GP50 as a promising early diagnostic antigen for Taenia multiceps infection in goats by indirect ELISA.

Science.gov (United States)

Huang, Xing; Xu, Jing; Wang, Yu; Guo, Cheng; Chen, Lin; Gu, Xiaobin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

2016-12-01

Coenurosis is caused by coenurus, the metacestode of Taenia multiceps, which mainly parasitizes the brain and spinal cord of cattle, sheep and goats. To date, no widely-approved methods are available to identify early coenurus infection. In this study, we identified a full-length cDNA that encodes GP50 (TmGP50) from the transcriptome of T. multiceps, and then cloned and expressed in E. coli. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of recombinant TmGP50 protein (rTmGP50) for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, we orally infected 20 goats with mature T. multiceps eggs. Praziquantel (10%) was given to 10 of the goats 45 days post-infection (p.i.). Blood samples were collected for 17 weeks p.i. from the 20 goats and anti-rTmGP50 antibodies were evaluated using the indirect ELISA established here. The TmGP50 contains an 897 bp open reading frame, in which signal sequence resides in 1 ~ 48 sites and mature polypeptide consists of 282 amino acid residues. Immunofluorescence staining showed that native TmGP50 was localized to the microthrix and parenchymatous zone of the adult parasite and coenurus, and the coenurus cystic wall. The indirect ELISA based on rTmGP50 exhibited a sensitivity of 95.0% and a specificity of 92.6% when detecting GP50 antibodies in sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmGP50 antibody was detectable from 2 to 17 weeks p.i. in the control group, while the antibody fell below the cut-off value about 3 weeks after praziquantel treatment. Our results indicate that recombinant TmGP50 is a suitable early diagnostic antigen for coenurus infection in goats.

Evaluation of monoclonal antibody-based sandwich direct ELISA (MSD-ELISA for antigen detection of foot-and-mouth disease virus using clinical samples.

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Kazuki Morioka

Full Text Available A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA method was previously developed for foot-and-mouth disease (FMD viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01. In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01. Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection.

Development of sensitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for monitoring bisphenol-A in canned foods and beverages.

Science.gov (United States)

Lu, Yang; Peterson, Joshua Richard; Gooding, John Justin; Lee, Nanju Alice

2012-06-01

Enzyme-linked immunosorbent assays (ELISAs) are investigated in this work for the detection of bisphenol-A (BPA), a plastic monomer and a critical contaminant in food and environment. A series of polyclonal antibodies generated in vivo using BPA-butyrate-protein conjugate and BPA-valerate-protein conjugate were evaluated on direct and indirect competitive assay formats with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate, and BPA-2-valerate). Two indirect ELISAs and one direct ELISA exhibiting high sensitivity and specificity for BPA were developed. The 50 % inhibition of antibody binding (IC(50)) values were 0.78 ± 0.01-1.20 ± 0.26 μg L(-1), and the limits of detection as measured by the IC(20) values were 0.10 ± 0.03-0.20 ± 0.04 μg L(-1). The assays were highly specific to BPA, only displaying low cross-reactivity (3-8 % for the indirect assays and 26 % for the direct assay) for 4-cumylphenol (4-CP), at pH 7.2. The degree of cross-reaction of 4-CP was influenced by the antibody/hapten conjugate combination, assay conditions, and the assay format. The assays were optimized for the analysis of BPA in canned vegetables, bottled water and carbonated drinks. The limits of quantification for these three evaluated sample types, based on the spike and recovery data, were 0.5, 2.5, and 100 μg L(-1), respectively.

Surveillance of bluetongue virus antibody in goats using a recombinant VP7-based indirect ELISA in the coastal saline area of West Bengal, India

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Raj K. Singh

2009-06-01

Full Text Available The authors describe the serological surveillance of bluetongue virus (BTV group-specific antibody in goats of the coastal saline (Sunderban area of West Bengal, India. A recombinant viral protein 7 (rVP7-based indirect enzyme-linked immunosorbent assay (ELISA was used to detect the antibody in sera. The bacterially expressed rVP7 was purified by affinity chromatography. The diagnostic performance of the assay was assessed by comparing it to the commercially available previously validated competitive ELISA. Using the control and 1 202 test sera, the cut-off value, sensitivity and specificity as well as other performance characteristics e.g. the Youden index, efficiency, positive and negative predictive value and prevalence were estimated. Field-collected goat sera (n = 1 202 were tested and a serological prevalence rate of 47% was observed in the study area.

A comparative study of an elisa test and an indirect immunofluorescence test for serological diagnosis of Babesia bovis infection

International Nuclear Information System (INIS)

Martins, J.R.; Cheong, F.H.; Correa, B.L.; Radley, D.E.; Cereser, V.H.

1998-01-01

Detection of antibodies to Babesia bovis in cattle is essential for the understanding of the epidemiology of babesiosis and this study was concerned with comparing the indirect fluorescent antibody with the ELISA. Both assays gave rise to 100% sensitivity whilst the ELISA was shown to be marginally more specific at 98%. The ease of use and low cost of the ELISA would make it the more obvious choice in conducting future serological surveys for this parasite. (author)

Evaluation of an indirect ELISA for the diagnosis of Babesia bovis in Uruguay

International Nuclear Information System (INIS)

Cardozo, H.; Solari, M.A.; Etchebarne, J.

1992-01-01

In initially establishing the FAO/IAEA indirect ELISA for the detection of antibodies to Babesia bovis, the optical density (OD) values of sera from known positive or negative local cattle were compared to the OD values obtained from the negative and positive reference sera provided with the ELISA kit. The percentage of false positive and negative sera were 2.53% and 2.97% respectively. The cut-off values for the negative reference serum in the kit were compared with those of a local negative population. These values were found to be similar. The specificity of the test was evaluated by testing 30 sera from animals experimentally infected with Anaplasma marginale and 30 sera from animals infected with Babesia bigemina. These were no cross-reaction either between A. marginale and B. bovis or between B. bigemina and B. bovis. A serological survey using this ELISA kit was carried out on animals from an enzootic area and an area free from the vecot Boophilus microplus. 53 out of 282 animals (18.8%) in the enzootic area were positive whilst all the animals (113) from the free area were negative. This study would indicate that the FAO/IAEA ELISA kit has a sensitivity of around 98% and specificity of 97%. (author). 8 refs, 2 figs, 2 tabs

Comparison of a direct and indirect ELISA for quantitating antisperm antibody in semen.

Science.gov (United States)

Lynch, D M; Howe, S E

1987-01-01

A direct and an indirect quantitative ELISA for antisperm antibody were compared using the spermatozoa and cell-free seminal fluid of 66 infertile males. The normal concentration of sperm binding immunoglobulin was less than or equal to 1.5 fg Ig per spermatozoon for the indirect seminal plasma assay and less than or equal to 1.5 fg Ig per spermatozoon by the direct assay. Of the 66 infertile males, 21% (14/66) had elevated levels of antisperm antibody in their seminal plasma and 26% (17/66) had elevated levels bound directly to their spermatozoa. The direct correlation between the results of these assays was 94%. A simple linear regression analysis between the indirect and direct measurements of antisperm antibody resulted in a correlation coefficient of r = 0.907. There was no statistically significant difference between results from the direct and indirect methods of the patients as a group. However, there was evidence of autospecificity in a small percentage of males who had elevated levels of antisperm antibody by the direct assay that was not detected by the indirect assay using pooled donor spermatozoa.

Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples

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Samira Moraes Cunha de Mesquita

2015-06-01

Full Text Available ABSTRACT. Mesquita S.M.C., Mansur F.J., Nascimento E.R., Barreto M.L. & Kimura L.M.S. [Standardization and application of indirect ELISA for diagnosis of Mycoplasma bovis in bovine blood serum samples.] Padroniza- ção e aplicação de ELISA indireto para diagnóstico de Mycoplasma bovis em amostras de soro sanguíneo bovino. Revista Brasileira de Medicina Veterinária, 37(2:101-107, 2015. Universidade Federal Fluminense, Faculdade de Veteriná- ria, Rua Vital Brazil Filho, 64, Vital Brazil, Niterói, RJ 24230-340, Brasil. E-mail: samira.veterinaria@gmail.com International researchers presented results indicating frequent involvement of Mycoplasma spp. as a causative agent of mastitis in cattle, associating its presence with significant economic losses to farmers. Mycoplasma bovis is the species most reported and relevant, because it causes more severe disease. The level of antibodies against M. bovis remains high for several months and can be detected by ELISA. The aim of this work was to develop an indirect ELISA with whole cell antigen of M. bovis (strain Donetta PG 45 with subsequent application in bovine blood serum samples for detection of antibodies against M. bovis. The immunization of cows A and B by inoculating an immunogen against M. bovis to obtain hyperimmune blood serum was the first stage of this work, then the stage of standardization of ELISA was proceeded. The concentration of 2 mg of antigen/mL for coating the microtiter plates was decided by statistical analyses. The optical density value 0,2 was determined as the limit of reactivity discrimination of samples (the cut-off point. The hyperimmune blood serum sample of the cow A (collected 30 days after immunization was chosen as the positive control and, the fetal calf serum was chosen as negative control of the assay. In addition, the ideal optimal dilutions found for blood serum samples was 1:400 and for conjugate was 1:10.000 and the substrate used was the ortho

Development and evaluation of an indirect ELISA for detection of exfoliative toxin ExhA, ExhB or ExhC produced by Staphylococcus hyicus

DEFF Research Database (Denmark)

Andresen, Lars Ole

1999-01-01

Immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) confirmed previous reports that the Staphylococcus hyicus exfoliative toxins ExhA and ExhB are metalloproteins, and further indicated that ExhC is also a metalloprotein. An indirect ELISA. was developed for the detection of toxigenic...... strains as an alternative method to the use of phage typing for selection of S. hyicus isolates to be used in autogenous vaccine against exudative epidermitis in pigs. The indirect ELISA was evaluated by investigating the presence of toxin among a total of 655 S. hyicus isolates from 69 pig skin samples......, one from each of the 69 pig herds with outbreak of exudative epidermitis. Toxigenic S. hyicus were detected in 74% of the cases by ELISA. From each of the five cases, in which initially no toxigenic S. hyicus were found, a further 40 S. hyicus-like colonies were tested in ELISA. Testing of this number...

ELISA indireto para detecção de IgG antivírus da doença de Newcastle em soro de codorna Indirect ELISA for the detection of IgG specific to Newcastle disease virus in quail serum

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D.D. Oliveira

2007-10-01

Full Text Available An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100%. These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation.

Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies.

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Emmanuel Bréard

Full Text Available A newly developed Enzym Like Immuno Sorbant Assay (ELISA based on the recombinant nucleocapsid protein (N of Schmallenberg virus (SBV was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515 in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT or an indirect immuno-fluorescence assay (IFA. The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.

Development & standardization of an in-house IgM indirect ELISA for the detection of parvovirus B19 infections

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Kumaran Vadivel

2017-01-01

Interpretation & conclusions: The in-house IgM indirect ELISA was found to be simple with high sensitivity and specificity when compared with nPCR and could be used as an alternative to expensive commercial kits in resource-poor settings.

Field trial of brucellosis competitive ELISA

International Nuclear Information System (INIS)

Perez, B.; Rojas, M.

1998-01-01

2990 sera samples from cattle were tested for antibodies to Brucella abortus using 8 serological tests for. The tests used were Rose Bengal (RBT), Buffer Plate Agglutination Test (BPAT), Complement Fixation (CFT), 2 Indirect and 2 Competitive Enzyme Linked Immunosorbent Assays (ELISA). Bacteriological evaluation from milk was done also. All tests were compared with respect to diagnostic specificity in vaccinated herds which were considered to be Brucella-free. The diagnostic specificity of the Indirect and Competitive ELISA was greater than 99,8%. Estimates of relative sensitivity were obtained from infected herds. The diagnostic sensitivity of the Indirect ELISA was greater than 95,8% and for the Competitive ELISA between 98,8 and 100 %, the last value refers to the Competitive ELISA Prototype II (SLPS antigen/M84 Mab), which was found highly suitable to differentiate vaccinated from brucella-infected cattle. The use of C-ELISA II for monitoring bovine populations under an eradication programme is recommended. (author)

ELISA-BASE: an integrated bioinformatics tool for analyzing and tracking ELISA microarray data

OpenAIRE

White, Amanda M.; Collett, James R.; Seurynck-Servoss, Shannon L.; Daly, Don S.; Zangar, Richard C.

2009-01-01

Summary:ELISA-BASE is an open source database for capturing, organizing and analyzing enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Software Environment (BASE) database system.

Validation of an indirect ELISA for the diagnosis of Babesia bovis in cattle in Yucatan, Mexico

International Nuclear Information System (INIS)

Dominguez, A.J.L.; Rodriguez, V.R.I.; Oura, C.; Cob, G.L.A.

