi Ruina; Zhong Xuefei; Wang Dan; Zhao Meiping; Li Yuanzong

2007-01-01

The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins

A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers

International Nuclear Information System (INIS)

Oram, J.D.; Crooks, A.J.

1979-01-01

A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods. (Auth.)

Desomorphine Screening Using Commercial Enzyme-Linked Immunosorbent Assays.

Science.gov (United States)

Winborn, Jessica; Kerrigan, Sarah

2017-06-01

Desomorphine ("Krokodil") is a semi-synthetic opioid that has drawn attention as a recreational drug, particularly in Russia, neighboring former Soviet Republics, Eastern and Central Europe. It has no accepted medicinal uses and is currently a schedule I drug in the United States. In clandestine environments, desomorphine is synthesized from codeine using red phosphorous, hydroiodic acid and gasoline. Residual starting materials in illicit preparations have been associated with severe dermatological effects and extensive tissue necrosis. Desomorphine is not well studied, and there are limited reports concerning its pharmacology or detection in biological matrices. Immunoassays are widely relied upon for both antemortem and postmortem toxicology screening. Although desomorphine is an opioid of the phenanthrene-type, its ability to bind to conventional opioid antibodies has not been described. In this report we describe the cross-reactivity of desomorphine using six commercially available enzyme-linked immunosorbent assays (Immunalysis Opiates Direct ELISA, Immunalysis Oxycodone/Oxymorphone Direct ELISA, Randox Opiate ELISA, OraSure Technologies OTI Opiate Micro-plate EIA, Neogen Opiate Group ELISA and Neogen Oxycodone/Oxymorphone ELISA). Cross-reactivites were highly variable between assays, ranging from 77 to desomorphine than those directed towards oxycodone. The Immunalysis Opiates Direct ELISA produced the greatest cross-reactivity, although several of the assays evaluated produced cross-reactivity of a sufficient magnitude to be effective for desomorphine screening. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Biosynthesis, purification and characterization of commercial enzyme by penicillium expansum link

International Nuclear Information System (INIS)

Ahmed, K.; Valeem, E.E.

2015-01-01

Ever growing biotechnological industry has motivated the research towards the comprehensive survey of microorganisms, which could be used in extreme conditions of industry. In the present work optimization parameters in submerged fermentation, purification and characterization of invertase from Penicillium expansum Link using agricultural wastes (sunflower waste, cotton stalk and rice husk) as well as agro-industrial wastes (date syrup and molasses) as sources of carbon. Maximum production of invertase (7.03 U/mL) was observed when the strain was grown on culture medium (CM1) containing yeast extract as a source of nitrogen, date syrup as a source of carbon after 48 h of incubation at initial pH 5.0, temperature 35 degree C, inoculum size of 6x106 conidia in 50 mL of culture medium and agitation rate of 150 rev/min. After optimization the enzyme was also purified partially and then characterized. Kinetic constants (Km 2.57 mM and Vmax 178.6 U/mL/min) were determined by Lineweaver-Burk Plot and molecular mass (110 kDa) by 10% SDS-PAGE. Invertase showed maximum activity at pH 5.5 (128.7 U/mL) and at the temperature of 60 degree C (114.6 U/mL). BaCl/sub 2/ (21.9%), MgSO/sub 4/ (42.6%), MnCl/sub 2/ (46.8%) and EDTA (8.3%) enhanced the relative activity of enzyme while HgCl2 (-90.9%), CuSO/sub 4/ (-82.3%) and CuCl/sub 2/ (-78.7%) were proved inhibitors. (author)

Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

Science.gov (United States)

This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

Science.gov (United States)

Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

2015-01-01

The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice.

Science.gov (United States)

Yim, Seol-Hwa; Hahn, Tae-Wook; Joo, Hong-Gu

2017-09-30

We previously demonstrated that Bordetella ( B .) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma ( M .) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Possible role of specific immunoglobulin M antibodies to Plasmodium falciparum antigens in immunoprotection of humans living in a hyperendemic area, Burkina Faso

DEFF Research Database (Denmark)

Boudin, C; Chumpitazi, B; Dziegiel, M

1993-01-01

of antibodies to crude extracts of Plasmodium falciparum (IgG or IgM antisomatic and IgG antiexoantigens) were tested by IFI or enzyme-linked immunosorbent assay and were followed up according to the fluctuations of the parasite densities. Specific IgG antibodies had the same evolution as parasite densities....... Group 3 was composed of immunoprotected adults. Specific IgM and IgG antibodies to crude extracts or a recombinant antigen (glutamate-rich protein) of P. falciparum were tested. Specific IgM antibodies were lower in group 1 (nonimmune) than in groups 2 (semiimmune) and 3 (immunoprotected). Furthermore...

Microbial genetic engineering and enzyme technology

Energy Technology Data Exchange (ETDEWEB)

Hollenberg, C.P.; Sahm, H.

1987-01-01

In a series of up-to-date contributions BIOTEC 1 has experts discussing the current topics in microbial gene technology and enzyme technology and speculating on future developments. Bacterial and yeast systems for the production of interferons, growth hormone or viral antigenes are described as well as the impact of gene technology on plants. Exciting is the prospect of degrading toxic compounds in our environment by microorganisms tuned in the laboratory. Enzymes are the most effective catalysts we know. They exhibit a very high substrate- and stereospecificity. These properties make enzymes extremely attractive as industrial catalysts, leading to new production processes that are non-polluting and save both energy and raw materials. (orig.) With 135 figs., 36 tabs.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

Energy Technology Data Exchange (ETDEWEB)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

International Nuclear Information System (INIS)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

1987-01-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [ 14 C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined

Changes of the antigenic and allergenic properties of a hen's egg albumin in a cake with gamma-irradiated egg white

Energy Technology Data Exchange (ETDEWEB)

Lee, J.-W.; Seo, J.-H.; Kim, J.-H.; Lee, S.-Y.; Kim, K.-S.; Byun, M.-W. E-mail: mwbyun@nanum.kaeri.re.kr

2005-04-01

Changes of the antigenicity and allergenicity of a hen's egg albumin (ovalbumin, OVA) in white layer cakes containing egg white gamma-irradiated with 10 or 20 kGy were monitored by an enzyme-linked immunosorbent assay (ELISA), individually formatted with mouse anti-OVA IgG (mouse IgG) and with egg allergic patients' IgE. Mouse IgG recognized OVA in the cakes with irradiated egg white better than that in the control with a non-irradiated one. Whereas, the detected concentrations of intact OVA in the control significantly decreased in the treatments, when determined by IgE-based ELISA. The results appeared to indicate that the antigenicity of the OVA increased, but that the allergenicity was decreased by irradiation and processing. Egg white irradiated for reducing the egg allergy could be used for producing a safer cake from the egg allergy.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

International Nuclear Information System (INIS)

Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

2011-01-01

Highlights: → Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. → An ideal artificial APCs system was successfully prepared in vivo. → Controlled release of IL-2 leads to much more T-cell expansion. → This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

Energy Technology Data Exchange (ETDEWEB)

Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

2011-08-05

Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

Directory of Open Access Journals (Sweden)

Franklin B Magalhães

Full Text Available Current strategies for the control of zoonotic visceral leishmaniasis (VL rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity although their performance was increased in the canine sera (48-91% sensitivity, with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84% and canine (88% sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

Development of a biomimetic enzyme-linked immunosorbent assay based on molecularly imprinted polymers on paper for the detection of carbaryl.

Science.gov (United States)

Zhang, Can; Cui, Hanyu; Han, Yufeng; Yu, Fangfang; Shi, Xiaoman

2018-02-01

A biomimetic enzyme-linked immunosorbent assay (BELISA) which was based on molecularly imprinted polymers on paper (MIPs-paper) with specific recognition was developed. As a detector, the surface of paper was modified with γ-MAPS by hydrolytic action and anchored the MIP layer on γ-MAPS modified-paper by copolymerization to construct the artificial antibody Through a series of experimentation and verification, we successful got the MIPs-paper and established BELISA for the detection of carbaryl. The development of MIPs-paper based on BELISA was applied to detect carbaryl in real samples and validated by an enzyme-linked immunosorbent assay (ELISA) based on anti-carbaryl biological antibody. The results of these two methods (BELISA and ELISA) were well correlated (R 2 =0.944). The established method of MIPs-paper BELISA exhibits the advantages of low cost, higher stability and being re-generable, which can be applied as a convenient tool for the fast and efficient detection of carbaryl. Copyright © 2017. Published by Elsevier Ltd.

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

Science.gov (United States)

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten

2014-01-01

The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (m......Abs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal...... diseases....

Optimization of Diagnostic Elisa - Based Tests for the Detection of Auto-Antibodies Against Tumor Antigens in Human Serum

Directory of Open Access Journals (Sweden)

Daria Štefatić

2008-08-01

Full Text Available Colorectal cancer is one of the most common cancer types worldwide and it continues to be a serious public health problem. Early detection and diagnosis are of great importance in cancer management. At present, diagnostic blood tests are based on the detection of tumor-associated markers such as carcinoembryonic antigen (CEA, the cancer antigen CA19-9 for gastrointestinal cancer, CA15-3 for breast cancer or CA125 for ovarian cancer. The lack of sensitivity and specificity of these markers prevents their general use in cancer screening of an average risk population. Therefore, new cancer biomarkers or better screening methods are necessary to improve the diagnostics of the disease. This study was directed to the optimization of a diagnostic, enzyme linked immunosorbent assay (ELISA based test to identify and validate new serum markers, such as extracellular Protein Kinase A (ecPKA and Nicotinamide A-Meth- yltransferase (NNMT. In this type of assay, the cancer antigens are quantified indirectly - by detecting the presence of auto-antibodies against tumor proteins in human serum. The result of the optimization and validation process was in the case of ecPKA a reproducible and stable assay. In case of NNMT the assay was probably not sensitive enough.

A sensitive label-free electrochemical immunosensor for detection of cytokeratin 19 fragment antigen 21-1 based on 3D graphene with gold nanopaticle modified electrode.

Science.gov (United States)

Zeng, Yan; Bao, Jing; Zhao, Yanan; Huo, Danqun; Chen, Mei; Yang, Mei; Fa, Huanbao; Hou, Changjun

2018-02-01

Previous studies have confirmed that cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) serves as a powerful biomarker in non-small cell lung cancer (NSCLC). Herein, we report for the first time a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker CYFRA21-1. In this work, three-dimensional graphene @ gold nanoparticles (3D-G@Au) nanocomposite was modified on the glassy carbon electrode (GCE) surface to enhance the conductivity of immunosensor. The anti-CYFRA21-1 captured and fixed on the modified GCE through the cross-linking of chitosan (CS), glutaraldehyde (GA) and anti-CYFRA21-1. The differential pulse voltammetry (DPV) peak current change due to the specific interaction between anti-CYFRA21-1 and CYFRA21-1 on the modified electrode surface was utilized to detect CYFRA21-1. Under optimized conditions, the proposed electrochemical immunosensor was employed to detect CYFRA21-1 and exhibited a wide linear range of 0.25-800ngmL -1 and low detection limit of 100pgmL -1 (S/N = 3). Moreover, the recovery rates of serum samples were in the range from 95.2% to 108.7% and the developed immunosensor also shows a good correlation (less than 6.6%) with enzyme-linked immunosorbent assay (ELISA) in the detection of clinical serum samples. Therefore, it is expected that the proposed immunosensor based on a 3D-G@Au has great potential in clinical medical diagnosis of CYFRA21-1. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of an enzyme-linked immunosorbent assay for the detection of ciguatoxin in fish tissue using chicken immunoglobulin Y.

Science.gov (United States)

Empey Campora, Cara; Hokama, Yoshitsugi; Yabusaki, Kenichi; Isobe, Minoru

2008-01-01

A sandwich enzyme-linked immunosorbent assay was developed to detect ciguatoxin (CTX) in fish tissue. The assay utilizes two antibodies, chicken immunoglobulin Y specific to the ABCD domain of CTX and a mouse monoclonal immunoglobulin G-horseradish peroxidase conjugate specific to the JKLM domain of CTX. The sensitivity, working range, cross reactivity, accuracy, precision, and reproducibility were examined.

A theoretical compartment model for antigen kinetics in the skin

NARCIS (Netherlands)

Römgens, A.M.; Bader, D.L.; Bouwstra, J.A.; Oomens, C.W.J.

2016-01-01

The skin is a promising location for vaccination with its abundant population of antigen capturing and presenting cells. The development of new techniques, such as the use of microneedles, can facilitate the delivery of vaccines into the skin. In recent years, many different types of microneedle

The link between CD8⁺ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate.

Science.gov (United States)

Lissina, Anna; Fastenackels, Solène; Inglesias, Maria C; Ladell, Kristin; McLaren, James E; Briceño, Olivia; Gostick, Emma; Papagno, Laura; Autran, Brigitte; Sauce, Delphine; Price, David A; Saez-Cirion, Asier; Appay, Victor

2014-02-20

Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag₂₆₃₋₂₇₂ KK10 epitope restricted by human leukocyte antigen (HLA)-B27. To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef₇₃₋₈₂ QK10 and Gag₂₀₋₂₉ RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag₂₆₃₋₂₇₂ KK10. For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

Design of a single-step immunoassay principle based on the combination of an enzyme-labeled antibody release coating and a hydrogel copolymerized with a fluorescent enzyme substrate in a microfluidic capillary device.

Science.gov (United States)

Wakayama, Hideki; Henares, Terence G; Jigawa, Kaede; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-11-21

A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

Enzyme-Linked Immunosorbent Assay Using a Virus Type-Specific Peptide Based on a Subdomain of Envelope Protein Erns for Serologic Diagnosis of Pestivirus Infections in Swine

Science.gov (United States)

Langedijk, J. P. M.; Middel, W. G. J.; Meloen, R. H.; Kramps, J. A.; de Smit, J. A.

2001-01-01

Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the Erns protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed. To identify type-specific pestivirus antibodies, a liquid-phase peptide ELISA, with a labeled, specific classical swine fever virus (CSFV) peptide and an unlabeled bovine viral diarrhea virus peptide to block cross-reactivity, was developed. Specificity and sensitivity of the liquid-phase peptide ELISA for CSFV were 98 and 100%, respectively. Because the peptide is a fragment of the Erns protein, it can be used to differentiate between infected and vaccinated animals when a vaccine based on the E2 protein, which is another pestivirus envelope protein, is used. PMID:11230402

Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin

DEFF Research Database (Denmark)

Petersen, H. H.; Nielsen, J. P.; Jensen, A.L.

2001-01-01

An enzyme-linked immunosorbent assay for quantification of haptoglobin in porcine serum was evaluated. Tbe detection limit when expressed as the estimated concentration of a blank sample was 0.0003 mg/ml. The precision of the assay was acceptable with intra-assay coefficients of variation below 4...... % and inter-assay coefficient of variation below 5 % for serum concentrations ranging from 1.0 mg/ml and above. For samples with a concentration below 0.8 mg/ml, the inter-assay coefficient of variation was above 10 %. The assay maintained linearity under dilution. Recovery was proportional. Haemolysis.......2. The maximum allowable analytical imprecision was 2.6 % and the maximum analytical inaccuracy was 9.9 %. The number of samples required to determine, the true haptoglobin value in an individual pig when accounting for the day-to-day fluctuation was 5. In conclusion, the haptoglobin assay was found...

Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay †

OpenAIRE

Wright, Sara F.; Morton, Joseph B.; Sworobuk, Janis E.

1987-01-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were no...

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Science.gov (United States)

Beauvais, Anne; Beau, Remi; Candoni, Anna; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Zanetti, Eleonora; Quadrelli, Chiara; Codeluppi, Mauro; Guaraldi, Giovanni; Pagano, Livio; Caira, Morena; Giovane, Cinzia Del; Maccaferri, Monica; Stefani, Alessandro; Morandi, Uliano; Tazzioli, Giovanni; Girardis, Massimo; Delia, Mario; Specchia, Giorgina; Longo, Giuseppe; Marasca, Roberto; Narni, Franco; Merli, Francesco; Imovilli, Annalisa; Apolone, Giovanni; Carvalho, Agostinho; Comoli, Patrizia; Romani, Luigina; Latgè, Jean Paul; Luppi, Mario

2013-01-01

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned

Enzyme-Linked Immunosorbent Assay Specific for (1→6) Branched, (1→3)-β-d-Glucan Detection in Environmental Samples

OpenAIRE

Milton, Donald K.; Alwis, K. Udeni; Fisette, Leslie; Muilenberg, Michael

2001-01-01

(1→3)-β-d-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1→6) branched, (1→3)-β-d-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1→3)-β-d-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall β-d-glucans...

Comparative analysis of minor histocompatibility antigens genotyping methods

Directory of Open Access Journals (Sweden)

A. S. Vdovin

2016-01-01

Full Text Available The wide range of techniques could be employed to find mismatches in minor histocompatibility antigens between transplant recipients and their donors. In the current study we compared three genotyping methods based on polymerase chain reaction (PCR for four minor antigens. Three of the tested methods: allele-specific PCR, restriction fragment length polymorphism and real-time PCR with TaqMan probes demonstrated 100% reliability when compared to Sanger sequencing for all of the studied polymorphisms. High resolution melting analysis was unsuitable for genotyping of one of the tested minor antigens (HA-1 as it has linked synonymous polymorphism. Obtained data could be used to select the strategy for large-scale clinical genotyping.

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

Science.gov (United States)

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).

Enzyme-linked immunosorbent assay characterization of Basal variation and heritability of systemic microfibrillar-associated protein 4

DEFF Research Database (Denmark)

Sækmose, Susanne Gjørup; Schlosser, Anders; Holst, René

2013-01-01

Microfibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation...... in systemic MFAP4 (sMFAP4) has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing...

One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides.

Science.gov (United States)

Rexer, Thomas F T; Schildbach, Anna; Klapproth, Jan; Schierhorn, Angelika; Mahour, Reza; Pietzsch, Markus; Rapp, Erdmann; Reichl, Udo

2018-01-01

Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20 mM MgCl 2 . The maximum reaction rate of GDP-mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl 2 producing 566 nmol GDP-mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc) 2 -Man 1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc) 2 -Man 1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Science.gov (United States)

Schildbach, Anna; Klapproth, Jan; Schierhorn, Angelika; Mahour, Reza; Pietzsch, Markus; Rapp, Erdmann; Reichl, Udo

2017-01-01

Abstract Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)2‐Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)2‐Man1 can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and

Evaluation of an antigen-capture EIA for the diagnosis of hepatitis E virus infection.

Science.gov (United States)

Zhao, C; Geng, Y; Harrison, T J; Huang, W; Song, A; Wang, Y

2015-11-01

An enzyme immunoassay (EIA) has been developed for hepatitis E virus (HEV) antigen (HEV-Ag) detection and marketed in China. This study aimed to evaluate the sensitivity of the assay and assess the value of HEV-Ag detection in the diagnosis of HEV infection in comparison with HEV RNA detection. Using serial dilutions of a genotype 4 HEV strain, significant correlation was found between the EIA (S/CO) and HEV RNA (IU/mL) concentration in the range 10(3.5) to 10(0.5) IU/mL HEV RNA, the Pearson correlation coefficient r approached 0.97. The EIA detection limit was 54.6 IU/mL, compared to 24 IU/mL for HEV RNA using real-time RT-PCR. In clinical samples from hepatitis E patients, the HEV-Ag and HEV RNA positivity rates were 55.6% (65/117) and 60.7% (71/117) in sera and 76.7% (56/73) and 84.9% (62/73) in stools, and the concordance of these two markers was 77.8% in sera and 80.8% in stools. In serum samples, the HEV-Ag positivity rate and the concordance between HEV-Ag and HEV RNA were inversely proportional to the presence of anti-HEV antibody. The presence of anti-HEV IgG could reduce the S/CO of the HEV-Ag EIA. These results reveal a significant correlation between the detection of HEV-Ag and HEV RNA. The sensitivity of the HEV-Ag EIA was lower than real-time RT-PCR but could be higher than conventional nested RT-PCR. Therefore, the detection of HEV-Ag in serum and faeces is valuable for the diagnosis and prognosis of HEV infection in developing regions where real-time RT-PCR is not available. © 2015 John Wiley & Sons Ltd.

Assessment of the repeatability and border-plate effects of the B158/B60 enzyme-linked-immunosorbent assay for the detection of circulating antigens (Ag-ELISA) of Taenia saginata.

Science.gov (United States)

Jansen, Famke; Dorny, Pierre; Berkvens, Dirk; Van Hul, Anke; Van den Broeck, Nick; Makay, Caroline; Praet, Nicolas; Gabriël, Sarah

2016-08-30

The monoclonal antibody-based circulating antigen detecting ELISA (B158/B60 Ag-ELISA) has been used elaborately in several studies for the diagnosis of human, bovine and porcine cysticercosis. Interpretation of test results requires a good knowledge of the test characteristics, including the repeatability and the effect of the borders of the ELISA plates. Repeatability was tested for 4 antigen-negative and 5 antigen-positive reference bovine serum samples by calculating the Percentage Coefficient of Variation (%CV) within and between plates, within and between runs, overall, for two batches of monoclonal antibodies and by 2 laboratory technicians. All CV values obtained were below 20% (except one: 24.45%), which indicates a good repeatability and a negligible technician error. The value of 24.45% for indicating the variability between batches of monoclonal antibodies for one positive sample is still acceptable for repeatability measures. Border effects were determined by calculating the %CV values between the inner and outer wells of one plate for 2 positive serum samples. Variability is a little more present in the outer wells but this effect is very small and no significant border effect was found. Copyright © 2016 Elsevier B.V. All rights reserved.

Diagnostic accuracy of an in-house ELISA using the intermediate species Leptospira fainei as antigen for diagnosis of acute leptospirosis in dogs.

Science.gov (United States)

Penna, Bruno; Marassi, Carla D; Libonati, Hugo; Narduche, Lorena; Lilenbaum, Walter; Bourhy, Pascale

2017-02-01

Diagnosis of animal leptospirosis is still challenging. The microscopic agglutination test, is the current method for diagnosing leptospirosis. However, this technique requires specific equipment, highly trained staff and the maintenance of live cultures of several reference strains of Leptospira for use as antigens. Recently, an ELISA (enzyme-linked immunosorbent assay) employing a Leptospira fainei serovar Hurstbridge based antigen for the early diagnostic of human leptospirosis was developed. In this study we estimate the diagnostic sensitivity and specificity of this test in identifying acute canine leptospirosis. A total of 271 serum samples divided into five panels and tested by MAT as a reference test, were used to evaluate the ELISA. Comparing acutely and non-acutely infected dogs, ELISA-Hb showed 95.6% sensitivity and 93% specificity. L. fainei-based ELISA is adequate for diagnosing acute canine leptospirosis, with high sensitivity and specificity and presenting practical advantages when compared to current techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Martin, Laurent; Chaabo, Ayman; Lasne, Françoise

2015-06-01

As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1-24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented. Copyright © 2014 John Wiley & Sons, Ltd.

Development of indirect sandwich ELISA for determination of excretory-secretory antigens of Fasciola hepatica

Directory of Open Access Journals (Sweden)

Libertad Alzamora-Gonzales

2016-05-01

Full Text Available Fasciolosis is a cosmopolitan parasitosis medical-veterinary importance caused by Fasciola hepatica, which affects sheep, goats and cattle; and it affects man accidentally causing an epidemic-endemic infection difficult to diagnose. The aim was to develop an indirect sandwich ELISA with 3 antibodies for detecting excretory-secretory antigens of Fasciola hepatica (ESFh. For the development of indirect sandwich ELISA were used, as capture antibody, mouse polyclonal antibodies anti ESFh and polyclonal antibodies rabbit anti-ESFh as detection antibody, at the concentrations of 10 and 5 µg/mL respectively. The conjugate used was mouse monoclonal anti- total immunoglobulins rabbit linked to peroxidase (1/1000. Were analized 31 sheep fecal samples, and the results were compared with those obtained by direct coproparasitological examination (DC and counterimmunoelectrophoresis (CIEP. The detection limit obtained for indirect sandwich ELISA was 100 ng/mL. The test had a 100% sensitivity, 96.6% specificity, positive and negative predictive values of 50% and 96.6% respectively, in relation to DC test. Comparing with CIEP the specificity obtained for indirect sandwich ELISA was 93.5% and a negative predictive value of 100%. We concluded that indirect sandwich ELISA designed is able to detect metabolic antigens in ovine feces samples and can be used for Fasciola hepatica diagnosis.

Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

International Nuclear Information System (INIS)

Patters, M.R.; Landsberg, R.L.; Johansson, L.-A.; Trummel, C.L.; Robertson, P.R.

1982-01-01

Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated 45 Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis. (author)

Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

Energy Technology Data Exchange (ETDEWEB)

Patters, M R; Landsberg, R L; Johansson, L A; Trummel, C L; Robertson, P R [Department of Periodontology, University of Connecticut, School of Dental Medicine, Farmington, Connecticut, U.S.A.

1982-01-01

Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated /sup 45/Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis.

Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium

International Nuclear Information System (INIS)

McGrath, B.C.; Osborn, M.J.

1991-01-01

Previous immunoelectron microscopic studies have shown that both the final intermediate in O-antigen synthesis, undecaprenol-linked O polymer, and newly synthesized O-antigenic lipopolysaccharide are localized to the periplasmic face of the inner membrane. In vivo pulse-chase experiments now provide further evidence that attachment of O antigen to core lipopolysaccharide, as well as polymerization of O-specific polysaccharide chains, takes place at the periplasmic face of the membrane. Mutants doubly conditional in lipopolysaccharide synthesis [kdsA(Ts) pmi] were constructed in which synthesis of core lipopolysaccharide and O antigen are temperature sensitive and mannose dependent, respectively. Periplasmic orientation of O antigen:core lipopolysaccharide ligase was established by experiments showing rapid chase of undecaprenol-linked O polymer, previously accumulated at 42 degrees C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30 degrees C. In addition, chase of the monomeric O-specific tetrasaccharide unit into lipopolysaccharide was found in similar experiments in an O-polymerase-negative [rfc kdsA(Ts) pmi] mutant, suggesting that polymerization of O chains also occurs at the external face of the inner membrane

Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

Science.gov (United States)

Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

2017-02-01

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization and enzyme-conjugation of a specific anti-L1 nanobody.

Science.gov (United States)

Minaeian, Sara; Rahbarizadeh, Fatemeh; Zarkesh Esfahani, Sayyed Hamid; Ahmadvand, Davoud

2012-01-01

Persistent infection of the human papillomaviruses (HPV) has been shown to result in cervical cancer and intraepithelial neoplasia. Early detection and screening programs are essential strategies against cervical cancer. A nanobody is the smallest antigen-binding fragment known and is derived from a camelid heavy-chain antibody. This tiny protein shows high solubility and stability. It can be produced cost-effectively with high yield production. In this study, we enriched a nanobody library against the L1 protein of HPV. Several colons were selected from this enriched library using monoclonal phage-enzyme linked immunosorbent assay (phage-ELISA) and analyzed for identification of nanobody genes. The expression of nanobody fragments was performed in Rosetta gami2. The C74 nanobody that showed strong binding to the L1 protein of HPV16 was selected, purified, and characterized by Western blotting and ELISA. The selected nanobody was tested for sensitivity, specificity, and affinity. A nanobody conjugated to horseradish peroxidase (HRP) was selected and used for detection of L1 protein of HPV16. This study demonstrates that the C74-HRP, due to its specificity and good binding affinity for a specific viral antigen, is a potential diagnostic tool that can be used as a promising reagent for the new generation of HPV diagnosis approaches.

Evaluation of efficacy of some serological tests used for diagnosis of ...

African Journals Online (AJOL)

These samples were subjected to the different serological tests including Rose Bengal plate antigen test, Tube Agglutination test, Rivanol test, Indirect Enzyme Linked Immunosorbent Assay and Competitive Enzyme Linked Immunosorbent Assay. Statistical analysis of the obtained results in different cattle groups was ...

Multiplexed evaluation of capture agent binding kinetics using arrays of silicon photonic microring resonators.

Science.gov (United States)

Byeon, Ji-Yeon; Bailey, Ryan C

2011-09-07

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

MHC-based detection of antigen-specific CD8(+) T cell responses

DEFF Research Database (Denmark)

Hadrup, Sine Reker; Schumacher, Nana Maria Pii

2010-01-01

The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different...

Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis

Science.gov (United States)

Gurung, Ratna B.; Purdie, Auriol C.; Whittington, Richard J.; Begg, Douglas J.

2014-01-01

Control of Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP) in ruminants using commercially available vaccine reduces production losses, mortality, fecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johne's disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE™ (ISA 50V2, 61VG, 71VG, and 201VG) adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ) release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE™ ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate. PMID:25077074

Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.

Directory of Open Access Journals (Sweden)

Ge Nie

Full Text Available BACKGROUND: Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. METHODOLOGY/PRINCIPAL FINDINGS: We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA based on IgY (egg yolk immunoglobulin against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA. The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15 in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000-9999, 86.7% (13 of 15 in cases of moderate infection (EPG 1000-4999 and 75.0% (9 of 12 in cases of light infection (EPG <1000 of clonorchiasis. Together 36 of total 42 (85.7% serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10 or cysticercosis (n = 10. Cross-reactivity was 6.7% (2 of 30 with schistosomiasis japonica and 10.0% (3 of 30 with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons. CONCLUSIONS: Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating

Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.

Science.gov (United States)

Nie, Ge; Wang, Ting; Lu, Shengjun; Liu, Wenqi; Li, Yonglong; Lei, Jiahui

2014-01-01

Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000-9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000-4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons. Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating antigen in human clonorchiasis.