1998-01-01

The ELISA kit provided by the FAO/IAEA for the diagnosis of Babesia bovis was validated. In order to determine the appropriate ELISA cut-off point that would serve as the threshold between positive and negative samples, 119 serum samples from a Mexican Babesia-free zone were analyzed. The optimal cut-off point chosen was at 12% of the reactivity of the high positive control serum sample (PP) which resulted in a specificity of 97%. One hundred and ninety-six cattle from Wisconsin, USA, were introduced into Yucatan, Mexico, of which 181 were vaccinated with an attenuated live Babesia bovis vaccine; 15 animals remained as unvaccinated controls. Before and after vaccination all animals were bled and tested by enzyme linked immunosorbent assay (ELISA) and indirect fluorescence antibody test (IFAT). Both tests showed a high degree of correlation in their results. To evaluate an immune response to vaccination the optimal cut-off point chosen was 12% PP resulting in a sensitivity 99% and a specificity 95%. We concluded that the ELISA test has proved to be useful in Yucatan, Mexico for serological surveys and monitoring the efficiency o vaccination programmes. (author)

A Monoclonal Antibody-Based ELISA for Multiresidue Determination of Avermectins in Milk

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Wenxiao Jiang

2012-06-01

Full Text Available Due to the widespread use and potential toxicity of avermectins (AVMs, multi-residue monitoring of AVMs in edible tissues, especially in milk, has become increasingly important. With the aim of developing a broad-selective immunoassay for AVMs, a broad-specific monoclonal antibody (Mab was raised. Based on this Mab, a homologous indirect enzyme-linked immunosorbent assay (ELISA for the rapid detection of AVMs in milk was developed. Under the optimized conditions, the IC₅₀ values in assay buffer were estimated to be 3.05 ng/mL for abamectin, 13.10 ng/mL for ivermectin, 38.96 ng/mL for eprinomectin, 61.00 ng/mL for doramectin, 14.38 ng/mL for emamectin benzoate. Detection capability (CCβ of the ELISA was less than 5 ng/mL and 2 ng/mL in milk samples prepared by simple dilution and solvent extraction, respectively. The optimized ELISA was used to quantify AVMs in milk samples spiked at different amounts. The mean recovery and coefficient of variation (CV were 95.90% and 15.42%, respectively. The Mab-based ELISA achieved a great improvement in AVMs detection. Results proved this broad-selective ELISA would be useful for the multi-residue determination of AVMs in milk without purification process.

Validation of an indirect ELISA for the diagnosis of Babesia bovis in El Salvador

International Nuclear Information System (INIS)

Molina, G.; Cardona, D.A.

1998-01-01

Validation and a preliminary serological study of Babesia bovis was made in El Salvador, using the indirect ELISA kit provided by the Joint FAO/IAEA Division of the International Atomic Energy Agency. Sera were collected from 545 cattle involving 10 regions of the country and various ages of cattle between 8 and 16 months. These were tested from May 1993 to February 1994. A 79.5% prevalence was found, but with a wide range from (5.8-100%), explained by different farm managing systems and different breeds. (author)

The time course of the specific antibody response by various ELISAs in pigs experimentally infected with Toxoplasma gondii

DEFF Research Database (Denmark)

Lind, Peter; Haugegaard, J.; Wingstrand, Anne

1997-01-01

With the aim of developing routine serological tests for monitoring the Toxoplasma infection status of Danish swine herds, four ELISAs based on tachyzoite antigen were set up: (1) an indirect ELISA for IgG-antibody; (2) a blocking ELISA for antibody to the membrane antigen, P-30; (3) an indirect ...

Performance of an ELISA and Indirect Immunofluorescence Assay in Serological Diagnosis of Zoonotic Cutaneous Leishmaniasis in Iran

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Bahador Sarkari

2014-01-01

Full Text Available Serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (VL and considered as a routine method for diagnosis of VL while these methods are not properly evaluated for diagnosis of cutaneous leishmaniasis (CL. This study aimed to assess the performance of indirect immunofluorescent-antibody test (IFA and enzyme-linked immunosorbent assay (ELISA for serodiagnosis of cutaneous leishmaniasis in Iran. Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera from healthy controls along with 50 sera from non-CL patients were collected. Antigen was prepared from promastigotes and amastigotes of Leishmania major. IFA was used to detect anti-Leishmania IgG while ELISA was used to detect anti-Leishmania IgM, total IgG, or IgG subclasses (IgG1 and 4. ELISA, for detection of total IgG and IgM, showed sensitivity of 83.6% and 84.7% and specificity of 62.7% and 54.6%, respectively. Sensitivity and specificity of ELISA for detecting IgG1 and IgG4 were 64%, 75% and 85%, 49%, respectively. Sensitivity and specificity of IFA were 91.6% and 81%. Conclusion. Findings of this study demonstrated that serological test, especially IFA, can be used for proper diagnosis of CL.

His-tag ELISA for the detection of humoral tumor-specific immunity

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Disis Mary L

2008-05-01

Full Text Available Abstract Background The application of high throughput molecular techniques such as SEREX are resulting in the identification of a multitude of tumor associated antigens. As newly identified antigens are incorporated into a variety of clinical trials, standardization of immunologic monitoring methods becomes increasingly important. We questioned whether mammalian cell expression of a histadine-linked human protein could be used to produce antigen suitable for detecting tumor-specific humoral immunity and whether such an assay could be amenable to standardization for clinical use. Methods We designed a his-tagged capture ELISA based on lysate from genetically engineered CHO cells for detection of antibodies to insulin-like growth factor binding protein 2, a novel tumor antigen. We performed technical and preliminary clinical validation studies, including comparison to a standard indirect ELISA based on commercially prepared recombinant antigen. Results The his-tagged capture ELISA could be standardized. Precision experiments resulted in CVs 2 values of 0.99. In comparison to Western blot analysis, his-tag and indirect ELISA accurately identified 88% and 93% of samples, respectively. Sample concordance between capture and indirect assays was highly significant (p = 0.003. Furthermore, significantly greater levels of IGFBP-2 antibody immunity were found in cancer patients compared to normal controls (p = 0.008. Conclusion A genetically engineered cell lysate based ELISA can be amenable to standardization and can detect increased levels of antibody immunity to tumor-associated antigen in cancer patients compared to non tumor-bearing healthy controls.

Serological response to an indirect and a competitive elisa in heifers vaccinated with Brucella abortus strain 19

International Nuclear Information System (INIS)

Carrasco, E.A.; Uzal, F.A.; Echaide, S.

1998-01-01

The different serologic techniques for bovine brucellosis diagnosis have different abilities to detect antibodies after vaccination with Brucella abortus strain 19. The humoral response in heifers vaccinated with Brucella abortus strain 19 was evaluated by using several serologic techniques. In the experimental field of INTA, Pilcaniyeu, Rio Negro province, sixteen 5 months old heifers were vaccinated subcutaneously with a standard dose (2ml, containing 20x10 9 to 10x10 9 living organisms) of Brucella abortus strain 19. Sera from all the heifers were obtained on 18 occasions (one 87 days before vaccination, one immediately before vaccination and on 16 occasions after vaccination, during 488 days) and analyzed by buffered plate antigen test, rose bengal test, standard tube agglutination test, 2-mercaptoetanol test, complement fixation test, indirect ELISA, and competitive ELISA. Prior vaccination, 100% of the heifers gave negative results in all the techniques used, while 100% of them gave positive reaction in the first sampling after vaccination to all the techniques, with the exception of standard tube agglutination test that showed agglutinating titters of 1/100 or higher (positive threshold) in only 71.4% of the heifers. The indirect ELISA technique showed a reducing percentage of positive animals up until 316 days after vaccination, after which positive results were obtained. The competitive ELISA gave positive results in a variable number of heifers up to 253 days after vaccination when 100% of the sera were negative to this technique. Buffered plate antigen test was the technique that gave positive results for a longest period, being 100% of the animals negative to this technique at 450 days after vaccination. The other serological techniques assayed gave positive results during variable periods of time, intermediate between standard tube agglutination test and buffered plate antigen test. Although the present results were obtained from a limited number of

Ducks as a potential reservoir for Pasteurella multocida infection detected using a new rOmpH-based ELISA.

Science.gov (United States)

Liu, Rongchang; Chen, Cuiteng; Cheng, Longfei; Lu, Ronghui; Fu, Guanghua; Shi, Shaohua; Chen, Hongmei; Wan, Chunhe; Lin, Jiansheng; Fu, Qiuling; Huang, Yu

2017-07-28

Pasteurella multocida is an important pathogen of numerous domestic poultry and wild animals and is associated with a variety of diseases including fowl cholera. The aim of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant outer-membrane protein H (rOmpH) for detection of anti-P. multocida antibodies in serum to determine their prevalence in Chinese ducks. The P. multocida ompH gene was cloned into pET32a, and rOmpH was expressed in Escherichia coli BL21 (DE3). Western blotting revealed that purified rOmpH was recognized by duck antisera against P. multocida, and an indirect ELISA was established. During analysis of serum samples (n=115) from ducks, the rOmpH ELISA showed 95.0% specificity, 100% sensitivity and a 92.0% κ coefficient (95% confidence interval 0.844-0.997) as compared with a microtiter agglutination test. Among 165 randomly selected serum samples, which were collected in 2015 and originated from six duck farms across Fujian Province, China, anti-P. multocida antibodies were detected in 22.42% of apparently healthy ducks, including 25 of 90 sheldrakes (27.8%), eight of 50 Peking ducks (16.0%) and four of 25 Muscovy ducks (16%). Overall, the data suggest that rOmpH is a suitable candidate antigen for the development of an indirect ELISA for detection of P. multocida in ducks; moreover, our results showed that ducks could serve as a potential reservoir for P. multocida infection.

Comparison of Rose Bengal Plate Agglutination, Standard tube agglutination and Indirect ELISA tests for detection of Brucella antibodies in Cows and Buffaloes

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S. N. Ghodasara

2010-04-01

Full Text Available A total of 180 serum samples (107 cows, 73 buffaloes from cases of abortion and various reproductive disorders were collected for detection of Brucella antibody by Rose Bengal Plate Agglutination Test (RBPT, Serum Tube Agglutination Test (STAT and indirect- ELISA (i-ELISA. The overall prevalence of brucellosis by RBPT, STAT and i-ELISA were 11.21%, 16.00% and 24.30% in cows 9.59%, 12.33% and 26.03% in buffaloes respectively. Overall seroprevalence of Brucellosis in cases of abortion, R.O.P. by RBPT, STAT and i-ELISA were 11.32%, 16.04% and 32.08% respectively. When three serological tests were compared, seropositivity was found highest by i-ELISA (25%, followed by STAT (14.45% and RBPT (10.56%. The results shows higher prevalence of brucellosis in cases of abortion and R.O.P., while at lower level from various reproductive disorders as detected serologically indicating endemicity of the infection in villages around Anand city, Gujarat. [Vet. World 2010; 3(2.000: 61-64

Comparative evaluation of competitive ELISA test in Colombian cattle

International Nuclear Information System (INIS)

Marino, O.; Rueda, E.; Sedano, L.; Zuniga, I.; Calderon, C.; Ortega, A.; Puentes, A.

1998-01-01

In order to contribute to the definition of the best ELISA test for screening and differential diagnosis of Brucella abortus to be applied for control programmes, a total of 2971 sera from Colombian cattle were tested for brucellosis. Conventional agglutination tests, Buffered Plate antigen test (BPAT) and Rose Bengal (RB) as well as Complement Fixation test (CFT) (Alton, et al. 1988) were used comparatively. Radial immunodiffusion test (RID) was also performed to all sera. The sera were also tested using four different ELISAs: indirect ELISA from FAO/IAEA and the indirect ELISA modified by Nielsen, et al. 1992 as well as two competitive ELISAs: one competitive ELISA used B. abortus O-polysaccharide antigen and an enzyme conjugated monoclonal to the O-polysaccharide for competition and detection. The second competitive ELISA used lipopolysaccharide (sLPS) antigen, a different monoclonal antibody for competition but also specific for the O-polysaccharide and a commercially available goat anti-mouse IgG enzyme conjugate for detection. The sera were analyzed based on its population status, 987 positive obtained from Brucella abortus infected herds based on clinical and/or bacteriological evidence and a high prevalence of brucellosis, CFT percentage of positive animals in the herd was greater than 5%. Eight hundred sixty six (866) negative sera from non-vaccinated cattle from a brucellosis free area and 1118 negative sera obtained from reglamentary vaccinated areas under a free herd program. Initial cut-off values were derived using negative serum samples. The diagnostic sensitivity and specificity was defined from frequency histograms based on this cut-off values and using 2x2 tables, corresponding confidence limits (95%) were calculated. The data were also analysed using signal detection analysis (ROC). Kappa statistics was determined for all tests and populations, accuracy was used as index of comparison to evaluate different assays. The data support the initial

TOXOCARIASIS: SEROLOGICAL DIAGNOSIS BY INDIRECT ANTIBODY COMPETITION ELISA Diagnóstico sorológico da toxocaríase através do método de ELISA indireto de competição

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Cáris Maroni NUNES

1999-03-01

Full Text Available Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%.A toxocaríase, uma emergente zoonose, é uma síndrome clínica decorrente da infecção humana por larvas de Toxocara canis e Toxocara cati, parasitas intestinais de cães e gatos, respectivamente. A dificuldade de detecção das larvas nos tecidos e a inespecificidade dos sinais clínicos tornam os testes sorológicos os meios diagnósticos mais adequados. Os antígenos excretores-secretores de T. canis empregados nos testes sorológicos, embora tenham contribuído para melhorar a especificidade destes, apresentam reações cruzadas com diversos parasitas

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

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Tanjina Akter

Full Text Available M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens.An Indirect Enzyme-Linked Immunosorbent Assay (ELISA for detection of M10 in biological fluids was developed using pharmaceutical grade synthetic M10 as a calibrator and commercially available anti-c-Met C12 antibody.M10 ELISA specifically detected in plasma M10, but not a scrambled peptide, following a single intraperitoneal administration of M10 (1mg/kg to mice. The detection limit was 9.6 ng/ml, and the measuring limit was between 15 ng/ml and 200 ng/ml. The recovery limits of M10 were between 80% and 120%; intra-assay coefficient of variation was between 5.3% and 6.3%; inter-assay coefficient of variation was between 5.0% and 8.0% over the buffer concentration tested in the range from 15 ng /ml to 250 ng /ml. The peak of M10 concentration following a single intraperitoneal injection (1mg/kg was achieved within 6 hours and declined to minimal levels by 48 hours. The experimentally obtained half-life for M10 was comparable to the theoretically predicted half-life for M10.We have established a highly sensitive ELISA to detect the antifibrotic peptide M10 in plasma samples, which should prove to be a novel tool to study the pharmacokinetics and efficacy of M10 in the treatment of fibroproliferative disorders.

Validation of indirect ELISA systems for the serodiagnosis of bovine trypanosomosis in endemic areas of Kenya

International Nuclear Information System (INIS)

Ouma, J.O.; Mwangi, J.M.; Mdachi, R.; Njiru, Z.K.; Ndung'u, J.M.

2000-01-01

The present study was aimed at validating the performance of four indirect ELISA systems developed for the detection of anti-trypanosomal antibodies in bovine serum. The assay systems employ the use of either native or denatured crude lysate antigens prepared from Trypanosoma congolense (Tc) and Trypanosoma vivax (Tv). Assay systems were designated as TcAGd, TcAGn, TvAGd or TvAGn depending on the trypanosome species from which the antigen was prepared (Tc or Tv) and whether the antigen was denatured (AGd) or native (AGn). The microtitre plates used were precoated with the above antigen preparations at the International Atomic Energy Agency laboratories in Vienna, Austria and shipped to Kenya. Diagnostic sensitivities and specificities were assessed using both known infected and uninfected bovine sera, respectively. All the positive samples were collected from cattle kept in trypanosomosis endemic areas of Galana and Ukunda in Coast province and Mfangano Island in Nyanza province of Kenya. Known negative sera were obtained from animals kept in a non-trypanosomosis endemic area in Muguga, near Nairobi, Kenya. Assay sensitivity ranged from 86% to 97%, while specificity was between 82% and 100% depending on the assay system used. Systems employing denatured antigens had slightly higher, diagnostic sensitivity and specificity. The study has demonstrated that antigen precoated plates are useful in circumventing the problem of antigen instability. However, further studies need to be undertaken using a larger sample size to determine if there are any significant differences between plates pre-coated with native and denatured antigens. The present version of indirect ELISA is a useful epidemiological tool and can be incorporated in mapping out the extent of disease. (author)

Comparative diagnostic efficacy of recombinant LLO and PI-PLC-based ELISAs for detection of listeriosis in animals.

Science.gov (United States)

Suryawanshi, Rahul D; Malik, Satya Veer Singh; Jayarao, Bhushan; Chaudhari, Sandeep P; Savage, Emily; Vergis, Jess; Kurkure, Nitin V; Barbuddhe, Sukhadeo B; Rawool, Deepak B

2017-06-01

The present study for the first time evaluates the serodiagnostic efficacy of two recombinant antigens namely, listeriolysin O (rLLO) and phosphatidyl-inositol phospholipase C (rPI-PLC). Indirect ELISA with the above recombinant antigens was used on samples collected from bovines (n=106), goats (n=138) and pigs (n=92) having either a history of abortion, emaciation and/or apparently healthy animals. Isolation of Listeria was attempted from the blood samples using USDA-FSIS method. On screening of test sera by rLLO-based ELISA, antibodies against anti-listeriolysin O (ALLO) were observed in goats (22.46%), bovines (15.10%) and pigs (16.31%). As advocated, after adsorption of positive serum samples with streptolysin O (SLO), the seropositivity for ALLO was marginally reduced (p>0.05) in goats (21.73%) and bovines (10.38%), whereas, in pigs the reduction (5.43%) was significant (pPLC-based ELISA revealed higher non-specific seropositivity for antilisterial antibodies in goats (45.65%), bovines (31.13%) and pigs (8.69%). Further, on comparing the seropositivity with isolation rate, of the 16 animals that were culturally-positive for L. monocytogenes, 15 showed ALLO positivity in unadsorbed as well as SLO-adsorbed sera by rLLO-based ELISA, however, rPI-PLC-based ELISA could detect seropositivity in only 5 animals. Moreover, rPI-PLC-based ELISA also showed seropositivity in those animals (7/30) that were culturally positive for other Listeria spp. In conclusion, rLLO can serve as a better antigen than rPI-PLC in ELISA for the serodiagnosis of listeriosis in animals; however, prior adsorption of test sera with SLO is required to avoid false positive results. Copyright © 2017 Elsevier B.V. All rights reserved.

Mobile phone based ELISA (MELISA).

Science.gov (United States)

Zhdanov, Arsenii; Keefe, Jordan; Franco-Waite, Luis; Konnaiyan, Karthik Raj; Pyayt, Anna

2018-04-30

Enzyme-linked immunosorbent assay (ELISA) is one of the most important technologies for biochemical analysis critical for diagnosis and monitoring of many diseases. Traditional systems for ELISA incubation and reading are expensive and bulky, thus cannot be used at point-of-care or in the field. Here, we propose and demonstrate a new miniature mobile phone based system for ELISA (MELISA). This system can be used to complete all steps of the assay, including incubation and reading. It weighs just 1 pound, can be fabricated at low cost, portable, and can transfer test results via mobile phone. We successfully demonstrated how MELISA can be calibrated for accurate measurements of progesterone and demonstrated successful measurements with the calibrated system. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

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Miriam F. Rebouças

2013-11-01

Full Text Available Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA, a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

Evaluation of a blocking ELISA for screening of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus

DEFF Research Database (Denmark)

Sørensen, K.J.; Bøtner, Anette; Madsen, E.S.

1997-01-01

A blocking Elisa was developed for the detection of antibodies against PRRS virus with a view to satisfying the need for examination of blood samples on a large scale. The test was evaluated in comparison with an indirect Elisa and the immunoperoxidase monolayer assay. The blocking Elisa...... was sensitive and specific. It had a higher capacity and was cheaper to perform than the immunoperoxidase monolayer assay and the indirect Elisa. It was comparable to the immunoperoxidase monolayer assay and better than the indirect Elisa in detecting antibodies formed early after infection, and it was superior...... to both the immunoperoxidase monolayer assay and the indirect Elisa in detecting antibodies at a late stage of infection....

USE OF AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA TO DETECT ANTIBODIES IN AYU (Plecogiossus altivelis VACCINATED BY IMMERSION ADMINISTRATION

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. 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

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA and indirect ELISA Detecção de anticorpos anti-Toxoplasma gondii por meio das técnicas de Imunofluorescência Indireta e ELISA Indireto em primatas experimentalmente e naturalmente infectados

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Andréa Bouer

2010-03-01

Full Text Available The Indirect Fluorescence Assay (IFA and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI with the IFA (IgG and IgM and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI. Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2% seropositive animals for this parasite and 149 (70.8% negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.Detectou-se anticorpos das classes IgG e IgM anti-Toxoplasma gondii em primatas experimentalmente e naturalmente infectados, utilizando-se como técnicas comparativas a RIFI e o ELISA-teste. No grupo dos primatas experimentalmente infectados, anticorpos de valor diagnóstico foram detectados a partir do 9º dia de infecção tanto na RIFI (IgG e IgM como no ELISA-IgG. O ELISA IgM detectou anticorpos a partir do 3º dia de infecção até o final do experimento (102 dias pós-infecção. Dos 209 soros dos primatas naturalmente infectados, de diversos zoológicos do Estado de São Paulo, 64,59 e 67,94% mostraram-se positivos na RIFI-IgG e no ELISA-IgG, respectivamente. O

An ELISA test for the detection of antibodies to Legionella pneumophila.

OpenAIRE

Wreghitt, T G; Nagington, J; Gray, J

1982-01-01

An enzyme-linked immunosorbent assay (ELISA) test has been developed to detect antibodies to Legionella pneumophila serogroup 1. There is good correlation between indirect fluorescent antibody (IFA) and ELISA titres but ELISA is more sensitive.

Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs.

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Kerstin Fischer

Full Text Available Hendra virus (HeV and Nipah virus (NiV belong to the genus Henipavirus in the family Paramyxoviridae. Henipavirus infections were first reported in the 1990's causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus Pteropus have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G proteins as well as the full-length NiV nucleocapsid (N protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses.

The use of monoclonal antibodies in competitive ELISA for the detection of antibodies to rinderpest and peste des petits ruminants viruses

International Nuclear Information System (INIS)

Anderson, J.; McKay, J.A.; Butcher, R.N.

1991-01-01

A monoclonal antibody against the haemagglutinin of rinderpest virus has been used in a competitive ELISA (C-ELISA) for the detection of antibodies to rinderpest virus in cattle, sheep, goat and game sera. Unlike the indirect ELISA and the virus neutralisation test (VNT), the C-ELISA detects only antibodies to rinderpest virus and gives no cross-reactivity with antibodies to peste des petits ruminants (PPR) virus. Antibodies to a wide range of strains of rinderpest virus have been detected using this assay, suggesting its suitability for both sero-monitoring and sero-surveillance. Analysis of C-ELISA results from the examination of field sera shows a much greater separation of negative and positive populations as compared to the indirect ELISA. A further monoclonal antibody against the H protein of PPR has also been found suitable for use in a C-ELISA for the detection of antibodies to PPR virus. The use of these two C-ELISA's has made possible rapid differential sero-diagnosis without recourse to cross-VNT testing. The use of monoclonal antibody-based assays will allow much greater standardisation of rinderpest and PPR diagnosis, and following field-trials the C-ELISA will replace the indirect ELISA for sero-monitoring throughout the Pan African Rinderpest Campaign. (author). 3 refs, 6 figs, 1 tab

Spatial distribution, risk factors and haemato-biochemical alterations associated with Theileria equi infected equids of Punjab (India) diagnosed by indirect ELISA and nested PCR.

Science.gov (United States)

Sumbria, Deepak; Singla, L D; Kumar, Sanjay; Sharma, Amrita; Dahiya, Rajesh K; Setia, Raj

2016-03-01

Equine piroplasmosis is a febrile, tick-borne disease of equids predominately caused by obligatory intra-erythrocytic protozoa Theileria equi in the Indian sub-continent. A cross-sectional study was carried out on 464 equids (426 horses and 38 donkeys/mules) in Punjab, India to assess the level of exposure to equine piroplasmosis by 18S rRNA gene nested polymerase chain reaction (nPCR) and equine merozoite antigen-2 (EMA2) indirect-ELISA (enzyme linked immunosorbent assay), to investigate risk factors and haemato-biochemical alterations associated with the infection. The endemicity of the disease was confirmed by positive PCR amplification in 21.77% and positive antibody titers in 49.78% equid samples. There was a fair agreement between these two diagnostic techniques (Kappa coefficient=0.326). The spatial distribution analysis revealed an increasing trend of T. equi prevalence from north-eastern to south-western region of Punjab by both the techniques correspondingly, which proffered a direct relation with temperature and inverse with humidity variables. The relatively prominent risk factor associated with sero-positivity was the presence of other domestic animals in the herd, while the propensity of finding a positive PCR amplification was higher in donkeys/mules, animal kept at unorganised farm or those used for commercial purposes as compared to their counterparts. There was a significant increase in globulins, gamma glutamyl-transferase, total bilirubin, direct bilirubin, indirect bilirubin, glucose levels and decrease in total erythrocyte count, haemoglobin, packed cell volume by animals, which were revealed positive by nPCR (may or may not positive by indirect-ELISA) and increase in creatinine, total bilirubin, direct bilirubin, glucose and decrease in total erythrocytes count by animals, which were revealed positive by indirect-ELISA (alone). To our knowledge, this study, for the first time, brings out a comprehensive report on the status on spatial

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres Indirect ELISA for the serological diagnosis of visceral leishmaniasis in wild canids

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Paulo R.B. Ferreira

2013-04-01

assay (ELISA for the serodiagnosis of visceral leishmaniasis (VL in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus, three from hoary foxes (Lycalopex vetulus, and two from crab-eating foxes (Cerdocyon thous. Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12 and three (03/12 seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001 and moderate (Kappa = 0.8; p<0.0015 agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.

Development of an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against wild-type porcine reproductive and respiratory syndrome from the vaccine strain TJM-F92 based on a recombinant Nsp2 protein.