High sensitivity, high surface area Enzyme-linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Singh, Harpal; Morita, Takahiro; Suzuki, Yuma; Shimojima, Masayuki; Le Van, An; Sugamata, Masami; Yang, Ming

2015-01-01

Enzyme-linked immunosorbent assays (ELISA) are considered the gold standard in the demonstration of various immunological reactions with an application in the detection of infectious diseases such as during outbreaks or in patient care. This study aimed to produce an ELISA-based diagnostic with an increased sensitivity of detection compared to the standard 96-well method in the immunologic diagnosis of infectious diseases. A '3DStack' was developed using readily available, low cost fabrication technologies namely nanoimprinting and press stamping with an increased surface area of 4 to 6 times more compared to 96-well plates. This was achieved by stacking multiple nanoimprinted polymer sheets. The flow of analytes between the sheets was enhanced by rotating the 3DStack and confirmed by Finite-Element (FE) simulation. An Immunoglobulin G (IgG) ELISA for the detection of antibodies in human serum raised against Rubella virus was performed for validation. An improved sensitivity of up to 1.9 folds higher was observed using the 3DStack compared to the standard method. The increased surface area of the 3DStack developed using nanoimprinting and press stamping technologies, and the flow pattern between sheets generated by rotating the 3DStack were potential contributors to a more sensitive ELISA-based diagnostic device.

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

Science.gov (United States)

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of an enzyme linked immunosorbent assay (ELISA) and a radioallergosorbent test (RAST) for detection of IgE antibodies to Brugia malayi

NARCIS (Netherlands)

Wahyuni, Sitti; van Ree, Ronald; Mangali, Andarias; Supali, Taniawati; Yazdanbakhsh, Maria; Sartono, Erliyani

2003-01-01

The enzyme linked immunosorbent assay (ELISA) for specific IgE antibodies to Brugia malayi was compared with the radioallergosorbent test (RAST) for use in immunoepidemiological studies of lymphatic filariasis. Sera used were from individuals (aged 5-82 years) living in an area endemic for lymphatic

Evaluation of a commercial competitive enzyme-linked immunosorbent assay for detection of avian influenza virus subtype H5 antibodies in zoo birds

DEFF Research Database (Denmark)

Jensen, Trine Hammer; Andersen, Jannie Holmegaard; Hjulsager, Charlotte Kristiane

2017-01-01

The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study com...

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes

DEFF Research Database (Denmark)

Gonzalez, Santiago F.; Lukacs-Kornek, Veronika; Kuligowski, Michael P.

2010-01-01

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including...

Enzymatic digestibility of peptides cross-linked by ionizing radiation

International Nuclear Information System (INIS)

Dizdaroglu, M.; Gajewski, E.; Simic, M.G.

1984-01-01

Digestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to γ-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail. (author)

The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis.

Science.gov (United States)

Wunsch, Marie; Hohmann, Christopher; Milles, Bianca; Rostermund, Christina; Lehmann, Paul V; Schroeter, Michael; Bayas, Antonios; Ulzheimer, Jochen; Mäurer, Mathias; Ergün, Süleyman; Kuerten, Stefanie

2016-04-23

There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.

The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis

Directory of Open Access Journals (Sweden)

Marie Wunsch

2016-04-01

Full Text Available There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV infection and brain inflammation in multiple sclerosis (MS. Here, we tested MS patients during relapse (n = 11 and in remission (n = 19 in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT and enzyme-linked immunosorbent assay (ELISA. Cytomegalovirus (CMV was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection

Science.gov (United States)

Cross, Robert W.; Boisen, Matthew L.; Millett, Molly M.; Nelson, Diana S.; Oottamasathien, Darin; Hartnett, Jessica N.; Jones, Abigal B.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shunichiro; Brown, Bethany L.; Pham, Ha; Rowland, Megan M.; Agans, Krystle N.; Geisbert, Joan B.; Heinrich, Megan L.; Kulakosky, Peter C.; Shaffer, Jeffrey G.; Schieffelin, John S.; Kargbo, Brima; Gbetuwa, Momoh; Gevao, Sahr M.; Wilson, Russell B.; Saphire, Erica Ollmann; Pitts, Kelly R.; Khan, Sheik Humarr; Grant, Donald S.; Geisbert, Thomas W.; Branco, Luis M.; Garry, Robert F.

2016-01-01

Background. Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013–2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. Methods. Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. Results. The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription–polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105–9.0 × 108 genomes/mL. Conclusions. The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015. PMID:27587634

Electrochemical immunoassay for the prostate specific antigen using a reduced graphene oxide functionalized with a high molecular-weight silk peptide

International Nuclear Information System (INIS)

Wang, Yanying; Qu, Ying; Li, Chunya; Wu, Kangbing; Liu, Guishen; Hou, Xiaodong; Huang, Yina; Wu, Wangze

2015-01-01

High molecular-weight silk peptide (SP) was used to functionalize the surface of nanosheets of reduced graphene oxide (rGO). The SP-rGO nanocomposite was then mixed with mouse anti-human prostate specific antigen monoclonal antibody (anti-PSA) and coated onto a glassy carbon electrode to fabricate an immunosensor. By using the hexacyanoferrate redox system as electroactive probe, the immunosensor was characterized by voltammetry and electrochemical impedance spectroscopy. The peak current, measured at the potential of 0.24 V (vs. SCE), is distinctly reduced after binding prostate specific antigen (PSA). Response (measured by differential pulse voltammetry) is linearly related to PSA concentration in the range from 0.1 to 5.0 ng · mL −1 and from 5.0 to 80.0 ng∙mL −1 , and the detection limit is 53 pg∙mL −1 (at an SNR of 3). The immunosensor was successfully applied to the determination of PSA in clinical serum samples, and the results were found to agree well with those obtained with an enzyme-linked immunosorbent assay. (author)

Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

DEFF Research Database (Denmark)

Chow, E.Y.W.; Wu, J.T.Y.; Jauho, E.S.

2004-01-01

In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested.......9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between...

Development and validation of an indirect Enzyme-linked Immunosorbent Assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants

NARCIS (Netherlands)

Heijden, van der H.M.J.F.; Bouwstra, R.J.; Mars, M.H.; Poel, van der W.H.M.; Wellenberg, G.J.; Maanen, van C.

2013-01-01

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood

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

Science.gov (United States)

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.

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.

Science.gov (United States)

Thålin, Charlotte; Daleskog, Maud; Göransson, Sophie Paues; Schatzberg, Daphne; Lasselin, Julie; Laska, Ann-Charlotte; Kallner, Anders; Helleday, Thomas; Wallén, Håkan; Demers, Mélanie

2017-06-01

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

Ether lipid vesicle-based antigens impart protection against experimental listeriosis

Directory of Open Access Journals (Sweden)

Ansari MA

2012-06-01

Full Text Available Mairaj Ahmed Ansari,1 Swaleha Zubair,2 Saba Tufail,1 Ejaj Ahmad,1 Mohsin Raza Khan,1 Zainuddin Quadri,1 Mohammad Owais,11Interdisciplinary Biotechnology Unit, 2Women's College, Aligarh Muslim University, Aligarh, UP, IndiaBackground: Incidence of food-borne infections from Listeria monocytogenes, a parasite that has adapted intracellular residence to avoid antibody onslaught, has increased dramatically in the past few years. The apparent lack of an effective vaccine that is capable of evoking the desired cytotoxic T cell response to obliterate this intracellular pathogen has encouraged the investigation of alternate prophylactic strategies. It should also be noted that Archaebacteria (Archae lipid-based adjuvants enhance the efficacy of subunit vaccines. In the present study, the adjuvant properties of archaeosomes (liposomes prepared from total polar lipids of archaebacteria, Halobacterium salinarum combined with immunogenic culture supernatant antigens of L. monocytogenes have been exploited in designing a vaccine candidate against experimental listeriosis in murine model.Methods: Archaeosome-entrapped secretory protein antigens (SAgs of L. monocytogenes were evaluated for their immunological responses and tendency to deplete bacterial burden in BALB/c mice challenged with sublethal listerial infection. Various immunological studies involving cytokine profiling, lymphocyte proliferation assay, detection of various surface markers (by flowcytometric analysis, and antibody isotypes (by enzyme-linked immunosorbent assay were used for establishing the vaccine potential of archaeosome-entrapped secretory proteins.Results: Immunization schedule involving archaeosome-encapsulated SAgs resulted in upregulation of Th1 cytokine production along with boosted memory in BALB/c mice. It also showed protective effect by reducing listerial burden in various vital organs (liver and spleen of the infected mice. However, the soluble form of the antigens (SAgs

Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

Science.gov (United States)

Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

2016-08-01

Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens.

Science.gov (United States)

Fernandes, Geisa Ferreira; Lopes-Bezerra, Leila Maria; Bernardes-Engemann, Andréa Reis; Schubach, Tânia Maria Pacheco; Dias, Maria Adelaide Galvão; Pereira, Sandro Antonio; de Camargo, Zoilo Pires

2011-01-27

The main objective of this study is to standardize an ELISA for the diagnosis of feline sporotrichosis. Sporothrix schenckii is the etiological agent of human and animal sporotrichosis. Cats may act as reservoirs for S. schenckii and can transmit the infection to humans by a bite or scratch. There are few methods for the serological diagnosis of fungal diseases in animals. In this paper, an ELISA test for the diagnosis of cat sporotrichosis is proposed, which detects S. schenckii-specific antibodies in feline sera. Two different kinds of antigens were used: "SsCBF", a specific molecule from S. schenckii that consists of a Con A-binding fraction derived from a peptido-rhamnomannan component of the cell wall, and a S. schenckii crude exoantigen preparation. The ELISA was developed, optimized, and evaluated using sera from 30 cats with proven sporotrichosis (by culture isolation); 22 sera from healthy feral cats from a zoonosis center were used as negative controls. SsCBF showed 90% sensitivity and 96% specificity in ELISA; while crude exoantigens demonstrated 96% sensitivity and 98% specificity. The ELISA assay described here would be a valuable screening tool for the detection of specific S. schenckii antibodies in cats with sporotrichosis. The assay is inexpensive, quick to perform, easy to interpret, and permits the diagnosis of feline sporotrichosis. Copyright © 2010 Elsevier B.V. All rights reserved.

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

Science.gov (United States)

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-03-01

Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

Advanced Low Energy Enzyme Catalyzed Solvent for CO₂ Capture

Energy Technology Data Exchange (ETDEWEB)

Zaks, Alex [Akermin Inc., St. Louis, MO (United States); Reardon, John [Akermin Inc., St. Louis, MO (United States)

2013-09-30

A proof-of-concept biocatalyst enhanced solvent process was developed and demonstrated in an integrated bench-scale system using coal post combustion flue gas. The biocatalyst was deployed as a coating on M500X structured packing. Rate enhancement was evaluated using a non-volatile and non-toxic 20 wt% potassium carbonate solution. Greater than 500-fold volumetric scale-up from laboratory to bench scale was demonstrated in this project. Key technical achievements included: 10-fold mass transfer enhancement demonstrated in laboratory testing relative to blank potassium carbonate at 45°C; ~ 7-fold enhancement over blank in bench-scale field testing at National Carbon Capture Center; aerosol emissions were below detection limits (< 0.8 ppm); 90% capture was demonstrated at ~19.5 Nm³/hr (dry basis); and ~ 80% CO₂ capture was demonstrated at ~ 30 Nm³/hr (dry basis) for more than 2800-hrs on flue gas with minimal detectible decline in activity. The regeneration energy requirement was 3.5 GJ/t CO₂ for this solvent, which was below the target of <2.1 GJ/t CO₂. Bench unit testing revealed kinetic limitations in the un-catalyzed stripper at around 85°C, but process modeling based on bench unit data showed that equivalent work of less than 300 kWh/t CO₂ including all CO₂ compression can be achieved at lower temperature stripping conditions. Cost analysis showed that 20% potassium carbonate in a basic solvent flow sheet with biocatalyst coated packing has economic performance comparable to the reference NETL Case-12, 30% MEA. A detailed techno-economic analysis indicated that addition of catalyst in the stripper could reduce the cost of capture by ~6% and cost of avoided CO₂ by ~10% below reference NETL Case-12. Based on these results, a directional plan was identified to reduce the cost of CO₂ capture in future work.

Mimicry of the immunodominant conformation-dependent antigenic site of hepatitis A virus by motifs selected from synthetic peptide libraries.

Science.gov (United States)

Mattioli, S; Imberti, L; Stellini, R; Primi, D

1995-09-01

Hepatitis A virus (HAV) is a positive-strand RNA virus with a genome length of approximately 7,480 nucleotides. Although HAV morphogenesis is thought to be similar to that of poliovirus, the prototype picornavirus, the complete characterization of the antigenic structure of this virus remains elusive. All the available evidences, however, support the existence, on HAV virions and empty capsids, of an immunodominant neutralization antigenic site which is conformation dependent and whose structure involves residues of both VP1 and VP3 capsid proteins. This particular feature and the difficulty of obtaining high virus yield in tissue cultures make HAV an ideal target for developing synthetic peptides that simulate the structure of its main antigenic determinant. To this end we utilized, in the present work, the divide-couple-recombine approach to generate a random library composed of millions of different hexapeptides. This vast library was screened with a well-characterized anti-HAV monoclonal antibody. By this strategy we identified a peptide that reacted specifically with monoclonal and polyclonal anti-HAV antibodies and, in mice, induced a specific anti-virus immune response. Furthermore, the peptide could also be used in an enzyme-linked immunosorbent assay for revealing a primary immunoglobulin M immune response in sera of acutely infected human patients. Interestingly, no sequence homology was found between the identified peptide and the HAV capsid proteins VP1 and VP3. Collectively, these data represent an additional important paradigm of a mimotope capable of mimicking an antigenic determinant with unknown tertiary structure.

Environmental quantification of Pasteuria penetrans endospores using in situ antigen extraction and immunodetection with a monoclonal antibody.

Science.gov (United States)

Schmidt, L M; Preston, J F; Dickson, D W; Rice, J D; Hewlett, T E

2003-05-01

Abstract Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.) that has attracted significant attention as a promising biocontrol agent. The inability to culture P. penetrans has invoked the need for a quantitative detection capability to facilitate biocontrol studies. A chemical extraction method using urea, dithiothreitol and CHES buffer (UDC) is shown to release soluble endospore envelope antigen from endospores present in complex matrices, generating an extract that can be used to determine the levels of spores when compared to a standard in an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody, MAb 2A41D10. Extractions can be performed in less than 1 h. Linear regression analysis routinely produced line fits with r(2)>0.90. Antigen extraction efficiency was not influenced by soil type. Three ELISA formats were analyzed for quantitative detection of P. penetrans endospores. A tertiary ELISA immunodetection system provided the lowest level of detection at approximately 300 spores per gram of soil. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots of soil extracts containing P. penetrans endospore antigen produced signature peptides bearing a common epitope characteristic of endospores of Pasteuria spp. MAb 2A41D10 was specific for Pasteuria spp. and did not react with extracts of Pasteuria-free soil or with spore extracts of native Gram-positive endospore-forming bacteria. Immunofluorescent microscopy revealed that MAb 2A41D10 recognizes an epitope uniformly distributed on the endospore surface. The development of a rapid extraction method and analysis of solubilized antigen by immunodetection has the potential for broad application in food and environmental microbiology.