Science.gov (United States)

Wang, X X; Wang, F X; Li, Z G; Wen, Y J; Wang, X; Song, N; Wu, H

2018-01-01

An accurate ELISA method to differentiate pigs infected with wild-type porcine reproductive and respiratory syndrome (PRRSV) strains from vaccinated ones would help to monitor PRRSV vaccination compliance. The recombinant protein GST-d120aa derived from the continuous deletion of 120 amino acids in the non-structural protein 2 region of the modified-live vaccine strain TJM-F92 was used to develop an indirect enzyme-linked immunosorbent assay (d120-ELISA) for differentiating serum antibodies against TJM-F92 from other PRRSV strains. At the optimized cut-off value which was calculated at an S/P of 0.25, it yielded a sensitivity of 90.7% and a specificity of 95.1%. Cross-reactivity tests suggested that the d120-ELISA was PRRSV-specific. Coefficient of variations of the repeatability tests ranged between 1.41-17.02%. The results suggest that the d120-ELISA is suitable for differentiating animals infected with wild-type strains from those immunized with MLV TJM-F92. Copyright © 2017. Published by Elsevier B.V.

An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats.

Science.gov (United States)

Wang, Yu; Nie, Huaming; Gu, Xiaobin; Wang, Tao; Huang, Xing; Chen, Lin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

2015-09-15

Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 μg/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis. Copyright © 2015 Elsevier B.V. All rights reserved.

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

Science.gov (United States)

Wang, Jing-Yu; Wu, Ning; Liu, Wan-Hua; Ren, Juan-Juan; Tang, Pan; Qiu, Yuan-Hao; Wang, Chi-Young; Chang, Ching-Dong; Liu, Hung-Jen

2014-01-01

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

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Seyed Ali Mirhosseini

Full Text Available ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA and polymerase chain reaction (PCR have been developed. In this study, recombinant FliC (rFliC was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC. The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Development of an ELISA and Immunochromatographic Strip for Highly Sensitive Detection of Microcystin-LR

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Liqiang Liu

2014-08-01

Full Text Available A monoclonal antibody for microcystin–leucine–arginine (MC-LR was produced by cell fusion. The immunogen was synthesized in two steps. First, ovalbumin/ bovine serum albumin was conjugated with 6-acetylthiohexanoic acid using a carbodiimide EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/ NHS (N-hydroxysulfosuccinimide reaction. After dialysis, the protein was reacted with MC-LR based on a free radical reaction under basic solution conditions. The protein conjugate was used for immunization based on low volume. The antibodies were identified by indirect competitive (icELISA and were subjected to tap water and lake water analysis. The concentration causing 50% inhibition of binding of MC-LR (IC50 by the competitive indirect ELISA was 0.27 ng/mL. Cross-reactivity to the MC-RR, MC-YR and MC-WR was good. The tap water and lake water matrices had no effect on the detection limit. The analytical recovery of MC-LR in the water samples in the icELISA was 94%–110%. Based on this antibody, an immunochromatographic biosensor was developed with a cut-off value of 1 ng/mL, which could satisfy the requirement of the World Health Organization for MC-LR detection in drinking water. This biosensor could be therefore be used as a fast screening tool in the field detection of MC-LR.

Detection of Fusarium verticillioides by PCR-ELISA based on FUM21 gene.

Science.gov (United States)

Omori, Aline Myuki; Ono, Elisabete Yurie Sataque; Bordini, Jaqueline Gozzi; Hirozawa, Melissa Tiemi; Fungaro, Maria Helena Pelegrinelli; Ono, Mario Augusto

2018-08-01

Fusarium verticillioides is a primary corn pathogen and fumonisin producer which is associated with toxic effects in humans and animals. The traditional methods for detection of fungal contamination based on morphological characteristics are time-consuming and show low sensitivity and specificity. Therefore, the objective of this study was to develop a PCR-ELISA based on the FUM21 gene for F. verticillioides detection. The DNA of the F. verticillioides, Fusarium sp., Aspergillus sp. and Penicillium sp. isolates was analyzed by conventional PCR and PCR-ELISA to determine the specificity. The PCR-ELISA was specific to F. verticillioides isolates, showed a 2.5 pg detection limit and was 100-fold more sensitive than conventional PCR. In corn samples inoculated with F. verticillioides conidia, the detection limit of the PCR-ELISA was 1 × 10 4 conidia/g and was also 100-fold more sensitive than conventional PCR. Naturally contaminated corn samples were analyzed by PCR-ELISA based on the FUM21 gene and PCR-ELISA absorbance values correlated positively (p PCR-ELISA developed in this study can be useful for F. verticillioides detection in corn samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

and Indigenous Indirect Enzyme-Linked Immunosorbent Assay

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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%.

DEVELOPMENT AND STANDARDIZATION OF AN INDIRECT ELISA FOR THE DIAGNOSIS MAEDI-VISNA IN SHEEP DESENVOLVIMENTO E PADRONIZAÇÃO DE UM ELISA INDIRETO PARA DIAGNÓSTICO DE MAEDI VISNA EM OVINOS

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Tânia Valeska Medeiros Dantas

2008-04-01

Full Text Available The objective of this work was to develop and standardize an indirect ELISA for diagnosis of Maedi Visna (MV infection. The antigen was produced from the supernatants of caprine synovial membrane (CSM cell monolayers inoculated with Maedi Visna virus (MVV, strain K1514, by cycles of freezing and thawed, and clarified by centrifugation at 3000 g for 40 min. The clarified suspension was precipitated with PEG 8000 and centrifugated at 12000 g for 60 min; the pellet was resuspended in buffer TNE and layered onto a sucrose cushion by centrifugation at 42000 g for 105 min. The pellet was resuspended in PBS containing phenylmethylsulphonyl fluoride (PMSF. The ELISA was performed in 96 wells plates, incubated for 1 h at 37 ºC. The reaction was detected by incubation with the enzyme substrate and o-phenylenediamine (OPD during 15 min.  The tests were compared using 175 sera samples. The concentration of the antigen was 2 µg/mL and the best dilution of the sera was 1:100. ELISA detected more positive samples (22.9% than AGID (6.3% and presented a better sensitivity than AGID and, although the specificity was below the expected, it could already be recomended in the diagnosis of MVV infection.KEY WORDS: ELISA, MaediVisna, serologic diagnosis, sheep lentivirus. O objetivo deste trabalho foi desenvolver e padronizar um ELISA indireto para diagnóstico de Maedi Visna (MV. Produziu-se o antígeno em sobrenadantes de cultivo celular de membrana sinovial caprina (MSC inoculado com o Maedi Visna Vírus (MVV cepa K1514, que passou por ciclos de congelamento e descongelamento, sendo logo após clarificado

Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection

International Nuclear Information System (INIS)

Li, Dongyang; Ying, Yibin; Wu, Jian; Niessner, Reinhard; Knopp, Dietmar

2013-01-01

We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL −1 ). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immuno detection. (author)

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

Directory of Open Access Journals (Sweden)

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.

Antibodies to UV irradiated DNA: the monitoring of DNA damage by ELISA and indirect immunofluorescence

Energy Technology Data Exchange (ETDEWEB)

Wani, A A; Gibson-D' Ambrosio, R E; D' Ambrosio, S M [Ohio State Univ., Columbus (USA). Dept. of Radiology

1984-10-01

The enzyme-linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV-irradiated single-stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC). Fifty per cent of the maximum antibody binding was observed at a 1-5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody, whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the UV dose to DNA and the concentration of antigen immobilized on the plate. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establishes that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated cells.

[A double antibody sandwich ELISA based assay for titration of severe fever with thrombocytopenia syndrome virus].

Science.gov (United States)

Liu, Lin; Zhang, Quan-Fu; Li, Chuan; Li, Jian-Dong; Jiang, Xiao-Lin; Zhang, Fu-Shun; Wu, Wei; Liang, Mi-Fang; Li, De-Xin

2013-06-01

To develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA. A double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method. The results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949). The novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.

Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

Comparative Diagnosis of Serum IgG1 and Coproantigen ELISA for Fasciolosis Detection of Goats in Mexico

Science.gov (United States)

Molina-Mendoza, Pedro; Hernández-Guzmán, Karina; Olivares-Pérez, Jaime; Sarracent-Pérez, Jorge; Zumaquero-Ríos, José

2016-01-01

The objective of present study was to determine the prevalence of natural caprine fasciolosis in the Mixteca region of Mexico using coproantigen and serum IgG1 ELISA tests for comparative purposes. A total of 1070 serum and faecal samples were analyzed for IgG1 antibodies and coproantigens, using ELISA with E/S products as antigen and a monoclonal antibody-based sandwich ELISA. Prevalence of 73.46% was found using the serological ELISA and a percentage of 77.20 was found for coproantigen ELISA. The diagnostic sensitivity and specificity for serum ELISA were 86.7% and 96.4%, and for the coproantigen ELISA they were 93.1% and 97.8%, respectively. The seropositive samples were further categorized as low, medium, or high positivity. Results show a great proportion of low and medium positive goats when the serum ELISA test was used. Correlation coefficients between coproantigens and seropositivity were statistically significant (P < 0.01) for low seropositivity (r = 0.93) and medium seropositivity (r = 0.84). The accuracy of faecal antigen ELISA was higher compared to indirect ELISA serological test. Two ELISAs were shown to be useful for demonstrating the current status of F. hepatica infection in the endemic areas and can be employed in studies on epidemiology as well as anthelmintics treatment for preventing economic loss and the risk of transmission to humans. PMID:27563665

Teste de ELISA indireto para o diagnóstico sorológico de pitiose Indirect ELISA for the serodiagnostic of pythiosis

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Janio M. Santurio

2006-03-01

Full Text Available A pitiose, doença granulomatosa de eqüinos causada pelo oomiceto Pythium insidiosum, tem como característica a evolução rápida seguida de morte dos animais. Estas mortes muitas vezes são causadas por diagnósticos errôneos ou demorados quando os doentes já não respondem ao tratamento. Este trabalho teve por objetivo a padronização do ensaio imunoenzimático indireto (ELISA para diagnóstico sorológico de pitiose em eqüinos e coelhos, visando a diminuição de erros e de tempo necessário para o diagnóstico. Para o desenvolvimento e validação do teste foram utilizadas 72 amostras de soro de eqüinos saudáveis e 44 soros de eqüinos com pitiose confirmada. Os resultados da validação do ELISA para eqüinos foram: sensibilidade 97,72%, especificidade 90,27%, valor preditivo positivo 86%, valor preditivo negativo 98,4% e eficiência de 93,1%. Para coelhos, o teste foi padronizado com 48 amostras de soro de animais saudáveis e 24 amostras de coelhos imunizados com antígenos de P. insidiosum. Os resultados foram: sensibilidade 91,66%, especificidade 95,83%, valor preditivo positivo 91,66%, valor preditivo negativo 95,83% e eficiência de 94,44%. Os resultados deste trabalho demonstram que o ensaio imunoenzimático indireto é um método seguro e eficaz para o diagnóstico sorológico da pitiose.Pythiosis is a granulomatous disease caused by the oomycete Pythium insidiosum that affects humans and animals, especially horses. Deaths are very often the consequence of incorrect or late diagnosis when animals no longer respond to treatment. This study aimed standardization of the ELISA assay for the serodiagnostic of pythiosis in horses and rabbits, in order to minimize errors and delays in the diagnosis of the disease. Sera of 72 healthy and 44 of by pythiosis affected horses were used for development and evaluation of the test. The ELISA for equine diagnostic showed 97.72% sensitivity, 90.27% specificity, 86% positive predictive value

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

DEFF Research Database (Denmark)

Dutaud, Dominique; Aubry, Laurent; Henry, Laurent

2002-01-01

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination...

Field trial of a brucellosis competitive enzyme linked immunoabsorbent assay (ELISA)

International Nuclear Information System (INIS)

Samartino, L.E.; Gregoret, R.J.; Sigal, G.

1998-01-01

The purpose of this study was to evaluate the performance of a competitive ELISA system for the diagnosis of bovine brucellosis in comparison to conventional aerological tests routinely used in Argentina. A total of 2.500 serum samples, comprising Brucella-free herds, vaccinated cattle and naturally infected animals, was tested by the following tests: buffered plate agglutination, Rose Bengal, 2-mercaptoethanol, complement fixation, and indirect and competitive ELISAs. Specificity and relative sensitivity at each test were determined. The competitive ELISA was considered suitable for detection of vaccinated animals and had higher specificity than the other tests. The results point to the potential use of the test as a complementary assay in the brucellosis control programme in Argentina. (author)

Comparing validation of four ELISA-systems for detection of Salmonella derby- and Salmonella infantis-infected pigs.