Onderzoek naar toepasbaarheid van Enzyme-Linked Immunosorbent Assay (ELISA) voor het bepalen van aflatoxinen in voedings- en voedermiddelen 1e interimrapport: Orientatie en taakstelling

NARCIS (Netherlands)

Egmond; H.P. van; Paulsch; W.E.; Sizoo; E.A.

1987-01-01

Immunochemische methoden van onderzoek voor het bepalen van mycotoxinen komen langzamerhand in de belangstelling te staan, vooral Enzyme-Linked Immunosorbent Assay (ELISA). Om een beeld te krijgen van de praktische en wetenschappelijke karakteristieken van ELISA's in het

Quantification of methanogenic biomass by enzyme-linked immunosorbent assay and by analysis of specific methanogenic cofactors

Energy Technology Data Exchange (ETDEWEB)

Gorris, L G.M.; Kemp, H A; Archer, D B

1987-01-01

The reliability and accuracy with which enzyme-linked immunosorbent assay (ELISA) and an assay of methanogenic cofactors detect and quantify methanogenic species were investigated. Both assays required standardization with laboratory cultures of methanogenic bacteria and were applied to mixtures of pure cultures and samples from anaerobic digesters. ELISA was shown to be a simple method for detecting and quantifying individual methanogenic species. The range of species which can be assayed is limited by the range of antisera available but, potentially, ELISA can be applied to all methanogens. Although the cofactor assay is not species-specific it can distinguish hydrogenotrophic and acetotrophic methanogens and is quantitative.

Enzyme-linked immunosorbent assays for Z-DNA.

Science.gov (United States)

Thomas, M J; Strobl, J S

1988-10-01

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e.l.i.s.a. was conducted in 48-well culture dishes at 37 degrees C using a rabbit polyclonal antiserum developed against Br-poly(dG-dC).poly(dG-dC), an alkaline phosphatase-conjugated second antibody, and p-nitrophenol as the substrate. Under conditions where antibody concentrations were not limiting, alkaline phosphatase activity was linear for 2 h. Dot blot e.l.i.s.a. conditions are described which allow quantification of Z-DNA [Br-poly(dG-dC).poly(dG-dC)] within the range 5-250 ng. Dot blot and transblot horseradish peroxidase e.l.i.s.a. are described that detect Z-DNA within supercoiled plasmid DNAs immobilized on diazophenylthioether (DPT) paper. In the transblot e.l.i.s.a., plasmid pUC8 derivatives containing 16, 24, or 32 residues of Z-DNA were electrophoresed in agarose gels and electrophoretically transferred to DPT paper. Z-DNA-antibody complexes were detected by the horseradish peroxidase-catalysed conversion of 4-chloro-1-naphthol to a coloured product that was covalently bound to the DPT paper. Z-DNA antibody reactivity was specific for supercoiled Z-DNA containing plasmids after removal of the antibodies cross-reactive with B-DNA by absorption onto native DNA-cellulose. The transblot e.l.i.s.a. was sensitive enough to detect 16 base pairs of alternating G-C residues in 100 ng of pUC8 DNA.

[Alanine solution as enzyme reaction buffer used in A to O blood group conversion].

Science.gov (United States)

Li, Su-Bo; Zhang, Xue; Zhang, Yin-Ze; Tan, Ying-Xia; Bao, Guo-Qiang; Wang, Ying-Li; Ji, Shou-Ping; Gong, Feng; Gao, Hong-Wei

2014-06-01

The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

International Nuclear Information System (INIS)

Abbas, A.K.; Haber, S.; Rock, K.L.

1985-01-01

Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

Immune activation by casein dietary antigens in bipolar disorder

NARCIS (Netherlands)

Severance, E.G.; Dupont, D.; Dickerson, F.B.; Stallings, C.R.; Origoni, A.E.; Krivogorsky, B.; Yang, S.; Haasnoot, W.; Yolken, R.H.

2010-01-01

Objectives: Inflammation and other immune processes are increasingly linked to psychiatric diseases. Antigenic triggers specific to bipolar disorder are not yet defined. We tested whether antibodies to bovine milk caseins were associated with bipolar disorder, and whether patients recognized

Dendritic cells induce specific cytotoxic T lymphocytes against prostate cancer TRAMP-C2 cells loaded with freeze- thaw antigen and PEP-3 peptide.

Science.gov (United States)

Liu, Xiao-Qi; Jiang, Rong; Li, Si-Qi; Wang, Jing; Yi, Fa-Ping

2015-01-01

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-β and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-γ, TNF-β and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

Effects of thermal processing on the enzyme-linked immunosorbent assay (ELISA) detection of milk residues in a model food matrix.

Science.gov (United States)

Downs, Melanie L; Taylor, Steve L

2010-09-22

Food products and ingredients are frequently tested for the presence of undeclared allergenic food residues (including milk) using commercial enzyme-linked immunosorbent assays (ELISAs). However, little is understood about the efficacy of these kits with thermally processed foods. This study evaluated the performance of three milk ELISA kits with a model food processed by several methods. A model food (pastry dough squares) was spiked with nonfat dry milk at several concentrations. The pastry squares were processed by boiling (100 °C for 2 min), baking (190 °C for 30 min), frying (190 °C for 2 min), and retorting (121 °C for 20 min with 17 psi overpressure). Samples were analyzed with three commercial ELISA kits: Neogen Veratox Total Milk, ELISA Systems β-lactoglobulin, and ELISA Systems casein. The detection of milk residues depended upon the type of processing applied to the food and the specific milk analyte targeted by the ELISA kit. Poor recoveries were obtained in all processed samples (2-10% of expected values) using the β-lactoglobulin kit. Better recoveries were obtained in boiled samples (44 and 59%, respectively) using the total milk and casein kits. However, these kits performed poorly with baked (9 and 21%) and fried (7 and 18%) samples. Moderate recoveries were observed in retorted samples (23 and 28%). The decreased detection in processed samples is likely due to protein modifications, including aggregation and Maillard reactions, which affect the solubility and immunoreactivity of the antigens detected by the ELISA methods. The observed decreases in ELISA detection of milk are dramatic enough to affect risk-assessment decisions. However, a lower detection of milk residues does not necessarily indicate decreased allergenicity. These ELISA kits are not acceptable for all applications, and users should understand the strengths and limitations of each method.

THE THEORETICAL AND PRACTICAL ASPECTS OF THE RECOMBINANT ANTIGENS FOR THE DIAGNOSIS OF BOVINE LEUKEMIA PRODUCTION

Directory of Open Access Journals (Sweden)

Shapovalova OV

2016-12-01

Full Text Available Introduction. Nowadays the problem of bovine leukemia (EBL effective diagnosis in countries where EBL is registered and the disease-free areas remains actual. The main diagnostic tests are immunodiffusion reaction (AGID and enzyme-linked immunosorbent assay (ELISA, which allow the identification of infected animals by the presence of antibodies to bovine leukemia virus (BLV both in serum and in milk samples. The effectiveness of these methods depends on the quality of diagnostic test systems used and determines by the cultural and recombinant virus antigens specificity. EBL recombinant antigens have certain advantages as they are more active and cheap. Purpose of the work. The analysis of theoretical and practical approaches in the bovine leukemia virus recombinant antigens development and its diagnostic potential evaluation. The article contains data from the literature on the recombinant antigens of bovine leukemia virus construction and use. Analysis of the literature showed that the recombinant proteins are widely used in the serological diagnosis of bovine leukemia. Numerous protocols of BLV gp51 and p24 immunodominant antigens preparation has been developed in heterologous systems (Saccharomyces cerevisiae, E. coli, vaccinia virus, baculovirus. In order to obtain recombinant antigens, the BLV provirus genome regions isolated from FLK-BLV cell culture, lymphocytes or tumor cells from naturally infected cattle are typically used. For the recombinant antigens labeled by hexahistidine or Srept II purification one-step immobilized-metal affinity chromatography IMAC and highly selective Strep-Tactin affinity chromatography methods are carried out. The end products activity and specificity are studied in the immunoblotting, ELISA and AGID diagnostic reactions. The ukrainian scientists’ publications are devoted to the clone E. coli HB101-2 transformed by the recombinant plasmid containing fully functional BLV env and gag genes nucleotide

Influenza A plasma and serum virus antibody detection comparison in dogs using blocking enzyme-linked immunosorbent assay

Directory of Open Access Journals (Sweden)

H. T. Lin

2015-05-01

Full Text Available Background and Aim: The influenza A virus (IAV is an important zoonotic pathogen with infections also reported in dogs. IAV infections can be detected through the presence of antibodies using the enzyme-linked immunosorbent assay (ELISA. Serum is the only standard sample source; however, there is no information on the availability of other sample sources for IAV antibody detection in dogs. Compared with serum, plasma is more widely employed in most animal hospitals. The object of this study is to investigate whether plasma collected in ethylenediaminetetraacetic acid (EDTA tubes (EDTA plasma or heparin tubes (heparin plasma could be used in the ELISA protocol instead of serum for IAV antibody detection in dogs. Materials and Methods: Totally, 82 matched EDTA plasma and serum sample pairs and 79 matched heparin plasma and serum sample pairs were employed using blocking enzyme-linked immunosorbent assay (bELISA. The agreement and correlation between the plasma (EDTA or heparin plasma and serum were assessed using the agreement index kappa (kD calculation and Pearson correlation coefficient, respectively. Results: The agreement index kD of EDTA plasma and serum was 1.0, and that of heparin plasma and serum was 0.85. The Pearson correlation coefficient of EDTA plasma and serum was 0.87 (p<0.01, and that of heparin plasma and serum was 0.82 (p<0.01. Conclusion: The results proved that plasma, especially EDTA plasma, could be substituted for serum in the bELISA test. This might greatly expand the clinical applicability of IAV antibody detection in dogs.

Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles.

Science.gov (United States)

Wilson, John T; Postma, Almar; Keller, Salka; Convertine, Anthony J; Moad, Graeme; Rizzardo, Ezio; Meagher, Laurence; Chiefari, John; Stayton, Patrick S

2015-03-01

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell

Evaluation of the antibody-detection ELISA using plates precoated with denatured T. congolense and T. vivax antigens for monitoring tsetse and trypanosomosis control in Zambia

International Nuclear Information System (INIS)

Machila, N.; Sinyangwe, L.; Eisler, M.C.

2000-01-01

An evaluation of the indirect enzyme-linked immunosorbent assay (ELISA) for the detection of anti-trypanosomal antibodies in bovine serum was conducted using ELISA plates pre-coated with denatured T. congolense and T. vivax antigen. The study was conducted on 280 samples from a parasitologically positive cattle population and 200 samples from a negative cattle population. The overwhelming majority of trypanosome infections in the parasitologically positive cattle were T. congolense. The Ab-ELISA was able to discriminate between the negative and positive cattle populations, at an optimum cut-off point of 40% positivity (T. congolense antigen), with 89% sensitivity and 86% specificity; and for the T. vivax antigen, the optimum cut-off point was at 30%, with 81% sensitivity and 70% specificity. However, the optical densities (OD) and percentage positivity (PP) values for sera from both the reference positive and negative cattle populations were unacceptably high particularly in the ELISA using T. congolense antigen. Furthermore the quality control sera used in the assay appear to have inappropriately low OD and PP values by comparison to typical sera from the reference positive and negative cattle populations. It is suggested that these ELISA's be re-titrated using more appropriate quality assurance sera. This should result in OD and PP values for sera from the reference populations failing within acceptable ranges. (author)

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

Science.gov (United States)

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Reactive oxygen species modulator 1, a novel protein, combined with carcinoembryonic antigen in differentiating malignant from benign pleural effusion.

Science.gov (United States)

Chen, Xianmeng; Zhang, Na; Dong, Jiahui; Sun, Gengyun

2017-05-01

The differential diagnosis of malignant pleural effusion and benign pleural effusion remains a clinical problem. Reactive oxygen species modulator 1 is a novel protein overexpressed in various human tumors. The objective of this study was to evaluate the diagnostic value of joint detection of reactive oxygen species modulator 1 and carcinoembryonic antigen in the differential diagnosis of malignant pleural effusion and benign pleural effusion. One hundred two consecutive patients with pleural effusion (including 52 malignant pleural effusion and 50 benign pleural effusion) were registered in this study. Levels of reactive oxygen species modulator 1 and carcinoembryonic antigen were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Results showed that the concentrations of reactive oxygen species modulator 1 both in pleural fluid and serum of patients with malignant pleural effusion were significantly higher than those of benign pleural effusion (both p pleural fluid reactive oxygen species modulator 1 were 61.54% and 82.00%, respectively, with the optimized cutoff value of 589.70 pg/mL. However, the diagnostic sensitivity and specificity of serum reactive oxygen species modulator 1 were only 41.38% and 86.21%, respectively, with the cutoff value of 27.22 ng/mL, indicating that serum reactive oxygen species modulator 1 may not be a good option in the differential diagnosis of malignant pleural effusion and benign pleural effusion. The sensitivity and specificity of pleural fluid carcinoembryonic antigen were 69.23% and 88.00%, respectively, at the cutoff value of 3.05 ng/mL, while serum carcinoembryonic antigen were 80.77% and 72.00% at the cutoff value of 2.60 ng/mL. The sensitivity could be raised to 88.17% in parallel detection of plural fluid reactive oxygen species modulator 1 and carcinoembryonic antigen concentration, and the specificity could be improved to 97.84% in serial detection.

An in-depth analysis of original antigenic sin in dengue virus infection.

Science.gov (United States)

Midgley, Claire M; Bajwa-Joseph, Martha; Vasanawathana, Sirijitt; Limpitikul, Wannee; Wills, Bridget; Flanagan, Aleksandra; Waiyaiya, Emily; Tran, Hai Bac; Cowper, Alison E; Chotiyarnwong, Pojchong; Chotiyarnwon, Pojchong; Grimes, Jonathan M; Yoksan, Sutee; Malasit, Prida; Simmons, Cameron P; Mongkolsapaya, Juthathip; Screaton, Gavin R

2011-01-01

The evolution of dengue viruses has resulted in four antigenically similar yet distinct serotypes. Infection with one serotype likely elicits lifelong immunity to that serotype, but generally not against the other three. Secondary or sequential infections are common, as multiple viral serotypes frequently cocirculate. Dengue infection, although frequently mild, can lead to dengue hemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary dengue infections, implying a role for the adaptive immune response in the disease. There is currently much effort toward the design and implementation of a dengue vaccine but these efforts are made more difficult by the challenge of inducing durable neutralizing immunity to all four viruses. Domain 3 of the dengue virus envelope protein (ED3) has been suggested as one such candidate because it contains neutralizing epitopes and it was originally thought that relatively few cross-reactive antibodies are directed to this domain. In this study, we performed a detailed analysis of the anti-ED3 response in a cohort of patients suffering either primary or secondary dengue infections. The results show dramatic evidence of original antigenic sin in secondary infections both in terms of binding and enhancement activity. This has important implications for dengue vaccine design because heterologous boosting is likely to maintain the immunological footprint of the first vaccination. On the basis of these findings, we propose a simple in vitro enzyme-linked immunosorbent assay (ELISA) to diagnose the original dengue infection in secondary dengue cases.

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

DEFF Research Database (Denmark)

Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders

2015-01-01

Background: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradica...

Low sensitivity of type VII collagen enzyme-linked immunosorbent assay in epidermolysis bullosa acquisita : serration pattern analysis on skin biopsy is required for diagnosis

NARCIS (Netherlands)

Terra, J. B.; Jonkman, M. F.; Diercks, G. F. H.; Pas, H. H.

BackgroundThe type VII collagen (coll VII) enzyme-linked immunosorbent assay (ELISA) has been reported to have high sensitivity (>93%) and specificity (>96%) for diagnosing epidermolysis bullosa acquisita (EBA) in patients who are seropositive on indirect immunofluorescence on salt-split skin (SSS).

Synthesis of S-linked oligoxylans

DEFF Research Database (Denmark)

Bonora, Beatrice

by several families of enzymes,collected under the name of Glycosyl Hydrolases (GHs). Among other methods,the use of enzyme inhibitors like thio-linked oligosaccharides has for a long timebeen a common tool to analyze and characterize these enzymes.In the present work the chemical synthesis of thio......-analogs of xylo- andarabinoxyloglycans is presented. Furthermore, the selection of a reliable method forthe incorporation of thiolinkages in the synthesis of oligoxylans is alsoinvestigated.Therefore, different strategies for assembling S-linked-disaccharides have beenapproached both involving 1-thioglycoside...... donors and thioacceptors. Advantagesand disadvantages concerning the different methods are described and evaluated inrelation to the synthesis of linear and branched oligoxylans....

Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

2015-01-01

Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL −1 with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO 2 particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors

Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

Energy Technology Data Exchange (ETDEWEB)

Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

2015-08-05

Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

Science.gov (United States)

Cross, Robert W; Boisen, Matthew L; Millett, Molly M; Nelson, Diana S; Oottamasathien, Darin; Hartnett, Jessica N; Jones, Abigal B; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Bornholdt, Zachary A; Fusco, Marnie L; Abelson, Dafna M; Oda, Shunichiro; Brown, Bethany L; Pham, Ha; Rowland, Megan M; Agans, Krystle N; Geisbert, Joan B; Heinrich, Megan L; Kulakosky, Peter C; Shaffer, Jeffrey G; Schieffelin, John S; Kargbo, Brima; Gbetuwa, Momoh; Gevao, Sahr M; Wilson, Russell B; Saphire, Erica Ollmann; Pitts, Kelly R; Khan, Sheik Humarr; Grant, Donald S; Geisbert, Thomas W; Branco, Luis M; Garry, Robert F

2016-10-15

 Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases.  Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance.  The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 10 5 -9.0 × 10 8 genomes/mL.  The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Evaluation of Serum Specific Antibody against Recombinant ESAT-6 Antigen in Patients with Tuberculosis and Comparing to Normal Controls

Directory of Open Access Journals (Sweden)

Homeira Izadi

2017-02-01

Full Text Available Background & Objective: Tuberculosis (TB is a zoonotic disease which is caused by Mycobacterium tuberculosis. Because of common structural and secretory antigens between pathogen and nonpathogenic mycobacterium, the specific diagnosis of TB is difficult. Therefore, it is very important to find a new method with high specificity and sensitivity for accurate and rapid diagnosis of tuberculosis. In this study, the serodiagnostic potential of Mycobacterium tuberculosis recombinant ESAT-6 in TB infected patients was evaluated by Enzyme Linked Immunosorbent Assay (ELISA. Materials & Methods: 55 TB patients with active disease and 28 healthy controls have been collected and evaluated in different dilutions in ELISA methods for the presence of specific anti-ESAT-6 antibody. The specificity and the sensitivity of this method was compared with the culture test. Results: TB patients have high levels of specific antibody against ESAT-6 antigens. The specificity and the sensitivity of this method was calculated as 80.90% and 85.45%, respectively. Conclusion: These findings provide useful information on the importance of ESAT-6 protein and suggested this serologic test as a good alternative method for rapid and prefect diagnosis of tuberculosis.

Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay.

Science.gov (United States)

Lv, Chao; Hong, Yang; Fu, Zhiqiang; Lu, Ke; Cao, Xiaodan; Wang, Tao; Zhu, Chuangang; Li, Hao; Xu, Rui; Jia, Bingguang; Han, Qian; Dou, Xuefeng; Shen, Yuanxi; Zhang, Zuhang; Zai, Jinli; Feng, Jintao; Lin, Jiaojiao

2016-03-09

Schistosomiasis is a huge threat to human and animal health. Apart from bovines, goats play an important role in the transmission of schistosomiasis in some endemic areas of China. An accessible, quality-assured goat schistosomiasis diagnostic technique is needed. Recently, our laboratory identified two recombinant diagnostic antigens, SjPGM and SjRAD23 via an immuno-proteomic method. The application of these two recombinant antigens to develop a higher sensitivity and specificity technique for the sheep schistosomiasis diagnosis is urgently needed. Epitopes of SjPGM and SjRAD23 were predicted and three polypeptides, two from SjRAD23 and one from SjPGM, were selected. Recombinant plasmids containing two to three DNA sequences encoding predicted polypeptides or large hydrophilic region of Sj23 (LHD-Sj23) were constructed and expressed. Eight recombinant schistosome antigens including four multi-epitope proteins and four recombinant single-molecule antigens as well as SEA, were assessed by ELISA in 91 sera from schistosome-infected goats, 44 sera from non-infected goats, 37 sera from Orientobilharzia-infected goats, and 12 from Haemonchus contortus-infected goats. ELISA tests showed that three multi-epitope proteins had higher sensitivity than the four single-molecule antigens (rSjRAD23, rSjPGM, rBSjRAD23-1, rBSj23) and the multi-epitope protein rBSjPGM-BSjRAD23-1-BSj23 had the highest sensitivity (97.8 %, 89/91) and maintained good specificity (100 %, 44/44) as well as low cross-reactivity with haemonchosis (8.33 %, 3/12) and orientobilharziasis (13.51 %, 5/37) in the diagnosis of goat schistosomiasis. In contrast, when SEA was applied as a diagnosis antigen, it had 100 % (91/91) sensitivity, 75 % (33/44) specificity, 25 and 83.78 % cross-reactivity with haemonchosis (3/12) and orientobilharziasis (31/37), respectively. The application of recombinant multi-epitope proteins may increase the sensitivity of diagnosis technique and retain high specificity of single

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

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Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

2018-01-16

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

Science.gov (United States)

Venkatachalam, Mahesh; Teuber, Suzanne S; Peterson, W Rich; Roux, Kenneth H; Sathe, Shridhar K

2006-02-22

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

In vivo collection of rare proteins using kinesin-based "nano-harvesters".

Energy Technology Data Exchange (ETDEWEB)

Bachand, Marlene; Bachand, George David; Greene, Adrienne Celeste; Carroll-Portillo, Amanda

2008-11-01

In this project, we have developed a novel platform for capturing, transport, and separating target analytes using the work harnessed from biomolecular transport systems. Nanoharvesters were constructed by co-organizing kinesin motor proteins and antibodies on a nanocrystal quantum dot (nQD) scaffold. Attachment of kinesin and antibodies to the nQD was achieved through biotin-streptavidin non-covalent bonds. Assembly of the nanoharvesters was characterized using a modified enzyme-linked immunosorbent assay (ELISA) that confirmed attachment of both proteins. Nanoharvesters selective against tumor necrosis factor-{alpha} (TNF-{alpha}) and nuclear transcription factor-{kappa}B (NF-{kappa}B) were capable of detecting target antigens at <100 ng/mL in ELISAs. A motility-based assay was subsequently developed using an antibody-sandwich approach in which the target antigen (TNF-{alpha}) formed a sandwich with the red-emitting nanoharvester and green-emitting detection nQD. In this format, successful sandwich formation resulted in a yellow emission associated with surface-bound microtubules. Step-wise analysis of sandwich formation suggested that the motility function of the kinesin motors was not adversely affected by either antigen capture or the subsequent binding of the detection nQDs. TNF-{alpha} was detected as low as {approx}1.5 ng/mL TNF-{alpha}, with 5.2% of the nanoharvesters successfully capturing the target analyte and detection nQDs. Overall, these results demonstrate the ability to capture target protein analytes in vitro using the kinesin-based nanoharvesters in nanofluidic environments. This system has direct relevance for lab-on-a-chip applications where pressure-driven or electrokinetic movement of fluids is impractical, and offers potential application for in vivo capture of rare proteins within the cytoplasmic domain of live cells.

Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

DEFF Research Database (Denmark)

Rimhanen-Finne, R.; Enemark, Heidi L.; Kolehmainen, J.

2007-01-01

The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5) oocysts/g, while the detection limit for Giardia ELISA was 10(4) cysts/g. The Cryptosporidium ELISA showed 94% specificity...... zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs...

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

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Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

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SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

2013-01-01

ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

Development of a Multianalyte Enzyme-Linked Immunosorbent Assay for Permethrin and Aroclors and Its Implementation for Analysis of Soil/Sediment and House Dust ExtractsExtracts

Science.gov (United States)

Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254, and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors ...

Sensitivity of hepatitis C virus core antigen and antibody combination assays in a global panel of window period samples

Science.gov (United States)

Laperche, Syria; Nubling, C. Micha; Stramer, Susan L.; Brojer, Ewa; Grabarczyk, Piotr; Yoshizawa, Hiroshi; Kalibatas, Vytenis; El Elkyabi, Magdy; Moftah, Faten; Girault, Annie; van Drimmelen, Harry; Busch, Michael P.; Lelie, Nico

2016-01-01

BACKGROUND Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective alternative to nucleic acid testing (NAT) for reducing the antibody-negative window period (WP). Later, a HCV antigen chemiluminescence immunoassay (CLIA) became available. STUDY DESIGN AND METHODS A panel composed of 337 HCV NAT–yield samples that were characterized for viral load (VL) and genotype was used to compare the sensitivity of two combination enzyme-linked immunosorbent assays (Monolisa, Bio-Rad; and Murex, formerly Abbott) and a HCV antigen CLIA (Abbott). Analytic sensitivity was compared with HCV RNA detection using Ultrio (Grifols) by testing serial dilutions of 10 genotype (gt)1 to gt4 samples. RESULTS HCV antigen CLIA detected 92.4% of samples, whereas Monolisa and Murex detected 38.3 and 47.5%, respectively. In the HCV RNA VL range of 105 to 107 IU/mL, Monolisa and Murex detected 38% to 56% of gt1, 85% to 78% of gt2, and 21% to 37% of gt3. The overall geometric mean 50% limit of detection (range) of Ultrio on gt1 to gt4 dilution series was 3.5 (1.2–7.7) copies/mL, compared to 3.3 × 106 (4.4 × 105-2.7 × 107), 3.4 × 106 (2.2 × 105–4.2 × 107), and 2728 (415–7243) copies/mL for Monolisa, Murex, and HCV antigen CLIA, respectively. CONCLUSION Analytical sensitivity of NAT was on average 1 million- and 780-fold higher than combination assays and HCV antigen CLIA, respectively. Relative sensitivities of combination assays differed for genotypes with Murex being more sensitive for gt1 and gt3 and Monolisa more sensitive for gt2. Although being less sensitive than NAT, combination assays could be considered in resource-limited settings since they detect 38% to 47% of seronegative WP donations. PMID:26013970

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

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Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

2015-05-01

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

Applicability of API ZYM to capture seasonal and spatial variabilities in lake and river sediments.

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Patel, Drashti; Gismondi, Renee; Alsaffar, Ali; Tiquia-Arashiro, Sonia M

2018-05-02

Waters draining into a lake carry with them much of the suspended sediment that is transported by rivers and streams from the local drainage basin. The organic matter processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and evaluate some of these variabilities in the sediments, we sampled six sites: three from the St. Clair River and three from Lake St. Clair in spring, summer, fall, and winter of 2016. At all sites and dates, we investigated the spatial and temporal variations in 19 extracellular enzyme activities using API ZYM. Our results indicated that a broad range of enzymes were found to be active in the sediments. Phosphatases, lipases, and esterases were synthesized most intensively by the sediment microbial communities. No consistent difference was found between the lake and sediment samples. Differences were more obvious between sites and seasons. Sites with the highest metabolic (enzyme) diversity reflected the capacity of the sediment microbial communities to breakdown a broader range of substrates and may be linked to differences in river and lake water quality. The seasonal variability of the enzymes activities was governed by the variations of environmental factors caused by anthropogenic and terrestrial inputs, and provides information for a better understanding of the dynamics of sediment organic matter of the river and lake ecosystems. The experimental results suggest that API ZYM is a simple and rapid enzyme assay procedure to evaluate natural processes in ecosystems and their changes.

Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

Science.gov (United States)

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.

Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

2013-03-01

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Zhao Hongwei; Xue Changguo; Nan Tiegui; Tan Guiyu; Li Zhaohu; Li, Qing X.; Zhang Qingchuan; Wang Baomin

2010-01-01

A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL -1 of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL -1 , respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

Use of enzyme immunoassays in disease diagnosis, with particular reference to rinderpest

International Nuclear Information System (INIS)

Crowther, J.R.

1986-01-01

The wider principles of the use of enzyme linked immunosorbent assays (ELISA) in diagnosis are illustrated. The development of an antirinderpest antibody assay is explained in order to demonstrate the relative ease with which the test can be set up and used in various countries. The various stages of the test can be standardized in central laboratories; for example, antigen (inactivated) can be prepared and passively attached to microplates. Purchasing and titration of conjugates and selection of negative control sera could also be done centrally. Thus 'kits' for the initiation of diagnosis could be rapidly made available. When assays have been used in specific countries to study antibody populations in large numbers of animals, it may be found necessary to modify the above procedures; for example, different negative sera may be necessary, reflecting the mean (and distribution) of the country. Relatively simple standardization steps to titrate antigen, conjugates and sera would allow assays to be set up in individual countries with no need for outside standardization. However, it may be useful to obtain sero-negative and sero-positive standards from an outside source which could be used on a worldwide basis for interlaboratory standardization. The provision of washing solution, blocking buffers, plates, pipettes and readers is the only requisite for the versatility of ELISA to be fully realized in the study of many disease agents. (author)

Detection and Antigenic Profiling of Undeclared Peanut in Imported Garlic Using an xMAP Multiplex Immunoassay for Food Allergens.