Science.gov (United States)

Roesler, Uwe; Szabo, Istvan; Matthies, Claudia; Albrecht, Kerstin; Leffler, Martin; Scherer, Kathrin; Nöckler, Karsten; Lehmann, Jörg; Methner, Ulrich; Hensel, Andreas; Truyen, Uwe

2011-01-01

The objective of this study was the comparative evaluation of four indirect Salmonella ELISA tests at study time approved in Germany to detect Salmonella infection in pigs.Three tests are based on a LPS-antigen mix and directed against specific IgG antibodies. The fourth test is based on a purified S. Typhimurium whole-cell lysate antigen and discriminates between Salmonella-specific IgM-, IgA-, and IgG- antibodies. In a longitudinal study, two groups of six weeks old hybrid piglets were orally infected with a porcine S. Infantis or S. Derby strain. Clinical and bacteriological parameters were monitored weekly during an observation period of 130 days after infection and serum samples were investigated in parallel with the respective ELISAs. Apparently, the LPS-based ELISA systems used in this study failed to recognize S. Infantis-infected pigs although those animals shed the pathogen in high amounts throughout the study until day 81 post infection (p. i.). In contrast, the isotype-specific Salmonella Typhimurium whole-cell-lysate based ELISA was capable of detecting Salmonella-infected pigs from day ten p. i. at all tested serotypes and revealed the highest sensitivity in detection of S. Infantis-infected pigs. Furthermore, it became apparent that the often used surveillance cut-off value of 40 OD% is not appropriate for intra-vitam detection of S. Infantis- and S. Derby-infected pigs. In contrast, the cut-off values of the ELISAs given by the suppliers result in considerable higher detection rates.

Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay.

Science.gov (United States)

Rücker, Hannelore; Amslinger, Sabine

2015-01-01

The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of

Development and Evaluation of a Novel ELISA for Detection of Antibodies against HTLV-I Using Chimeric Peptides.

Science.gov (United States)

Mosadeghi, Parvin; Heydari-Zarnagh, Hafez

2018-04-01

We aimed to develope a peptide-based indirect ELISA to detect antibodies against Human T-lymphotropic virus type I (HTLV-I). Two chimeric peptides (CP-1 and CP-2) were designed using linear immunodominant epitopes of gp-46-I, and gp21-I proteins, according to the sequence from Uniprot database. These peptides were studied initially in the ELISA using infected sera. The most promising peptideCP-1, was used to develop a peptide ELISA for detection of HTLV-I infected sera. The optimal conditions for CP-1ELISA were: the optimum coating buffer was 100mM NaHCO3, pH 9.6; coating peptide concentration was 10 µg/mL; the optimal blocking buffer was5% fetal bovine serum (FBS); the secondary antibody concentration was 1:2000; and serum dilution was 1:20. 20serum samples from HTLV-I infected patients were evaluated by ELISA developed. CP-1 showed high antigenicity while lacking any cross-reactivity with normal human sera. The results of evaluations indicated that in comparison with commercial ELISA, CP-1 ELISA showed good sensitivity and specificity. With further validation, CP-1as described in the present study could be introduced as novel reliable and cost-effective candidates for the high-specific screening of HTLV-I/-II infections in endemic regions.

Implementation and Setup of a System Dedicated Logbook based on ELisA

CERN Document Server

Van Tonder, Raynette

2016-01-01

ELisA is a web tool used by the ATLAS community as a daily logbook facility in order to record and share information concerning the experiment's operation and deployment. A few subsystems, notably the FTK subsystem, of the ATLAS experiment would like to setup a logbook based on the ELisA logbook for their own private usage. This new logbook would be completely separated from the main ATLAS logbook and will only be accessed from the general public network. In this project the implementation and setup of the ELisA based logbook will be discussed as well as various modifications that were made to the new logbook. These modifications were specified by the FTK users in order to make the logbook relevant to the FTK subsystem. Once the logbook was functioning as expected and the users were satisfied with the modifications, the FTK logbook was installed on a FTK dedicated machine where it is currently being used by members of the FTK group.

Seroprevalence of equine granulocytic anaplasmosis and lyme borreliosis in Canada as determined by a point-of-care enzyme-linked immunosorbent assay (ELISA).

Science.gov (United States)

Schvartz, Gili; Epp, Tasha; Burgess, Hilary J; Chilton, Neil B; Pearl, David L; Lohmann, Katharina L

2015-06-01

Equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB) are an emerging concern in Canada. We estimated the seroprevalence of EGA and equine LB by testing 376 convenience serum samples from 3 provinces using a point-of-care SNAP(®) 4Dx(®) ELISA (IDEXX Laboratories, Westbrook, Maine, USA), and investigated the agreement between the point-of-care ELISA and laboratory-based serologic tests. The estimated seroprevalence for EGA was 0.53% overall (0.49% in Saskatchewan, 0.71% in Manitoba), while the estimated seroprevalence for LB was 1.6% overall (0.49% in Saskatchewan, 2.86% in Manitoba). There was limited agreement between the point-of-care ELISA and an indirect fluorescent antibody test for EGA (kappa 0.1, PABAK 0.47) and an ELISA/Western blot combination for LB (kappa 0.23, PABAK 0.71). While the SNAP(®) 4Dx(®) ELISA yielded expected seroprevalence estimates, further evaluation of serologic tests for the purposes of disease exposure recognition may be needed.

Elisa Miller | NREL

Science.gov (United States)

Elisa Miller Photo of Elisa Miller Elisa Link-Miller Researcher III-Chemistry Elisa.Miller@nrel.gov | 303-384-6777 Dr. Elisa Miller-Link studies the surface of semiconductors that are applicable for , and other nanocrystalline films. Elisa came to NREL in 2013 as an NREL Director's Fellowship recipient

Evaluation of immunoradiometric and ELISA versions of a microtitre plate assay for Bacillus anthracis spores

Energy Technology Data Exchange (ETDEWEB)

Phillips, A P; Martin, K L; Cross, N L [Chemical Defence Experimental Establishment, Porton (UK); Drake, R G [Glasgow Univ. (UK). Inst. of Biochemistry

1984-05-11

Solid-phase indirectly-labelled antibody assays for Bacillus anthracis spores heat-fixed on polystyrene microtitre plates were compared as immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA) versions. Signal-to-noise ratios were usually higher in the IRMA than in the ELISA performed under parallel conditions but replicates were more varied in the IRMA. The antigen detection threshold and resolution limit calculated after regression analysis were broadly comparable in the 2 types of assay.

The impact of milk handling procedures on Ostertagia ostertagi antibody ELISA test results.

Science.gov (United States)

Vanderstichel, Raphaël; Dohoo, Ian; Stryhn, Henrik

2010-04-19

The impact of various milk handling stressors were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) test measuring Ostertagia ostertagi antibodies in milk from dairy cattle (Svanovir). An indirect ELISA has the ability to determine the amount of milk production losses related to intestinal parasitism. The ELISA test recommends fresh defatted milk, however, milk collected from Dairy Herd Improvement (DHI) programs in North America undergo many stressors, including, heating, freezing and are not defatted. Normalized optical density ratios (ODRs) were compared between fresh defatted milk and milk subjected to one or more stressors with a linear mixed model accounting for differences in variation between the fresh and the frozen samples. Concordance correlation coefficients were also analyzed for comparisons to other similar studies. After accounting for random cow and container effects, the treatment factors interacted with each other (p<0.001). Biologically interesting contrasts were created to explain the interaction. The estimated difference in ODR between the milk samples handled according to recommendations of the manufacturers of Svanovir and the whole milk samples that were subjected to the most extreme treatment (heated, frozen, thawed, and re-frozen for 4 weeks) was 0.062 (p<0.001). This difference represented less than 5% of the range, and was thus considered biologically negligible. Frozen whole milk processed by DHI programs, the most likely method of collecting on-farm samples in North America, will likely yield reliable results for the indirect ELISA tests, particularly, Svanovir.

Image-based ELISA on an activated polypropylene microtest plate--a spectrophotometer-free low cost assay technique.

Science.gov (United States)

Parween, Shahila; Nahar, Pradip

2013-10-15

In this communication, we report ELISA technique on an activated polypropylene microtest plate (APPµTP) as an illustrative example of a low cost diagnostic assay. Activated test zone in APPµTP binds a capture biomolecule through covalent linkage thereby, eliminating non-specific binding often prevalent in absorption based techniques. Efficacy of APPµTP is demonstrated by detecting human immunoglobulin G (IgG), human immunoglobulin E (IgE) and Aspergillus fumigatus antibody in patient's sera. Detection is done by taking the image of the assay solution by a desktop scanner and analyzing the color of the image. Human IgE quantification by color saturation in the image-based assay shows excellent correlation with absorbance-based assay (Pearson correlation coefficient, r=0.992). Significance of the relationship is seen from its p value which is 4.087e-11. Performance of APPµTP is also checked with respect to microtiter plate and paper-based ELISA. APPµTP can quantify an analyte as precisely as in microtiter plate with insignificant non-specific binding, a necessary prerequisite for ELISA assay. In contrast, paper-ELISA shows high non-specific binding in control sera (false positive). Finally, we have carried out ELISA steps on APPµTP by ultrasound waves on a sonicator bath and the results show that even in 8 min, it can convincingly differentiate a test sample from a control sample. In short, spectrophotometer-free image-based miniaturized ELISA on APPµTP is precise, reliable, rapid, and sensitive and could be a good substitute for conventional immunoassay procedures widely used in clinical and research laboratories. Copyright © 2013 Elsevier B.V. All rights reserved.

Highly sensitive and specific determination of mercury(II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody

Energy Technology Data Exchange (ETDEWEB)

Wang, Yuzhen; Li, Yuan [Sichuan University, College of Chemistry, Chengdu (China); Yang, Hong [Soochow University, College of Pharmacy, Suzhou (China); Pschenitza, Michael; Niessner, Reinhard; Knopp, Dietmar [Technical University Munich, Chair for Analytical Chemistry, Institute of Hydrochemistry and Chemical Balneology, Munich (Germany); Deng, Anping [Sichuan University, College of Chemistry, Chengdu (China); Soochow University, College of Chemistry, Chemical Engineering and Materials Science, Suzhou (China)

2012-07-15

Mercury is one of the most toxic heavy metals present in the environment. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of Hg{sup 2+} was developed. A new bifunctional ligand, 6-mercaptonicotinic acid (MNA), which contains a pyridine ring bearing a carboxylic group and a mercapto group, was selected for the preparation of immunogen. After immunization of mice and performing the hybridoma technique, the obtained mAb was characterized for its binding affinity and selectivity for Hg{sup 2+}. Based on this novel mAb, an ELISA was established. At optimal experimental conditions, the standard curve of the ELISA for Hg{sup 2+} was constructed in concentration range of 0.1-100 ng mL{sup -1}. The values of IC{sub 50} and LOD of the assay were found to be 1.12 and 0.08 ng mL{sup -1}. The cross-reactivity was lower than 2 % with MNA, CH{sub 3}Hg, and CH{sub 3}Hg-MNA and was 11.5 % and 4.4 % for Hg{sup +} and Au{sup 3+}, respectively. No cross-reactivity was found with other metal ions such as Cu{sup 2+}, Sn{sup 2+}, Ni{sup 2+}, Mn{sup 2+}, Pb{sup 2+}, Zn{sup 2+}, Cd{sup 2+}, Fe{sup 2+}, Co{sup 2+}, Mg{sup 2+}, Ca{sup 2+}, and anions such as Cl{sup -}, NO{sub 3} {sup -}, NO{sub 2} {sup -}, HCO{sub 3} {sup -}, F{sup -}, and SO{sub 4} {sup 2-}, indicating that the assay displays not only high sensitivity but also high selectivity. Different kinds of samples including water, milk, green vegetable, kelp, facial cleanser, and night cream were spiked with Hg{sup 2+} and the extracts were analyzed by ELISA. Acceptable recovery rates of 80.0-113.0 % and coefficients of variation of 1.9-18.6 % were obtained. A linear relationship between ELISA and cold-vapor atomic fluorescence spectroscopy (CV-AFS) as indicated by a correlation coefficient of 0.97 for liquid samples (water samples) and 0.98 for other samples was obtained. The proposed mAb-based ELISA provides a

Canine specific ELISA for coagulation factor VII

DEFF Research Database (Denmark)

Knudsen, Tom; Kjelgaard-Hansen, Mads; Tranholm, Mikael

2011-01-01

available to date. In this study, a canine specific ELISA for measurement of FVII:Ag in plasma was developed and validated. The FVII:Ag ELISA correctly diagnosed homozygous and heterozygous hereditary FVII deficiency. Together with activity based assays, such as FVII:C, the FVII:Ag ELISA should be valuable...

Validation of a commercial ELISA for the detection of bluetongue virus (BTV) specific antibodies in individual milk samples of Dutch dairy cows

NARCIS (Netherlands)

Kramps, J.A.; Maanen, van K.; Mars, M.H.; Popma, J.K.; Rijn, van P.A.

2008-01-01

recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum

Comparsion of an immunochromatographic strip with ELISA for simultaneous detection of thiamphenicol, florfenicol and chloramphenicol in food samples.

Science.gov (United States)

Guo, Lingling; Song, Shanshan; Liu, Liqiang; Peng, Juan; Kuang, Hua; Xu, Chuanlai

2015-09-01

Rapid and sensitive indirect competitive enzyme-linked immunosorbent assays (ic-ELISA) and gold nanoparticle immunochromatographic strip tests were developed to detect thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in milk and honey samples. The generic monoclonal antibody for TAP, FF and CAP was prepared based on a hapten [D-threo-1-(4-aminophenyl)-2- dichloroacetylamino-1,3-propanediol], and the haptenwas linked to a carrier protein using the diazotization method. After the optimization of several parameters (coating, pH, sodium chloride content and methanol content), the ic-ELISA was established. The quantitative working range for TAP was 0.11-1.36 ng/mL, with an IC50 of 0.39 ng/mL. The optimized ELISA showed cross-reactivity to CAP (300%) and FF (15.6%), with IC50 values of 0.13 and 2.5 ng/mL, respectively. The analytical recovery of TAP, FF and CAP in milk and honey samples in the ic-ELISA ranged from 81.2 to 112.9%. Based on this monoclonal antibody, a rapid and sensitive immunochromatographic test strip was also developed. This strip had a detection limit of 1 ng/mL for TAP, FF and CAP in milk and honey samples. Moreover, the test was completed within 10 min. Our results showed that the proposed ic-ELISA and immunochromatographic test strip method are highly useful screening tools for TAP, FF and CAP detection in milk and honey samples. Copyright © 2015 John Wiley & Sons, Ltd.

Diagnostic Accuracy of Recombinant Immunoglobulin-like Protein A-Based IgM ELISA for the Early Diagnosis of Leptospirosis in the Philippines.

Directory of Open Access Journals (Sweden)

Emi Kitashoji

Full Text Available Leptospirosis is an important but largely under-recognized public health problem in the tropics. Establishment of highly sensitive and specific laboratory diagnosis is essential to reveal the magnitude of problem and to improve treatment. This study aimed to evaluate the diagnostic accuracy of a recombinant LigA protein based IgM ELISA during outbreaks in the clinical-setting of a highly endemic country.A prospective study was conducted from October 2011 to September 2013 at a national referral hospital for infectious diseases in Manila, Philippines. Patients who were hospitalized with clinically suspected leptospirosis were enrolled. Plasma and urine were collected on admission and/or at discharge and tested using the LigA-IgM ELISA and a whole cell-based IgM ELISA. Sensitivity and specificity of these tests were evaluated with cases diagnosed by microscopic agglutination test (MAT, culture and LAMP as the composite reference standard and blood bank donors as healthy controls: the mean+3 standard deviation optical density value of healthy controls was used as the cut-off limit (0.062 for the LigA-IgM ELISA and 0.691 for the whole cell-based IgM ELISA. Of 304 patients enrolled in the study, 270 (89.1% were male and the median age was 30.5 years; 167 (54.9% were laboratory confirmed. The sensitivity and ROC curve AUC for the LigA-IgM ELISA was significantly greater than the whole cell-based IgM ELISA (69.5% vs. 54.3%, p<0.01; 0.90 vs. 0.82, p<0.01 on admission, but not at discharge. The specificity of LigA-IgM ELISA and whole cell-based IgM ELISA were not significantly different (98% vs. 97%. Among 158 MAT negative patients, 53 and 28 were positive by LigA- and whole cell-based IgM ELISA, respectively; if the laboratory confirmation was re-defined by LigA-IgM ELISA and LAMP, the clinical findings were more characteristic of leptospirosis than the diagnosis based on MAT/culture/LAMP.The newly developed LigA-IgM ELISA is more sensitive than the

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.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

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Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

Generation of Monoclonal Antibodies against Ag85A Antigen of Mycobacterium tuberculosis and Application in a Competitive ELISA for Serodiagnosis of Bovine Tuberculosis

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Zhengzhong Xu

2017-06-01

Full Text Available The Ag85 complex functions as the main secretory protein of Mycobacterium tuberculosis (M. tuberculosis and BCG. This complex is composed of the proteins, Ag85A, Ag85B, and Ag85C, with Ag85A thought to play the largest role within the complex. However, the lack of commercially available monoclonal antibodies (mAbs against Ag85A still hinders the biological and applicative research on this protein. In this study, we developed and identified anti-Ag85A mAbs, and five hybridoma cells were established. Using the indirect immunofluorescence test, we found that two anti-Ag85A mAbs did not cross-react with Ag85B and/or Ag85C. In addition, we showed that all of the mAbs tested in this study are able to react with endogenous Ag85A protein in BCG and rBCG:Ag85A using indirect ELISA and Western blot analyses. A competitive ELISA (cELISA based on mAb 3B8 was developed, the analyses of clinic serum samples from cattle with bovine tuberculosis (TB and healthy cattle demonstrated that the sensitivity of the cELISA was 54.2% (26/48 and the specificity was 83.5% (167/200. This study demonstrated that the mAbs against Ag85A will provide useful reagents for further investigation into the function of the Ag85 complex and can be used for serodiagnosis of bovine TB.

Detection of Ganoderic Acid A in Ganoderma lingzhi by an Indirect Competitive Enzyme-Linked Immunosorbent Assay.

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Sakamoto, Seiichi; Kohno, Toshitaka; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

2016-05-01

Ganoderma is a genus of medicinal mushroom traditionally used for treating various diseases. Ganoderic acid A is one of the major bioactive Ganoderma triterpenoids isolated from Ganoderma species. Herein, we produced a highly specific monoclonal antibody against ganoderic acid A (MAb 12 A) and developed an indirect competitive ELISA for the highly sensitive detection of ganoderic acid A in Ganoderma lingzhi, with a limit of detection of 6.10 ng/mL. Several validation analyses support the accuracy and reliability of the developed indirect competitive ELISA for use in the quality control of Ganoderma based on ganoderic acid A content. Furthermore, quantitative analysis of ganoderic acid A in G. lingzhi revealed that the pileus exhibits the highest ganoderic acid A content compared with the stipe and spore of the fruiting body; the best extraction efficiency was found when 50 % ethanol was used, which suggests the use of a strong liquor to completely harness the potential of Ganoderma triterpenoids in daily life. Georg Thieme Verlag KG Stuttgart · New York.

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

DEFF Research Database (Denmark)

Nielsen, Ragnhild; van den Bosch, Johannes F.; Plambeck, Tamara

2000-01-01

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apr I, Apr II and Apr III. The toxins are important virulence factors. In the present study, ELISAs with purified Apr I, Apr II and Apr III, respectively...... of exotoxin is not revealed serologically in the ELISA test....

Science with the space-based interferometer eLISA. II. Gravitational waves from cosmological phase transitions

International Nuclear Information System (INIS)

Caprini, Chiara; Hindmarsh, Mark; Helsinki Univ.; Huber, Stephan

2016-04-01

We investigate the potential for the eLISA space-based interferometer to detect the stochastic gravitational wave background produced by strong first-order cosmological phase transitions. We discuss the resulting contributions from bubble collisions, magnetohydrodynamic turbulence, and sound waves to the stochastic background, and estimate the total corresponding signal predicted in gravitational waves. The projected sensitivity of eLISA to cosmological phase transitions is computed in a model-independent way for various detector designs and configurations. By applying these results to several specific models, we demonstrate that eLISA is able to probe many well-motivated scenarios beyond the Standard Model of particle physics predicting strong first-order cosmological phase transitions in the early Universe.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA.

Science.gov (United States)

Meegan, Jenny; Dunn, J Lawrence; Venn-Watson, Stephanie K; Smith, Cynthia R; Sidor, Inga; Jensen, Eric D; Van Bonn, William G; Pugh, Roberta; Ficht, Thomas; Adams, L Garry; Nielsen, Klaus; Romano, Tracy A

2012-12-03

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

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

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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.

An Evidence-Based Approach to Detection by DASI-ELISA and RT-PCR in Dormant Period

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Antonio Olmos

2008-01-01

Full Text Available An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASI-ELISA and RT-PCR.

Selection of specific inhibitor peptides in enzyme-linked immunosorbent assay (ELISA) of cardiac troponin I using immuno-dominant epitopes as competitor.

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Rezaee, Majid Asiabanha; Rasaee, Mohammad Javad; Mohammadnejad, Javad

2017-01-01

Human cardiac troponin I (cTni) is the gold marker for early diagnosis of myocardial infarction. In this regard, four immune-dominant epitopes of cTni were predicted and their 3D structures were determined. Thereafter, the competitive performance of the peptides was monitored with the developed polyclonal antibody-based indirect competitive ELISA; a half-maximal inhibitory concentration (IC50) of 0.49 (µg/mL) and detection limit of 0.037 (µg/mL) were achieved for recombinant cTni. The competitive ELISA determined sensitivity levels of 0.306, 0.141, 0.960, and 0.155 (µg/mL), respectively, for each peptide as competitor. We indicated that two of the selected epitopes have significant sensitivity scales and inhibition ability.

Development of enzyme-linked immunosorbent assay (ELISA) for glutathione S-transferase (GST-S) protein in the intertidal copepod Tigriopus japonicus and its application for environmental monitoring.

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Rhee, Jae-Sung; Kim, Bo-Mi; Jeong, Chang-Bum; Leung, Kenneth Mei Yee; Park, Gyung Soo; Lee, Jae-Seong

2013-11-01

To utilize the GST-S protein as a useful biomarker for environmental contamination, we developed a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) in the intertidal copepod Tigriopus japonicus. Two polyclonal antibodies, TJ-GST-S1 and TJ-GST-S2, were raised against two TJ-GST-S synthetic peptides. Also a recombinant TJ-GST-S protein was purified as a standard for ELISA development. Each polyclonal antibody was tested by Western blot analysis and indirect ELISA. Of two polyclonal antibodies, TJ-GST-S2 ELISA was further employed due to its wide range of detection and the limit of specificity compared to those of TJ-GST-S1 ELISA system. After exposure to 4 metals (Ag, As, Cd, and Cu) to T. japonicus, the amount of TJ-GST-S protein was significantly elevated in a concentration-dependent manner. Also, TJ-GST-S protein was upregulated at relative high concentrations of B[α]P, PCB, and TBT. In this paper, we suggest that T. japonicas ELISA for TJ-GST-S2 is useful as a potential indicator system for marine contaminants. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of methods to reduce background using the Python-based ELISA_QC program.

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Webster, Rose P; Cohen, Cinder F; Saeed, Fatima O; Wetzel, Hanna N; Ball, William J; Kirley, Terence L; Norman, Andrew B

2018-05-01

Almost all immunological approaches [immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blot], that are used to quantitate specific proteins have had to address high backgrounds due to non-specific reactivity. We report here for the first time a quantitative comparison of methods for reduction of the background of commercial biotinylated antibodies using the Python-based ELISA_QC program. This is demonstrated using a recombinant humanized anti-cocaine monoclonal antibody. Several approaches, such as adjustment of the incubation time and the concentration of blocking agent, as well as the dilution of secondary antibodies, have been explored to address this issue. In this report, systematic comparisons of two different methods, contrasted with other more traditional methods to address this problem are provided. Addition of heparin (HP) at 1 μg/ml to the wash buffer prior to addition of the secondary biotinylated antibody reduced the elevated background absorbance values (from a mean of 0.313 ± 0.015 to 0.137 ± 0.002). A novel immunodepletion (ID) method also reduced the background (from a mean of 0.331 ± 0.010 to 0.146 ± 0.013). Overall, the ID method generated more similar results at each concentration of the ELISA standard curve to that using the standard lot 1 than the HP method, as analyzed by the Python-based ELISA_QC program. We conclude that the ID method, while more laborious, provides the best solution to resolve the high background seen with specific lots of biotinylated secondary antibody. Copyright © 2018. Published by Elsevier B.V.

Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

Science.gov (United States)

Wang, Sheng-Yu; Li, Zhe; Wang, Xian-Jiang; Lv, Sha; Yang, Yun; Zeng, Lian-Qiang; Luo, Fang-Hong; Yan, Jiang-Hua; Liang, Da-Feng

2014-10-01

Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

Development and application of radioimmunoassays and enzyme immunoassays in microbiological and immunological diagnosis. 2. Comparative studies for the detection of toxoplasma antibodies with ELISA, RIA and other serological methods

Energy Technology Data Exchange (ETDEWEB)

Mueller, W A; Struy, H; Holzwarth, F [Medizinische Akademie, Magdeburg (German Democratic Republic)

1982-06-01

Comparative studies of indirect immunofluorescence test (IT), complement binding reaction (CBR), enzyme- and radioimmunoassay (ELISA, RIA) for the detection of toxoplasma antibodies in sera of 513 patients are reported. The precision dependent on time, showed coefficients of variation from 3% to 12% (IFT 3%, CBR 10%, ELISA 12%, RIA 7%). The correlation of IFT and ELISA as well as RIA was relatively unfavourable (coefficient of correlation IFT/ELISA r = 0.52, IFT/RIA r = 0.54, RIA/ELISA r = 0.60). The ELISA is the most sensitive method for the detection of antibodies. The specificity of the Toxo-ELISA has to be improved by application of suitable fractions of antigens.

Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

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Elza FT Belo

Full Text Available Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic. The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs were absorbed by nitrocellulose membrane and blocked with a 5% skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

Comparative evaluation of antibody positive titer by ELISA and IFA in Theileria annulata vaccinated cattle in Iran

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Hashemi-Fesharki R.