Science.gov (United States)

Pedersen, Ronnie O; Peters, Tim; Panda, Rakhi; Wehling, Paul; Garber, Eric A E

2017-07-01

A shipment of imported garlic powder was suspected of containing peanut. Samples (subs) collected from the shipment displayed considerable variability in peanut antigenicity when analyzed by enzyme-linked immunosorbent assay (ELISA). This raised questions regarding whether peanut was actually present, the amount present, and the basis for the variability in antigenic content. Analyses that used an xMAP multiplex assay for the detection of peanut and additional food allergens generated responses that were characteristic of peanut. Specifically, the relative intensities of two different peanut-specific antibodies coupled to beads (peanut-37 and -38) and the antigen profiles were identical to garlic controls spiked with peanut. In addition, the xMAP data did not indicate the presence of other allergens. Quantitative analyses indicated an approximately fivefold variation in peanut concentration among different subs. In contrast, within a sub, the apparent peanut concentration appeared constant. Particle size analyses of the garlic powder subs indicated a single distribution profile, with a peak at 380 μm. ELISA analysis of sieve-fractionated garlic powder from one of the subs indicated that slightly less than half of the detectable peanut was smaller than 212 μm, with the remainder almost evenly split between 212 and 300 μm and >300 μm. Modeling to predict possible oral exposure levels of peanut other than those directly measured requires additional research on the physicochemical properties of peanut and garlic, along with information on the production of the garlic powder.

Characterising Complex Enzyme Reaction Data.

Directory of Open Access Journals (Sweden)

Handan Melike Dönertaş

Full Text Available The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG. Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

Science.gov (United States)

Cortina, María E; Balzano, Rodrigo E; Rey Serantes, Diego A; Caillava, Ana J; Elena, Sebastián; Ferreira, A C; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

2016-06-01

Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Global inhibition of DC priming capacity in the spleen of self-antigen vaccinated mice requires IL-10

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Douglas Matthew Marvel

2014-02-01

Full Text Available DC in the spleen are highly activated following intravenous vaccination with a foreign antigen, promoting expansion of effector T cells, but remain phenotypically and functionally immature after vaccination with a self-antigen. Up-regulation or suppression of expression of a cohort of pancreatic enzymes 24-72 hours post-vaccination can be used as a biomarker of stimulatory versus toleragenic DC, respectively. Here we show, using MUC1 transgenic mice (MUC1.Tg and a vaccine based on the MUC1 peptide which these mice perceive as a self-antigen, that the difference in enzyme expression that predicts whether DC will promote immune response or immune tolerance, is seen as early as 4-8 hours following vaccination. We also identify early production of IL-10 as a predominant factor that both correlates with this early time point and controls DC function. Pre-treating mice with an antibody against the IL-10 receptor (IL-10R prior to vaccination results in DC that up-regulate CD40, CD80, and CD86 and promote stronger IFNγ+ T cell responses. This study suggests that transient inhibition of IL-10 prior to vaccination could improve responses to cancer vaccines that utilize self-tumor antigens.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

DEFF Research Database (Denmark)

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

Epitope mapping of the domains of human angiotensin converting enzyme.

Science.gov (United States)

Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

2006-06-01

Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

Micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

Energy Technology Data Exchange (ETDEWEB)

Layton, G T [Royal Infirmary, Manchester (UK)

1980-10-01

Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10/sup -3/ units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus.

Evaluation and comparison of radio-, fluorescence, and enzyme-linked immunoassays for serum thyroxine

International Nuclear Information System (INIS)

Kaplan, L.A.; Gau, N.; Fearn, J.; Steain, E.A.; Chen, I.W.; Maxon, H.; Volle, C.

1981-01-01

We have compared three analytical systems for the measurement of serum thyroxine: enzyme-linked immunoassay (EIA), fluorescent immunoassay (FIA) and a radioimmunoassay (RIA). These were evaluated with respect to their precision, accuracy, analytical sensitivity and sample throughput. The RIA is more sensitive than the EIA (10 μg/L vs. 35 μg/L). Both systems have excellent precision (X=86 μg/L, C.V.sub(RIA)=C.V.sub(EIA)=4.6 percent). Both the EIA and RIA demonstrate good accuracy with recovery of between 97-98 percent of added thyroxine. The FIA has an apparent sensitiviity between that of the RIA and EIA (25 μg/L), but a precision consistently lower than the other two systems (C.V. =7.4 percent, X=86 μg/L). Patients' results by RIA compared well with those from EIA (r=0.91,P 0.05). Although not fully automated, the EIA performed on the Abbott ABA-100 analyzer has a sample throughput equal to the automated RIA system (Micromedic, Concept 4)

Design considerations of a hollow microneedle-optofluidic biosensing platform incorporating enzyme-linked assays

Science.gov (United States)

Ranamukhaarachchi, Sahan A.; Padeste, Celestino; Häfeli, Urs O.; Stoeber, Boris; Cadarso, Victor J.

2018-02-01

A hollow metallic microneedle is integrated with microfluidics and photonic components to form a microneedle-optofluidic biosensor suitable for therapeutic drug monitoring (TDM) in biological fluids, like interstitial fluid, that can be collected in a painless and minimally-invasive manner. The microneedle inner lumen surface is bio-functionalized to trap and bind target analytes on-site in a sample volume as small as 0.6 nl, and houses an enzyme-linked assay on its 0.06 mm2 wall. The optofluidic components are designed to rapidly quantify target analytes present in the sample and collected in the microneedle using a simple and sensitive absorbance scheme. This contribution describes how the biosensor components were optimized to detect in vitro streptavidin-horseradish peroxidase (Sav-HRP) as a model analyte over a large detection range (0-7.21 µM) and a very low limit of detection (60.2 nM). This biosensor utilizes the lowest analyte volume reported for TDM with microneedle technology, and presents significant avenues to improve current TDM methods for patients, by potentially eliminating blood draws for several drug candidates.

Direct immunofluorescence and enzyme-linked immunosorbent assays for evaluating chlorinated hydrocarbon degrading bacteria

Energy Technology Data Exchange (ETDEWEB)

Brigmon, R.L.; Franck, M.M.; Brey, J.; Fliermans, C.B. [Westinghouse Savannah River, Aiken, SC (United States). Environmental Biotechnology Section; Scott, D.; Lanclos, K. [Medical Coll. of Georgia, Augusta, GA (United States)

1997-06-01

Immunological procedures were developed to enumerate chlorinated hydrocarbon degrading bacteria. Polyclonal antibodies (Pabs) were produced by immunizing New Zealand white rabbits against 18 contaminant-degrading bacteria. These included methanotrophic and chlorobenzene (CB) degrading species. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Pabs. Direct fluorescent antibodies (DFAs) were developed with these Pabs against select methanotrophic bacteria isolated from a trichloroethylene (TCE) contaminated landfill at the Savannah River Site (SRS) and cultures from the American Type Culture Collection (ATCC). Analysis of cross reactivity testing data showed some of the Pabs to be group specific while others were species specific. The threshold of sensitivity for the ELISA is 105 bacteria cells/ml. The DFA can detect as few as one bacterium per ml after concentration. Results from the DFA and ELISA techniques for enumeration of methanotrophic bacteria in groundwater were higher but not significantly different (P < 0.05) compared to indirect microbiological techniques such as MPN. These methods provide useful information on in situ community structure and function for bioremediation applications within 1--4 hours of sampling.

Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange.

Science.gov (United States)

Curtis, C F

2001-02-24

This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable.

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

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Anna-Janina Behrens

2016-03-01

Full Text Available The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664 maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Oviedo, J M; Valiño, F; Plasencia, I

2001-01-01

We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...... used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein...

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

Science.gov (United States)

Restrepo, B I; Obregón-Henao, A; Mesa, M; Gil, D L; Ortiz, B L; Mejía, J S; Villota, G E; Sanzón, F; Teale, J M

2000-05-01

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

Energy Technology Data Exchange (ETDEWEB)

Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R. (Thyroid Research Unit, The Montreal General Hospital Research Institute, Montreal, Quebec (Canada)); Medeiros-Neto, G.; Iacona, A.; Lima, N. (Thyroid Clinic, Hospital das Clinicas, Sao Paulo (Brazil))

1989-01-01

We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in {sup 51}Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of {sup 51}Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. (Abstract Truncated)

The in vivo effect of N-nitrosomorpholine on the activity of enzymes ...

African Journals Online (AJOL)

Neoplasm antigens outnumber the enzymes which are utilized to determine the cancer. Cancer development in the living organisms chronologically follows the cytotoxic, organotoxic and mutagenic alterations. Generally, the first symptom for chemical carcinogens is a metabolical response in connection with the ...

Subcellular localization of pituitary enzymes

Science.gov (United States)

Smith, R. E.

1970-01-01

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

Carcinoma-associated antigens

International Nuclear Information System (INIS)

Bartorelli, A.; Accinni, R.

1981-01-01

This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125 I-labeled forms of said antigens and methods of detecting said antigens in serum or plasma. The invention also relates to a diagnostic kit containing standardised antigens or antisera or marked forms thereof for the detection of said antigens in human blood, serum or plasma. (author)

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil.

Science.gov (United States)

Matos, Carlos António; Gonçalves, Luiz Ricardo; Alvarez, Dasiel Obregón; Freschi, Carla Roberta; Silva, Jenevaldo Barbosa da; Val-Moraes, Silvana Pompeia; Mendes, Natalia Serra; André, Marcos Rogério; Machado, Rosangela Zacarias

2017-01-01

Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

Science.gov (United States)

Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

2007-04-01

Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

International Nuclear Information System (INIS)

Sadeghi, H.; Da Silva, A.M.; Klein, C.

1988-01-01

The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [ 3 H]myristate, [ 3 H]palmitate, and [ 14 C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [ 14 C]ethanolamine but not with [ 3 H]myristate of [ 3 H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

Identification of a vesicular-arbuscular mycorrhizal fungus by using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Science.gov (United States)

Wright, S F; Morton, J B; Sworobuk, J E

1987-09-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

Immunity to tumour antigens.

Science.gov (United States)

Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C

2005-01-01

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

A Subpath-based Logit Model to Capture the Correlation of Routes

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Xinjun Lai

2016-06-01

Full Text Available A subpath-based methodology is proposed to capture the travellers’ route choice behaviours and their perceptual correlation of routes, because the original link-based style may not be suitable in application: (1 travellers do not process road network information and construct the chosen route by a link-by-link style; (2 observations from questionnaires and GPS data, however, are not always link-specific. Subpaths are defined as important portions of the route, such as major roads and landmarks. The cross-nested Logit (CNL structure is used for its tractable closed-form and its capability to explicitly capture the routes correlation. Nests represent subpaths other than links so that the number of nests is significantly reduced. Moreover, the proposed method simplifies the original link-based CNL model; therefore, it alleviates the estimation and computation difficulties. The estimation and forecast validation with real data are presented, and the results suggest that the new method is practical.

Seroprevalence of Hepatitis B Surface Antigen and Occupational Risk Factors Among Health Care Workers in Ekiti State, Nigeria.

Science.gov (United States)

Alese, Oluwole Ojo; Alese, Margaret Olutayo; Ohunakin, Afolabi; Oluyide, Peter Olumuyiwa

2016-02-01

Hepatitis B virus (HBV) infection is contracted from blood and other body fluid making healthcare workers (HCW) prone to the infection especially in the developing world. Though it is a vaccine preventable disease, the level of awareness and universal precaution among HCW is low in sub-Saharan African and Asia. The study was aimed at determining the seroprevalence of hepatitis B surface antigen and occupational risk factors among health care workers at Ekiti State University Teaching Hospital, Ado Ekiti. One hundred and eighty-seven (187) blood samples were collected from volunteer subjects who comprised of medical doctors, nurses, health attendants, and porters who are in regular contact with blood, body fluids and patients after informed consent. Well detailed and structured questionnaires were used to obtain demographic and other relevant data from the subjects. Blood samples were tested by Enzyme Linked Immunosorbent assay (ELISA) for hepatitis B surface antigen. Out of the 187 HCWs there were 91 males (48.7%) and 96 (51.3%) females. Only 2 participants tested positive to hepatitis B surface antigen with a prevalence of 1.1%. Also, only 30 (16.0%) of the participants had been fully vaccinated against the infection while the remaining 157(84.0%) had no adult vaccination. It is obvious that the awareness of the infection is low among the HCWs studied thus the need to incorporate screening for HbsAg and vaccination against HBV into the periodic/pre-employment health intervention programmes by employers to help in the protection of HCWs and control the spread of the virus.

Diagnostic value of tissue polypeptide specific antigen in serum for patients with pancreatic carcinoma

International Nuclear Information System (INIS)

Lu Yun; Liu Xingdang; Li Ding; Dei Shouzhen

2009-01-01

Objective: To compare the diagnostic value of tissue polypeptide specific antigen (TPS) in serum, with the carbohydrate antigens (CA) 19-9, CA50, CA125 and CA242 in pancreatic carcinoma. Methods: Serum TPS was measured with enzyme linked immunosorbent assay (ELISA). CA19-9 and CA125 were measured with chemiluminescent immunoassay. CAS0 and CA242 were measured with immunoradiometric assay in 33 patients with pancreatitis, 34 patients with pathologically proven pancreatic carcinoma, and 35 patients with non-pancreatic malignancies. Statistic analysis was carried out with SPSS 9.0 software. Results: Patients with pancreatic carcinoma had relatively higher levels of TPS [(386.5 ± 315. 1) U/L] and CA19-9 [(10 820.9 ± 389.7) kU/L] when compared with patients with pancreatitis [(86. 2 ± 28.1) U/L and (61.5 ± 24.7) kU/L, respectively; F = 936. 42, P < 0. 001 ; F = 2217. 09, P < 0. 001], with a sensitivity and a specificity of 70.6% (48/68) and 57.4% (39/68), respectively, for TPS, and 82.4% (28/34) and 77.9% (53/68), respectively, for CA19-9. Diagnostic performance was further improved when TPS was assayed in combination with CA19-9, CA50, CA125 and CA242. Conclusion: Serum TPS has an incremental value in complementing CA19-9 in the diagnosis of pancreatic carcinoma. (authors)

Occurrence of West Nile Virus Antibodies in Wild Birds, Horses, and Humans in Poland

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Jowita Samanta Niczyporuk

2015-01-01

Full Text Available Serum samples of 474 wild birds, 378 horses, and 42 humans with meningitis and lymphocytic meningitis were collected between 2010 and 2014 from different areas of Poland. West Nile virus (WNV antibodies were detected using competition enzyme linked immunosorbent assays: ELISA-1 ID Screen West Nile Competition, IDvet, ELISA-2 ID Screen West Nile IgM Capture, and ELISA-3 Ingezim West Nile Compac. The antibodies were found in 63 (13.29% out of 474 wild bird serum samples and in one (0.26% out of 378 horse serum samples. Fourteen (33.33% out of 42 sera from patients were positive against WNV antigen and one serum was doubtful. Positive samples obtained in birds were next retested with virus microneutralisation test to confirm positive results and cross-reactions with other antigens of the Japanese encephalitis complex. We suspect that positive serological results in humans, birds, and horses indicate that WNV can be somehow closely related with the ecosystem in Poland.