2006-03-01

Full Text Available An enzyme linked immunosorbent assay (ELISA was used to evaluate antibody positive titer in vaccinated and non-vaccinated cattle using schizont infected myeloid cells as an antigen. The result was compared with indirect fluorescent antibody level in the same animals. For this study 116 milking cows, 95 vaccinated and 21 non-vaccinated, were bleeded in order to prepare sera. They were tested with both ELISA and IFA tests. 94 sera had positive antibody titer and 22 sera were negative through ELISA test but, with IFA test, only 89 sera showed positive antibody titer and 27 were negative. Thereby, it was concluded that the sensitivity and specificity of ELISA test in comparison with IFA test was 95.5 % and 66.6 % respectively. This study generally indicated that ELISA could be an effective test for seroepidemiological investigations of bovine tropical theileriosis, and it is considered to be valid as an additional test to distinguish the vaccinated from the non vaccinated cattle in order to schedule vaccination programs.

Cellphone-based hand-held microplate reader for point-of-care ELISA testing (Conference Presentation)

Science.gov (United States)

Berg, Brandon; Cortazar, Bingen; Tseng, Derek; Ozkan, Haydar; Feng, Steve; Wei, Qingshan; Chan, Raymond Y.; Burbano, Jordi; Farooqui, Qamar; Lewinski, Michael; Di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

2016-03-01

Enzyme-linked immunosorbent assay (ELISA) in a microplate format has been a gold standard first-line clinical test for diagnosis of various diseases including infectious diseases. However, this technology requires a relatively large and expensive multi-well scanning spectrophotometer to read and quantify the signal from each well, hindering its implementation in resource-limited-settings. Here, we demonstrate a cost-effective and handheld smartphone-based colorimetric microplate reader for rapid digitization and quantification of immunoserology-related ELISA tests in a conventional 96-well plate format at the point of care (POC). This device consists of a bundle of 96 optical fibers to collect the transmitted light from each well of the microplate and direct all the transmission signals from the wells onto the camera of the mobile-phone. Captured images are then transmitted to a remote server through a custom-designed app, and both quantitative and qualitative diagnostic results are returned back to the user within ~1 minute per 96-well plate by using a machine learning algorithm. We tested this mobile-phone based micro-plate reader in a clinical microbiology lab using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests on 1138 remnant patient samples (roughly 50% training and 50% testing), and achieved an overall accuracy of ~99% or higher for each ELISA test. This handheld and cost-effective platform could be immediately useful for large-scale vaccination monitoring in low-infrastructure settings, and also for other high-throughput disease screening applications at POC.

Validation and use of an ELISA kit for the diagnosis of Babesia bovis in Cuba

International Nuclear Information System (INIS)

Blandino, T.; Alonso, M.; Barrera, M.; Mendoza, E.

1998-01-01

Babesia bovis, the most important etiological agent causing bovine babesiosis, is widely distributed in Cuba and affects mainly adult cattle. A survey of the prevalence of the disease in cattle using an ELISA kit (FAO/IAEA) revealed that 34.2% of the animals between 6 and 18 months of age were positive to Babesia bovis, whereas 69.9% on the cattle older than 18 months were positive. Antibodies to Babesia bovis were detected in 96.9% of calves vaccinated with an attenuated Babesia bovis vaccine. A good correlation was found between the results of ELISA kit with those from indirect immunofluorescence and immunoperoxidase tests developed in Cuba. (author)

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.

Efficacy demonstration of tetanus vaccines by double antigen ELISA.

Science.gov (United States)

Rosskopf, U; Noeske, K; Werner, E

2005-09-01

This paper describes a double antigen ELISA (DAE) for rapid, specific and reliable assessment of the antitetanus immune status of horses and sheep. Compared with the indirect ELISA, the double antigen ELISA has the advantage of species-independent testing of sera. Thanks to its test design, it is more specific since the detected antibodies are forced to bind tetanus toxoid twice. In addition, it is very sensitive to tetanus antibodies, enabling the detection of low antibody titres, in range which is relevant for the assessment of the protective status (tetanus toxin neutralising antibodies). The detection limit of the DAE for tetanus antibodies is in the order of 10(-4) EU/ml. A comparison of in vitro results of individual sera with in vivo titres showed that horse sera with titres of 0.04 and 0.05 EU/ml in the DAE showed titres of > 0.05 IU and 0.034 IU/ml respectively during in vivo testing thus indicating good agreement. For tested sheep sera which were rated > 0.05 IU/ml in vivo, the corresponding titre in the DAE was 0.24 EU/ml. Clear tetanus antitoxin establishment of protective ELISA limits requires further comparative examination of sera with low titres (tetanus vaccines ad us. vet. As a consequence, the toxin neutralisation test (still being the standard method of choice for quantifying tetanus toxin neutralising antitoxin titres) could be replaced, since it requires too great a number of animals per test and involves considerable suffering for the animals. The test described here reduces the use of mice and guinea pigs within vaccine efficacy testing. In addition, it involves less exposure of the laboratory personnel to toxin.

Validation of Geno-Sen's scrub typhus real time polymerase chain reaction kit by its comparison with a serological ELISA Test

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Velmurugan Anitharaj

2017-01-01

Full Text Available Background: In the recent past, scrub typhus (ST has been reported from different parts of India, based on Weil-Felix/enzyme-linked immunosorbent assay (ELISA/indirect immunofluorescence assay (IFA. Molecular tests are applied only by a few researchers. Aims: Evaluation of a new commercial real time polymerase chain reaction (PCR kit for molecular diagnosis of ST by comparing it with the commonly used IgM ELISA is our aim. Settings and Design: ST has been reported all over India including Puducherry and surrounding Tamil Nadu and identified as endemic for ST. This study was designed to correlate antibody detection by IgM ELISA and Orientia tsutsugamushi DNA in real time PCR. Materials and Methods: ST IgM ELISA (InBios Inc., USA was carried out for 170 consecutive patients who presented with the symptoms of acute ST during 11 months (November, 2015– September, 2016. All 77 of these patients with IgM ELISA positivity and 49 of 93 IgM ELISA negative patients were subjected to real time PCR (Geno-Sen's ST real time PCR, Himachal Pradesh, India. Statistical Analysis: Statistical analysis for clinical and laboratory results was performed using IBM SPSS Statistics 17 for Windows (SPSS Inc., Chicago, USA. Chi-square test with Yates correction (Fisher's test was employed for a small number of samples. Results and Conclusion: Among 77 suspected cases of acute ST with IgM ELISA positivity and 49 IgM negative patients, 42 and 7 were positive, respectively, for O. tsutsugamushi 56-kDa type-specific gene in real time PCR kit. Until ST IFA, the gold standard diagnostic test, is properly validated in India, diagnosis of acute ST will depend on both ELISA and quantitative PCR.

Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

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Xiaoxiang Fu

2015-08-01

Full Text Available Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA was then developed. The median inhibitory concentration (IC50 of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.

Phospho-SMC1 in-Cell ELISA based Detection of Ataxia Telangiectasia

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Majid Zaki dizaji

2016-12-01

Full Text Available BackgroundAtaxia telangiectasia (A-T is a common genetically inherited cause of early childhood-onset ataxia. The infrequency of this disease, vast phenotype variation, disorders with features similar to those of A-T, and lack of definite laboratory test, make diagnosis difficult.  In addition, there is no rapid reliable laboratory method for identifying A-T heterozygotes, who susceptible to ionizing radiation (IR, atherosclerosis, diabetes, and cancers. We used SMC1pSer966 (pSMC1 in-cell colorimetric ELISA to diagnosis and screen in A-T families.Materials and Methods: With informed consent, 2cc peripheral blood was collected from the 15 A-T patients, their parents, and 24 healthy controls with no family history of malignancy, diabetes, and atherosclerosis. Extracted peripheral blood mononuclear cells (PBMCs were cultured in poly-L-Lysine treated 96-well plate with density of 70,000 cells per well. SMC1 phosphorylation was evaluated with cell-based ELISA kit 1 hour after 5 Gy IR and the pSMC1data normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH.Results: SMC1 phosphorylation was significantly low in A-T`s PBMC (mean + standard deviation [SD]: 0.075 + 0.034 in comparison to carriers (mean + SD: 0.190 + 0.060 and healthy controls (mean + SD: 0.312 +0.081, but unluckily could only discriminate A-T patients (Area Under the Curve -receiver operating characteristic [AUC-ROC]: 1.00, 1.00-1.00. This method in spite of rapidness and simplicity showed poor imprecision (22.49% coefficient of variation [CV] for intraday imprecision.Conclusion: It seems pSMC1 assessment by in-cell ELISA can be used for detection of A-T patients, but it may not sensitive enough for identification of carriers. This ELISA test is very simple, rapid, and requires less than 2cc blood. Thus it may be proposed for the early differential diagnosis of A-T as an alternative method.

Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor

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Xuan Weng

2016-06-01

Full Text Available The risks associated with the presence of hidden allergens in food have increased the need for rapid, sensitive, and reliable methods for tracing food allergens in commodities. Conventional enzyme immunosorbent assay (ELISA has usually been performed in a centralized lab, requiring considerable time and sample/reagent consumption and expensive detection instruments. In this study, a microfluidic ELISA platform combined with a custom-designed optical sensor was developed for the quantitative analysis of the proteins wheat gluten and Ara h 1. The developed microfluidic ELISA biosensor reduced the total assay time from hours (up to 3.5 h to 15–20 min and decreased sample/reagent consumption to 5–10 μL, compared to a few hundred microliters in commercial ELISA kits, with superior sensitivity. The quantitative capability of the presented biosensor is a distinctive advantage over the commercially available rapid methods such as lateral flow devices (LFD and dipstick tests. The developed microfluidic biosensor demonstrates the potential for sensitive and less-expensive on-site determination for rapidly detecting food allergens in a complex sample system.

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

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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.

Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies Detection.

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Francesco Pisani

Full Text Available Serological markers of Nuromyelitis Optica (NMO, an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4. We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs. Many tests have been developed to detect AQP4-IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA, which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91% compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used.

Biomimetic ELISA detection of malachite green based on magnetic molecularly imprinted polymers.

Science.gov (United States)

Li, Lu; Lin, Zheng-Zhong; Peng, Ai-Hong; Zhong, Hui-Ping; Chen, Xiao-Mei; Huang, Zhi-Yong

2016-11-01

A direct competitive enzyme-linked immunosorbent assay (ELISA) method was used for the detection of malachite green (MG) with a high sensitivity and selectivity using magnetic molecularly imprinted polymers (MMIPs) as a bionic antibody. MMIPs were prepared through emulsion polymerization using Fe 3 O 4 nanoparticles as magnetic nuclei, MG as a template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent and span-80/tween-80 as mixed emulsifiers. The MMIPs were characterized by scanning electron micrographs (SEM), thermal-gravimetric analyzer (TGA), Fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometer (VSM), respectively. A high magnetic saturation value of 54.1emug -1 was obtained, resulting in rapid magnetic separation of MMIPs with an external magnet. The IC 50 of the established ELISA method was 20.1μgL -1 and the detection limit (based on IC 85 ) was 0.1μgL -1 . The MMIPs exhibited high selective binding capacity for MG with cross-reactivities less than 3.9% for MG structural analogues. The MG spiking recoveries were 85.0%-106% with the relative standard deviations less than 4.7%. The results showed that the biomimetic ELISA method by using MMIPs as bionic antibody could be used to detect MG rapidly in fish samples with a high sensitivity and accuracy. Copyright © 2016 Elsevier B.V. All rights reserved.

A membrane-based ELISA assay for the herbicide Isoproturon in soil samples

OpenAIRE

Baskeyfield, Damian E. H.; Davis, Frank; Magan, Naresh; Tothill, Ibtisam E.

2012-01-01

A membrane based enzyme linked immunosorbent assay (MELISA) for the detection of a common herbicide, isoproturon is described. A heterogeneous competitive ELISA was the format chosen for isoproturon detection. An immunoassay system with a horseradish peroxidase (HRP) labeled polyclonal antibody preparation was developed and characterized before suitable sensitivity and selectivity for isoproturon were attained. After development as a microtiter plate immunoassay, the system was transferred to...

Assessment of an ELISA for serodiagnosis of active pulmonary tuberculosis in a Cuban population

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Julio Cesar Ayala

2015-10-01

Full Text Available Objective: To explore the serodiagnostic potential of the five recombinant Mycobacterium tuberculosis antigens CFP-10 (Rv3874, ESAT-6 (Rv3875, APA (Rv1860, PstS-1 (Rv0934, Ag85A (Rv3804c and their combination in a Cuban population with active pulmonary tuberculosis. Methods: The serodiagnostic potential of the recombinant antigens rESAT-6, rCFP-10, rAPA, rPstS-1 produced in Escherichia coli, rAg85A produced in Streptomyces lividans and the combination of the five proteins was evaluated by an indirect ELISA. Humoral immune response was analysed in a group of 140 patients with active pulmonary tuberculosis (smear-, Mantoux- and culture-positive and in a control group consisting of 34 bacillus CalmetteGuerin vaccinated, Mantoux-negative, healthy subjects. Results: With the exception of CFP-10, the use of the separate recombinant antigens or the antigenic cocktail in ELISA-based serodiagnosis resulted in a significant difference in the mean optical densitiy values between sera of patients and healthy subjects. The highest sensitivity of the assay using single antigens, being 58.57%, was achieved with rPstS-1 compared to 27.14% with rCFP-10, 31.65% with Ag85A, 42.86% with rAPA and 44.29% with rESAT-6. Single antigen ELISAs provided high specificity values ranging from 94.12% to 97.06%. A cocktail of the aforementioned antigens increased the sensitivity to 87.14% and the specificity to 97.06%. Conclusions: An ELISA using a multi-antigen mix containing recombinant immuno-dominant antigens of Mycobacterium tuberculosis, namely, rCFP-10, rESAT-6, rAPA, rPstS-1 and rAg85, increases the sensitivity and specificity compared with that using the single antigens and shows potential as a complementary tool for the diagnosis of active pulmonary tuberculosis in Cuba.