Isolation and characterization of antigen-Ia complexes involved in T cell recognition

DEFF Research Database (Denmark)

Buus, S; Sette, A; Colon, S M

1986-01-01

Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is he......Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes...... with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance...

Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

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Marco Tamba

2010-01-01

Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

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Yuan-Cheng Cao

2015-01-01

Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

Energy Technology Data Exchange (ETDEWEB)

Zhao Hongwei [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Xue Changguo [Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027 (China); Nan Tiegui; Tan Guiyu; Li Zhaohu [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Li, Qing X. [Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822 (United States); Zhang Qingchuan, E-mail: zhangqc@ustc.edu.cn [Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027 (China); Wang Baomin, E-mail: wbaomin@263.com [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China)

2010-08-31

A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL{sup -1} of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL{sup -1}, respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

Development of an enzyme-linked immunosorbent assay for the detection of dicamba.

Science.gov (United States)

Clegg, B S; Stephenson, G R; Hall, J C

2001-05-01

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in water. The CI-ELISA has a detection limit of 2.3 microg L(-1) and a linear working range of 10--10000 microg L(-1) with an IC(50) value of 195 microg L(-1). The dicamba polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with a dicamba metabolite, 5-hydroxydicamba, and structurally related chlorobenzoic acids. The assay was used to estimate quantitatively dicamba concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, a C(18) (reversed phase) column concentration step was devised prior to analysis, and the detection limits were increased by at least by 10-fold. After the sample preconcentration, the detection limit, IC(50), and linear working range were 0.23, 19.5, and 5-200 microg L(-1), respectively. The CI-ELISA estimations in water correlated well with those from gas chromatography-mass spectrometry (GC-MS) analysis (r(2) = 0.9991). This assay contributes to reducing laboratory costs associated with the conventional GC-MS residue analysis techniques for the quantitation of dicamba in water.

and Indigenous Indirect Enzyme-Linked Immunosorbent Assay

Directory of Open Access Journals (Sweden)

Annapurna S. Agasthya

2012-01-01

Full Text Available Brucellosis is one of the most important reemerging zoonoses in many countries. Brucellosis is caused by Gram-negative coccobacillus belonging to genus Brucella. Human brucellosis often makes the diagnosis difficult. The symptoms and clinical signs most commonly reported are fever, fatigue, malaise, chills, sweats headaches, myalgia, arthralgia, and weight loss. Some cases have been presented with only joint pain, lower backache, and involuntary limb movement, burning feet, or ischemic heart attacks. The focus of this work was to develop a highly sensitive and specific indirect ELISA by using smooth lipopolysaccharide antigen of Brucella abortus 99 to detect anti-Brucella antibodies at Project Directorate on Animal Disease Monitoring and Surveillance. Serum samples collected from 652 individuals in whom fever was not the major symptom but the complaint was of joint pain, headache, lower backache, and so forth, were screened by Rose Bengal plate agglutination test (RBPT and standard tube agglutination test (STAT. Subsequent testing of sera by indigenous indirect ELISA detected 20 samples positive (3.6% seroprevalence, and indirect ELISA was found to be more sensitive than RBPT and STAT. The seroprevalence in South Karnataka was 2.14%, and in North Karnataka it was 0.92%.

Potential Impact of Seasonal Malaria Chemoprevention on the Acquisition of Antibodies Against Glutamate-Rich Protein and Apical Membrane Antigen 1 in Children Living in Southern Senegal

DEFF Research Database (Denmark)

Ndiaye, Magatte; Sylla, Khadime; Sow, Doudou

2015-01-01

Seasonal malaria chemoprevention (SMC) is defined as the intermittent administration of full treatment courses of an antimalarial drug to children during the peak of malaria transmission season with the aim of preventing malaria-associated mortality and morbidity. SMC using sulfadoxine-pyrimetham......Seasonal malaria chemoprevention (SMC) is defined as the intermittent administration of full treatment courses of an antimalarial drug to children during the peak of malaria transmission season with the aim of preventing malaria-associated mortality and morbidity. SMC using sulfadoxine......-pyrimethamine (SP) combined with amodiaquine (AQ) is a promising strategy to control malaria morbidity in areas of highly seasonal malaria transmission. However, a concern is whether SMC can delay the natural acquisition of immunity toward malaria parasites in areas with intense SMC delivery. To investigate this......, total IgG antibody (Ab) responses to Plasmodium falciparum antigens glutamate-rich protein R0 (GLURP-R0) and apical membrane antigen 1 (AMA-1) were measured by enzyme-linked immunosorbent assay in Senegalese children under the age of 10 years in 2010 living in Saraya and Velingara districts (with SMC...

Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry.

Science.gov (United States)

Bayraç, Ceren; Öktem, Hüseyin Avni

2017-02-01

To monitor the specificity of Staphylococcus aureus aptamer (SA-31) against its target cell, we used enzyme-linked aptamer assay. In the presence of target cell, horseradish peroxidase-conjugated streptavidin bound to biotin-labeled SA-31 showed specific binding to S  aureus among 3 different bacteria with limit of detection of 10 3 colony-forming unit per milliliter. The apparent K a was 1.39 μM -1  ± 0.3 μM -1 . The binding of SA-31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K a , ΔH, and ΔG). Since binding of aptamer to its targets solely depends on its 3-dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA-31 to its target on surface of bacteria. At 4°C, SA-31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA-31 slightly varied from K a  = 1.56 μM -1  ± 0.69 μM -1 at 25°C to K a  = 1.03 μM -1  ± 0.9 μM -1 at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 μM -1  ± 0.38 μM -1 at 50mM, 1.60 μM -1  ± 0.11 μM -1 at 137mM, and 3.28 μM -1  ± 0.46 μM -1 at 200 mM of salt concentration. In this study, it was demonstrated that enzyme-linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of serum and salivary antibodies in children vaccinated with oral live or parenteral inactivated poliovirus vaccines of different antigen concentrations.

Science.gov (United States)

Zaman, S; Carlsson, B; Jalil, F; Mellander, L; Van Wezel, A L; Böttiger, M; Hanson, L A

1991-12-01

A new antigen-rich inactivated poliovirus vaccine (IPV) in ordinary (IPV1), double (IPV2) and quadruple (IPV4) antigen concentrations was given in 2 doses to 6 and 18 week old Pakistani infants. The immune responses to poliovirus types 1 and 3 were compared to those in infants given three doses of oral poliovirus vaccine (OPV) at 6, 12 and 18 weeks of age. Enzyme-linked immunosorbent assay, ELISA, was used to estimate IgG and IgA in serum and secretory IgA (SIgA) in saliva. Two to three years later, a follow-up of the serum antibody response was carried out in the same infants using a microneutralization test. Serum IgG antibody responses to poliovirus type 1 antigen after two doses of IPV1, IPV2 and IPV4 were not significantly higher than the response after three doses of OPV at 21 weeks of age (p greater than 0.05). The serum IgG responses to poliovirus type 3 were similar to those against type 1 in all the groups. Mean neutralizing antibody titres to poliovirus type 1 was significantly higher in the IPV2 group than the rest of the groups (p less than 0.01). For type 3, these titres were highest but not significantly, in the IPV4 group (p greater than 0.05). This study shows that two doses of a new antigen-rich IPV can give similar immediate serum antibody responses as OPV but higher late responses. SIgA antibodies in saliva were more efficiently induced by OPV after three doses than after 2 doses of IPV (p less than 0.05).

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

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Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

2015-10-23

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

Science.gov (United States)

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

Serum biotin in Japanese children: Enzyme-linked immunosorbent assay measurement.

Science.gov (United States)

Wakabayashi, Kenji; Kodama, Hiroko; Ogawa, Eishin; Sato, Yasuhiro; Motoyama, Kahoko; Suzuki, Mitsuyoshi

2016-09-01

Biotin deficiency has been reported in Japanese infants fed special formulas for medical reasons, including those with milk allergy and congenital metabolic diseases, because these formulas contain little biotin. Serum biotin measurement is useful for diagnosing biotin deficiency. We applied a simple and rapid method to analyze serum biotin, and established normal ranges for children and adults. Serum biotin in 188 healthy Japanese children aged 0-4 years and in 25 healthy adults was analyzed using a Biotin ELISA Kit (immundiagnostik). The effects of various conditions on the measurement of serum biotin were also examined. Median biotin in children aged 0-4 years was 10.4 ng/dL (IQR, 7.9-13.4 ng/dL), and that in adults was 12.9 ng/dL (IQR, 10.8-15.8 ng/dL). Normal range was 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults (calculated using two-sided 95%CI). Measurements obtained with this method were not affected by frozen storage, freeze-thaw, or hemolysis, indicating that serum biotin can be analyzed accurately under these conditions, with a possible application to plasma samples. Serum biotin was significantly lower in children than in adults, with the normal range being 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults. This simple and accurate enzyme-linked immunosorbent assay method is useful for diagnosing biotin deficiency. © 2016 The Authors. Pediatrics International published by John Wiley & Sons Australia, Ltd on behalf of Japan Pediatric Society.

USE OF AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA TO DETECT ANTIBODIES IN AYU (Plecogiossus altivelis VACCINATED BY IMMERSION ADMINISTRATION

Directory of Open Access Journals (Sweden)

. Sukenda

2007-05-01

Full Text Available ABSTRACTAn indirect enzyme-linked immunosorbent assay (ELISA was used to detect serum antibody in ayu, Plecoglossus altivelis, immunized against Pseudomonasplecoglossicida by immersion vaccination.  First, the procedure of the ELISA was optimized and the sensitivity was checked.  Secondly, the formalin-killed cells (FKC of P. plecoglossicida was administered to ayu by immersion vaccination.  Two weeks after vaccination, fish were divided into two groups, one group was given booster.  The level of specific antibody production of both boostered and vaccinated only fish were statistically higher than unvaccinated control fish at the time of each blood collection.  However, the differences between the boostered and vaccinated only fish were not statistically significant.Keywords :  immunization, Pseudomonas plecoglossicida, ayu, ELISA ABSTRAKIndirect enzyme-linked immunosorbent assay (ELISA digunakan untuk mendeteksi antibodi pada ayu, Plecoglossus altivelis, yang diimunisasi dengan cara perendaman untuk melawan infeksi Pseudomonas plecoglossicida.  Pertama, prosedur ELISA dioptimasikan dan sensitivitas dari metode ini juga diperiksa.  Kemudian, bakteri Plecoglossus altivelis yang sudah dimatikan dengan formalin diberikan ke ikan ayu dengan vaksinasi perendaman.  Dua minggu setelah vaksinasi, ikan dibagi menjadi dua kelompok, satu kelompok diberi vaksinasi kedua.  Produksi antibodi spesifik dari ikan-ikan yang divaksinasi satu kali dengan vaksinasi dua kaii secara statistik lebih tinggi dibandingkan dengan control.  Akan tetapi, tidak ada perbedaan produksi antibodi antara ikan yarig divaksanisi satu kali dengan divaksinasi dua kali.Kata kunci :  imunisasi, Pseudomonasplecoglossicida, ayu, ELISA

Early Diagnosis of Congenital Trypanosoma cruzi Infection, Using Shed Acute Phase Antigen, in Ushuaia, Tierra del Fuego, Argentina

Science.gov (United States)

Mallimaci, María Cristina; Sosa-Estani, Sergio; Russomando, Graciela; Sanchez, Zunilda; Sijvarger, Carina; Alvarez, Isabel Marcela; Barrionuevo, Lola; Lopez, Carlos; Segura, Elsa Leonor

2010-01-01

Chagas' disease, or American trypanosomiasis, is caused by the protozoan parasite Trypanasoma cruzi. It is estimated that 15,000 new cases of congenital T. cruzi transmission occur in the Americas each year. The aim of this study was to estimate the rate of congenital T. cruzi infection in infants born to infected women living in Ushuaia, Argentina, as well to assess a serologic test using Shed Acute Phase Antigen (SAPA) for a timely diagnosis of congenital infection. The rate of congenital infection among children in the study was 4.4% (3/68). Our results show that for infants younger than 30 days of age, matched blood samples from mother and infant were capable of identifying congenital transmission of infection using an enzyme-linked immunosorbent assay with SAPA. For infants older than 3 months, congenital infection could be ruled out using the same procedure. PMID:20064996

Antibodies reactive to Plasmodium falciparum serine repeat antigen in children with Burkitt lymphoma from Ghana.

Science.gov (United States)

Guech-Ongey, Mercy; Yagi, Masanori; Palacpac, Nirianne Marie Q; Emmanuel, Benjamin; Talisuna, Ambrose O; Bhatia, Kishor; Stefan, D Cristina; Biggar, Robert J; Nkrumah, Francis; Neequaye, Janet; Tougan, Takahiro; Horii, Toshihiro; Mbulaiteye, Sam M

2012-04-15

The role of protective immunity to Plasmodium falciparum (Pf) malaria in Burkitt lymphoma (BL) is unknown. We investigated the association between BL and antibodies reactive to SE36 antigen, a recombinant protein based on P. falciparum serine repeat antigen 5 gene, targeted by protective malaria immune responses. Cases were children (0-14 years) enrolled at the Korle-Bu Teaching Hospital, Accra, Ghana, during 1965-1994 with BL confirmed by histology or cytology (92% of cases). Controls were apparently healthy children enrolled contemporaneous to the cases from the nearest neighbor house to the case house and were age,- sex-frequency-matched to the cases. Anti-SE36 IgG antibodies were measured using enzyme-linked absorbent immunoassays (ELISAs). SE36 titers were estimated by extrapolating ELISA optical density readings to a standard fitting curve. Anti-SE36 titers were log-transformed for analysis. Odds ratios (ORs) and two-sided 95% confidence intervals (95% CIs) were estimated using unconditional logistic regression. The mean log endpoint dilution titers were 0.63 logs lower in cases than in controls (8.26 [SD 1.68] vs. 8.89 [SD 1.75], Student's t-test, p = 0.019). Lower titers were observed in cases than controls aged 0-4 years (p = 0.05) and in those aged 5-14 years (p = 0.06). Low and medium tertiles of anti-SE36 IgG antibodies were associated with increased OR for BL ([OR 1.67, 95% CI 1.21-2.31] and [OR 1.33, 95% CI 0.96-1.86], respectively, p(trend) = 0.002) in analyses adjusting for age, sex, calendar period and test plate. Our findings suggest that compared to similarly aged children enrolled from the same community, children with BL in Ghana have lower antibodies to SE36 antigen. Copyright © 2011 UICC.

A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

International Nuclear Information System (INIS)

Layton, G.T.

1980-01-01

Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10 -3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

Serum levels of fetal antigen 1 in extreme nutritional States

DEFF Research Database (Denmark)

Andries, Alin; Niemeier, Andreas; Støving, Rene K

2012-01-01

Objective. Recent data suggest that fetal antigen (FA1) is linked to disorders of body weight. Thus, we measured FA1 serum levels in two extreme nutritional states of morbid obesity (MO) and anorexia nervosa (AN) and monitored its response to weight changes. Design. FA1 and insulin serum...

Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT).

Science.gov (United States)

Francis, R J; Mather, S J; Chester, K; Sharma, S K; Bhatia, J; Pedley, R B; Waibel, R; Green, A J; Begent, R H J

2004-08-01

MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.

Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

International Nuclear Information System (INIS)

Francis, R.J.; Chester, K.; Sharma, S.K.; Bhatia, J.; Pedley, R.B.; Green, A.J.; Begent, R.H.J.; Mather, S.J.; Waibel, R.

2004-01-01

MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99m Tc-carbonyl [ 99m Tc(H 2 O) 3 (CO) 3 ] + (abbreviated to TcCO) mediated labelling of 99m Tc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

Beyond financial value capturing? Interactions between value capturing and cooperation at the interface of road infrastructure and land use planning

NARCIS (Netherlands)

Heeres, Niels; Lenferink, Sander; Tillema, Taede; Arts, Jos

2016-01-01

To deal with tensions between functional interrelatedness and institutional fragmentation, road infrastructure planning is increasingly interested in integrated planning approaches. From a value capture (VC) perspective, integration offers possibilities for financially linking road infrastructure

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

Science.gov (United States)

Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

2016-01-01

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

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

Directory of Open Access Journals (Sweden)

Nikhil S. Gopal

2017-01-01

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

Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Akiyama, Mitoshi; Takaishi, Masatoshi; Murakami, Yoshie; Ueda, Ryuzo; Yamakido, Michio; Tsubokura, Tokuo.

1989-09-01

Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

Seroprevalence of Human Immunodeficiency Virus, Hepatitis B ...

African Journals Online (AJOL)

surface antigen (HBsAg), syphilis and HCV from the antenatal records. The data were extracted by two trained assistants. Hepatitis B surface antigen and antibodies to HCV were determined using Clinotech diagnostic enzyme linked immunosorbent assay (ELISA) test kits (Clinotech Laboratories,. USA; batch/lot no. for ...

Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins.

Science.gov (United States)

Carmichael, W W; An, J

1999-01-01

Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

DEFF Research Database (Denmark)

Götz, C; Koenig, M G; Issinger, O G

1995-01-01

by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains.

Science.gov (United States)

Tano, Yoshio; Shimizu, Hiroyuki; Martin, Javier; Nishimura, Yorihiro; Simizu, Bunsiti; Miyamura, Tatsuo

2007-10-10

A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1-3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

ELISA testing for soy antigens in dry dog foods used in dietary elimination trials.

Science.gov (United States)

Willis-Mahn, Christine; Remillard, Rebecca; Tater, Kathy

2014-01-01

The use of elimination diet trials is necessary in the diagnosis of food allergies and intolerances. The objective of this study was to determine in vitro if four over-the-counter (OTC) dry dog foods carrying a "no soy" claim and seven veterinary therapeutic dry dog foods designed for food elimination trials were suitable for a soybean elimination trial. A 100 g sample of each diet plus one soy positive and one soy negative control diet were submitted for enzyme-linked immunosorbent assay testing to an independent food laboratory. The positive control diet contained >25 ppm soy protein antigens and the negative control contained 25 ppm. Three veterinary therapeutic diets had less than the lowest detectable limit of soy protein and four were positive (>2.5 ppm). OTC dog food diets that claim to contain "no soy" may contain high concentrations of soy protein and, therefore, should not be used in soy elimination trials in suspect food allergic dogs. The veterinary therapeutic diet selected for a soy elimination trial needs to be carefully chosen based on diet history.

Comparison between the 13C-urea breath test and stool antigen test for the diagnosis of childhood Helicobacter pylori infection

International Nuclear Information System (INIS)

Kato, Seiichi; Minoura, Takanori; Iinuma, Kazuie; Konno, Mutsuko; Tajiri, Hitoshi; Matsuhisa, Takeshi

2004-01-01

As noninvasive tests for Helicobacter pylori infection, the 13 C-urea breath test (UBT) and stool antigen test have been widely used. In children, however, there are few studies reporting which test shows superior performance. The purpose of this study was to compare the 13 C-UBT and stool antigen test for their accuracy in diagnosing. H. pylori infection in children. A total of 123 Japanese children, ages 2 to 17 years (mean, 12 years) who underwent gastric biopsies for H. pylori infection were studied. The diagnoses included gastritis (n=55), gastric ulcer (n=5), duodenal ulcer (n=20), iron-deficiency anemia (n=7), and other conditions (n=36). The cutoff value of the 13 C-UBT was defined to be 3.5 per mille. The stool antigen test was performed using the H. pylori stool antigen (HpSA) enzyme-linked immunosorbent assay (ELISA) (Premier Platinum HpSA). In 16 patients who received eradication therapy, the 13 C-UBT and HpSA were repeated 2 months after treatment. Based on biopsy tests, 60 children were infected with H. pylori and 63 children were not. For the 13 C-UBT, the sensitivity, specificity, and accuracy were 95.0% (95% confidence interval [CI], 86.1%-99.0%), 98.4% (95% CI, 91.5%-100%), and 96.4% (95% CI, 93.6%-99.9%), respectively. For the HpSA, the sensitivity, specificity, and accuracy were 983.% (95% CI, 90.8%-100%), 98.4% (95% CI, 91.2%-100%), and 98.3% (95% CI, 96.0%-100%), respectively. There were no significant differences between the performance of these two tests. In the assessment of H. pylori eradication, the results of 13 C-UBT and HpSA agreed with those of biopsy tests. The 13 C-UBT and the HpSA are equally accurate for the diagnosis of active H. pylori infection in Japanese children. (author)

Reduction-sensitive polymer-shell-coated nanogels for intracellular delivery of antigens

NARCIS (Netherlands)

Li, Dandan; Chen, Yinan|info:eu-repo/dai/nl/377279048; Mastrobattista, Enrico|info:eu-repo/dai/nl/228061105; Van Nostrum, Cornelus F.|info:eu-repo/dai/nl/134498690; Hennink, Wim E.|info:eu-repo/dai/nl/070880409; Vermonden, Tina|info:eu-repo/dai/nl/275124517

2017-01-01

Nowadays, layer-by-layer assembled microsized particles receive interest as drug delivery systems. In the present study, we report nanosized hydrogels loaded with a protein antigen that are coated with a disulfide cross-linked polymer shell. These disulfide bonds are stable in the nonreducing

Evaluation of electrochemiluminescene immunoassay and enzyme-linked immunosorbent assay for serum HBsAb detection

International Nuclear Information System (INIS)

Ma Caiyun; Jiang Li; Ge Gaoxia; Zhang Xiaojie

2005-01-01

Electrochemiluminescene immunoassay (ECLIA) and enzyme-linked immunosorbent (ELISA) were used to detect the different concentrations of serum HBsAb, in order to compare the results of ECLIA and ELISA. The result showed that intra-assay coefficient of variation of ECLIA was about 0.95% for high value, 1.13% for mean values and 2.63% for low value, while that of ELISA was about 5.76%, 12.8% and 15.9%, respectively. The interassay coefficient of ECLIA was about 4.03% for high values, 4.93% for mean values and 7.34% for low values, while that of ELISA was about 10.1%, 19.6% and 25.2%, respectively. The analytical sensitivity of ECLIA was about 4.0IU/L, while that of ELISA is about 19.0IU/L. Only in 3 samples, the results measured by ECLIA and ELISA were different (ECLIA: positive; ELISA: negative) among 165 samples. It is concluded that the met hod of ECLIA is more efficient than ELISA for detection of HBsAb in serum. (authors)

Evaluation of Two Commercial Enzyme-Linked Immunosorbent Assay Kits for the Detection of Human Circulating Metrnl.

Science.gov (United States)

Zheng, Si-Li; Li, Zhi-Yong; Zhang, Zheng; Wang, Dong-Sheng; Xu, Jian; Miao, Chao-Yu

2018-04-01

Metrnl is a newly discovered secreted protein with neurotrophic activity and metabolic effect, while in earlier studies its circulating level in human was not explored. We evaluated two commercial enzyme-linked immunosorbent assay kits (DY7867-05, R&D Systems and SK00478-02, Aviscera Bioscience) for the detection of human circulating Metrnl. The DY7867-05 kit showed superiority over the SK00478-02 kit since it generated better curve fitting degree, smaller variation among tests, higher inter-assay reproducibility and better specificity, and could effectively detect human Metrnl in six types of blood samples. Subsequent analysis was performed using the DY7867-05 kit. Sample storage conditions were investigated. No gender difference in circulating Metrnl levels was found, while people with newly diagnosed type 2 diabetes mellitus (T2DM) had significantly lower Metrnl levels compared to the healthy controls.

Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein e(rns) for serologic diagnosis of pestivirus infections in swine

NARCIS (Netherlands)

Langedijk, J.P.; Middel, W.G.; Meloen, R.H.; Kramps, J.A.; Smit, de J.A.

2001-01-01

Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure

Enzyme-linked electrochemical DNA ligation assay using magnetic beads

Czech Academy of Sciences Publication Activity Database

Stejskalová, Eva; Horáková Brázdilová, Petra; Vacek, J.; Bowater, R. P.; Fojta, Miroslav

2014-01-01

Roč. 406, č. 17 (2014), s. 4129-4136 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GPP206/11/P739; GA ČR(CZ) GAP206/11/1638; GA AV ČR(CZ) IAA400040901 Institutional support: RVO:68081707 Keywords : Electrochemistry * Enzyme labeling * DNA ligase Subject RIV: BO - Biophysics Impact factor: 3.436, year: 2014

Novel CO{sub 2} capture. Final CRADA Report.

Energy Technology Data Exchange (ETDEWEB)

Snyder, S. W.; Energy Systems

2009-11-30

The goal of this work was to use electrochemically driven pH control to develop a second generation, enzyme-based contained liquid membrane (CLM) permeator to extract CO{sub 2} from a variety of coal-based flue gas streams more efficiently than does the CLM current design, while achieving performance coincident with DOE targets of less than 45% Cost of electricity (COE) in 2007 and less than 20% COE in 2012. Central to this goal the CLM would be alkaline (>pH 8) at the feed gas side and acid (capture and release using Argonne's resin-wafer electrode ionization (RW-EDI) system integrated with Carbozyme's carbonic anhydrase (CA) enzyme. Argonne developed RW-EDI for pH controlled desalination of process streams (e.g. Patents 7,452,920 & 7,306,934). In the current work, Argonne captured CO{sub 2} as HCO{sub 3}{sup -} and released it as CO{sub 2}. The goal is to both capture CO{sub 2} from a simulated flue gas stream and release it within the DOE targets for increase in COE. Initial performance results indicate that the 2012 COE targets are achievable with the developed technology. The design is subject to patent-hold. This task was funded in an exploratory phase, so no process optimization was attempted. Argonne believes that with optimization this performance could be significantly improved.

Photoperiodism and Enzyme Activity

Science.gov (United States)

Queiroz, Orlando; Morel, Claudine

1974-01-01

Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

Energy Technology Data Exchange (ETDEWEB)

Booth, J C; Hannington, G; Bakir, T M.F.; Stern, H; Kangro, H; Griffiths, P D; Heath, R B [Saint George' s Hospital Medical School, London (UK); Saint Bartholomew' s Hospital, London (UK))

1982-12-01

The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems.

Enzyme Engineering for In Situ Immobilization.

Science.gov (United States)

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Seroprevalence of Hepatitis B Surface Antigen and Occupational Risk Factors Among Health Care Workers in Ekiti State, Nigeria

Science.gov (United States)

Alese, Oluwole Ojo; Ohunakin, Afolabi; Oluyide, Peter Olumuyiwa

2016-01-01

Introduction Hepatitis B virus (HBV) infection is contracted from blood and other body fluid making healthcare workers (HCW) prone to the infection especially in the developing world. Though it is a vaccine preventable disease, the level of awareness and universal precaution among HCW is low in sub-Saharan African and Asia. Aim The study was aimed at determining the seroprevalence of hepatitis B surface antigen and occupational risk factors among health care workers at Ekiti State University Teaching Hospital, Ado Ekiti. Materials and Methods One hundred and eighty-seven (187) blood samples were collected from volunteer subjects who comprised of medical doctors, nurses, health attendants, and porters who are in regular contact with blood, body fluids and patients after informed consent. Well detailed and structured questionnaires were used to obtain demographic and other relevant data from the subjects. Blood samples were tested by Enzyme Linked Immunosorbent assay (ELISA) for hepatitis B surface antigen. Results Out of the 187 HCWs there were 91 males (48.7%) and 96 (51.3%) females. Only 2 participants tested positive to hepatitis B surface antigen with a prevalence of 1.1%. Also, only 30 (16.0%) of the participants had been fully vaccinated against the infection while the remaining 157(84.0%) had no adult vaccination. Conclusion It is obvious that the awareness of the infection is low among the HCWs studied thus the need to incorporate screening for HbsAg and vaccination against HBV into the periodic/pre-employment health intervention programmes by employers to help in the protection of HCWs and control the spread of the virus. PMID:27042489

Cellular Immune Responses for Squamous Cell Carcinoma Antigen Recognized by T Cells 3 in Patients with Hepatocellular Carcinoma.

Directory of Open Access Journals (Sweden)

Kiichiro Kaji

Full Text Available Squamous cell carcinoma antigen recognized by T cells 3 (SART3, a tumor-associated antigen expressed in many cancers, functions in tumor rejection. In this study, we investigated its usefulness as an immunotherapeutic target in hepatocellular carcinoma (HCC.The expression of SART3 in hepatoma cell lines and HCC tissues was investigated by immunofluorescence and immunohistochemical analyses. Two peptides derived from SART3 (SART3109 and SART3315 were used for immunological analysis. T-cell responses were investigated by interferon-gamma (IFN-γ enzyme-linked immunospot and cytotoxic T lymphocyte (CTL assays using peripheral blood mononuclear cells (PBMCs in 47 patients, and tumor-infiltrating lymphocytes in 8 of 47 patients with HCC. The safety of immunotherapy using a SART3-derived peptide was investigated by vaccinations of SART3109 in 12 patients with HCC (trial registration: UMIN000005677.The immunofluorescence and immunohistochemical analyses showed that SART3 was expressed in six HCC cell lines, and in HCC tissues including of alpha-fetoprotein-negative individuals. SART3-specific CTLs were generated by stimulating PBMCs with the peptides, and they showed cytotoxicity against HCC cells expressing the protein. Of the 47 HCC patients, 25.5% and 10.6% showed significant responses to SART3109 and SART3315, respectively. The infiltration of SART3109-specific IFN-γ-producing CTLs into the tumor site was confirmed. In the vaccination study, no severe adverse events were observed, and the peptide-specific CTLs were newly induced in four of five patients tested.SART3 is an immunotherapeutic candidate, and peptides from this antigen may be applied in HCC immunotherapy.UMIN000005677.

Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes.

Science.gov (United States)

Gard, A L; Warrington, A E; Pfeiffer, S E

1988-05-01

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.