Biomimetic ELISA detection of malachite green based on molecularly imprinted polymer film.

Science.gov (United States)

Li, Lu; Peng, Ai-Hong; Lin, Zheng-Zhong; Zhong, Hui-Ping; Chen, Xiao-Mei; Huang, Zhi-Yong

2017-08-15

A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of malachite green (MG) using a molecularly imprinted polymer (MIP) film as bionic antibody. The MIP film, based on the self-polymerization of dopamine, was fabricated on the surfaces of a 96-well microplate. It showed specific recognition for MG in aqueous solution. A direct competitive ELISA method was established with the sensitivity reaching 10.31μgL -1 and the detection limit being 0.3μgL -1 . The cross-reactivity of two structural analogues to MG was less than 10%. The average recovery tested by MG standard spiking was 88.8% for bass and 90.4% for water, and the relative standard deviations were less than 3.6%. All the above results indicated that the developed method could be used to detect MG in fish and water samples rapidly, specifically and accurately. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bulk tank milk ELISA for detection of antibodies to Mycobacterium avium subsp paratuberculosis: Correlation between repeated tests and within-herd antibody-prevalence

DEFF Research Database (Denmark)

Nielsen, Søren Saxmose; Toft, Nils

2014-01-01

Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were...... Danish Holstein herds over a period of one year. All samples were tested using a commercial indirect ELISA for detection of MAP specific antibodies. The individual cow's results were dichotomised and used to estimate the within-herd antibody prevalence at each test-date. These prevalences were...... to 0.60 when corrected for the within-herd antibody prevalence. Although the test-results were relatively consistent and correlated with the within-herd prevalence, the magnitude of the test-values makes it difficult to use the BTM-ELISA for surveillance of MAP infections in practice....

ELISA-based assay for IP-10 detection from filter paper samples

DEFF Research Database (Denmark)

Drabe, Camilla Heldbjerg; Blauenfeldt, Thomas; Ruhwald, Morten

2014-01-01

IP-10 is a small pro-inflammatory chemokine secreted primarily from monocytes and fibroblasts. Alterations in IP-10 levels have been associated with inflammatory conditions including viral and bacterial infections, immune dysfunction, and tumor development. IP-10 is increasingly recognized as a b...... as a biomarker that predicts severity of various diseases and can be used in the immunodiagnostics of Mycobacterium tuberculosis and cytomegalovirus infection. Here, we describe an ELISA-based method to detect IP-10 from dried blood and plasma spot samples....

Development and optimization of an ESAT6-ELISA-based detection system of Mycobacterium tuberculosis complex, suitable for bovine TB eradication

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Rouhollah Keshavarz

2015-01-01

Conclusion: Performing the ELISA test based on ESAT-6 antigen shows that this test can be a suitable way for screening beside the tuberculin test for accurate detection. It is noticed that the specificity of the ELISA test was determined more than the tuberculin test, especially since the culture and PCR are gold standards. Therefore, incorrect sampling can change the specificity of the tuberculin test. The results of this kit can encourage designing of an ELISA kit for the detection of human TB.

Control charts for identifying systematic errors using control sera to detect antibody to Salmonella in an indirect ELISA

DEFF Research Database (Denmark)

Bak, H.; Barfod, Kristen

2008-01-01

This study evaluated the preparation of Shewhart's control charts using the concept of rational subgroups for monitoring the Salmonella antibody ELISA used for surveillance of Danish pig herds. Control charts were prepared for a buffer control sample, a negative serum sample and a positive serum...

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.

Evaluation of the radioimmunoassay, indirect enzyme linked immunosorbent assay, and dot blot assay for the identification of Xanthomonas campestris pv. phaseoli

Energy Technology Data Exchange (ETDEWEB)

Malin, E; Belden, E L; Roth, D

1985-09-01

A radioimmunoassay (RIA), an indirect competitive enzyme-linked immunosorbent assay (ELISA), and a dot-blot modification of the ELISA were evaluated for detection and identification of Xanthomonas campestris pv. phaseoli (X. c. pv. phaseoli). RIA and the dot blot tests were specific for X. c. pv. phaseoli; however, significant cross reactions occurred in the indirect competitive ELISA when using anti-X. c. pv. phaseoli antiserum against other closely related bacteria. The sensitivity level of all procedures for X. c. pv. phaseoli was approximately l0/sup 5/ colony forming unitsmL. All procedures were unsatisfactory in reliably detecting low levels of X. c. pv. phaseoli directly from extracts of bean seed. However when used in conjunction with ilution plating the dot blot assay and the RIA would be useful in specifically identifying X. c. pv. phaseoli. The relative merits of these tests for identification of X. c. pv. phaseoli are discussed.

No serological evidence for Zika virus infection and low specificity for anti-Zika virus ELISA in malaria positive individuals among pregnant women from Madagascar in 2010.

Science.gov (United States)

Schwarz, Norbert Georg; Mertens, Eva; Winter, Doris; Maiga-Ascofaré, Oumou; Dekker, Denise; Jansen, Stephanie; Tappe, Dennis; Randriamampionona, Njary; May, Jürgen; Rakotozandrindrainy, Raphael; Schmidt-Chanasit, Jonas

2017-01-01

It was previously reported that a malaria infection may interfere with the specificity of a commercial ELISA test against Zika virus (ZIKV). We analyzed 1,216 plasma samples from healthy, pregnant women collected in two sites in Madagascar in 2010 for ZIKV antibodies using a commercial ELISA and for Plasmodium infection by PCR. This screen revealed six putative ZIKV-positive samples by ELISA. These results could not be confirmed by indirect immunofluorescence assays or virus neutralization tests. Four of these six samples were also positive for P. falciparum. We noted that the frequency of malaria positivity was higher in ZIKV-ELISA positive samples (50% and 100% in the two study sites) than ZIKV-negative samples (17% and 10%, respectively), suggesting that malaria may have led to false ZIKV-ELISA positives.

Detecting diclofenac in livestock carcasses in India with an ELISA: A tool to prevent widespread vulture poisoning

International Nuclear Information System (INIS)

Saini, Mohini; Taggart, Mark A.; Knopp, Dietmar; Upreti, Suchitra; Swarup, Devendra; Das, Asit; Gupta, Praveen K.; Niessner, Reinhard; Prakash, Vibhu; Mateo, Rafael; Cuthbert, Richard J.

2012-01-01

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), has caused catastrophic vulture declines across the Indian sub-continent. Here, an indirect ELISA is used to detect and quantify diclofenac in 1251 liver samples from livestock carcasses collected across India between August 2007 and June 2008, one to two years after a ban on diclofenac manufacture and distribution for veterinary use was implemented. The ELISAs applicability was authenticated with independent data obtained using LC–ESI/MS. Of 1251 samples, 1150 (91.9%) were negative for diclofenac using both methods, and 60 (4.8%) were positive at 10–4348 and 10–4441 μg kg −1 when analysed by ELISA and LC–ESI/MS, respectively. The residue level relationship in the 60 positive samples was highly significant (p 2 = 0.644). Data suggest that this immunological assay could be used not only for cost effective sample screening, but also for residue level semi-quantification. - Highlights: ► An ELISA is validated for use in diclofenac monitoring. ► We compare ELISA and LC–ESI/MS data for 1251 samples. ► Results indicate the ELISA can be reliably used for screening and semi-quantification. ► In 2007–2008, in India, around 1 in 20 ungulate carcasses contained detectable diclofenac. - The prevalence of diclofenac in carcasses available to vultures in India has declined from around 1:10 to 1:20 since restrictions on veterinary use were first put in place in 2006.

(ELISA) kit for diagnosis copro-antigens of Giardia lamblia

African Journals Online (AJOL)

STORAGESEVER

2010-08-02

Aug 2, 2010 ... methods based on antigen scanning of parasites such as enzyme linked immunosorbent assay (ELISA), ... samples. To design this method, a pure antibody against parasite as well as an antibody conjugated to a ..... school children in Santiago, Chile by capture ELISA for the detection of fecal Giardia ...

Introduction and use of ELISA based technologies for the diagnosis and monitoring of foot-and-mouth disease in Malaysia

International Nuclear Information System (INIS)

Karuppanan, P.; Naheed, M.

2000-01-01

Continued outbreaks of foot-and-mouth disease (FMD) in northern Malaysia drove the decision to establish a diagnostic surveillance capability at the regional laboratory in Kota Bharu. Based on using ELISA based diagnostic systems the laboratory was equipped for the detection of both the conservative virus and a serological response in animals. Considerable detail was given on the subsequent testing that was carried out clearly demonstrating the value both of the ELISA technology but also of what can be achieved at reasonable costs for conducting routine surveillance of FMD. (author)

Latent class analysis of bulk tank milk PCR and ELISA testing for herd level diagnosis of Mycoplasma bovis

DEFF Research Database (Denmark)

Nielsen, Per Kantsø; Petersen, Mette Bisgaard; Nielsen, Liza Rosenbaum

2015-01-01

of this study was to evaluate the herd-level diagnostic performance of an indirect ELISA test by comparison to a real-time PCR test when diagnosing M. bovis in cattle herds of bulk tank milk. Bulk tank milk samples from Danish dairy herds (N=3437) were analysed with both the antibody detecting BIO K 302 M...

Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods.

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Margaret M Billingsley

Full Text Available Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA. In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS decorated with antibodies specific to epidermal growth factor receptor (EGFR as a model system (EGFR-NS. We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that

Detección de anticuerpos anti-Brucella spp. en cerdos mediante técnicas de aglutinación y ELISA indirecto en las provincias de Buenos Aires y La Pampa: Argentina Detection of anti-Brucella spp. antibodies in swine by agglutination techniques and indirect ELISA in the Buenos Aires and La Pampa provinces: Argentina

Directory of Open Access Journals (Sweden)

H.A. Castro

2006-04-01

Full Text Available En nuestro país no existe un programa de control sobre brucelosis porcina y su verdadera situación epidemiológica es desconocida. El objetivo de nuestro trabajo fue detectar la presencia de anticuerpos anti-Brucella spp. en porcinos provenientes de criaderos del sudoeste de la provincia de Buenos Aires y del este de la provincia de La Pampa. La toma de muestras de sangre se realizó en el momento del faenado de los animales. La detección de anticuerpos se efectuó mediante las técnicas de aglutinación con antígeno tamponado en placa (BPA, seroaglutinación en tubo (SAT, aglutinación con 2-ME (2-ME y ELISA indirecto, con dos antígenos diferentes: el antígeno CYT (fracción citoplasmática de B. abortus S19 y el antígeno CP (extracto citoplasmático libre de lipopolisacárido. Del total de las muestras analizadas (n=325, el 17,8% fue positivo para BPA, el 13,8% fue positivo para SAT y sólo el 8,0% fue positivo para 2-ME. Mediante ELISA-CYT, este porcentaje se elevó a 21,0%, mientras que a través del ELISA-CP sólo se halló un 10,0% de muestras reactivas. Estos resultados son compatibles con los informados en los escasos reportes previos para todo el país y sugieren la necesidad de extender los estudios a otras zonas, donde sea habitual la cría de cerdos.Porcine brucellosis is one of the most important zoonoses in this country. Currently, there is no control program for porcine brucellosis in Argentina and the epidemiological situation is still unknown. The purpose of our study was to detect anti-Brucella spp. antibodies in swine in the southwest of the Buenos Aires province and the east of the La Pampa province. Blood samples were obtained when animals were slaughtered. The presence of anti-brucella antibodies was studied by the buffered plate agglutination test (BPA, the tube agglutination test (SAT, the 2-mercaptoethanol (2-ME agglutination test and indirect ELISA tests, using the cytosolic fraction from Brucella abortus S19

Comportamento do método quimioluminescente-ELISA em relação a resultados considerados discordantes por meio de três técnicas convencionais para diagnóstico da doença de Chagas Behavior of the chemiluminescent ELISA method in relation to results considered discordant via three conventional techniques for diagnosing Chagas disease

Directory of Open Access Journals (Sweden)

Cláudia Regina De Marchi

2007-02-01

Full Text Available Quando utilizadas, em conjunto, a hemaglutinação indireta, a imunofluorescência indireta e ELISA para diagnóstico sorológico da doença de Chagas por vezes ocorrem resultados considerados discordantes, por não haver concordância entre o que indicam essas técnicas. A disponibilidade do método quimioluminescente-ELISA permitiu executá-lo com 200 soros que examinados pelos três testes citados que motivaram a obtenção de resultados discordantes. Com o método quimioluminescente-ELISA sucederam 193 negativos e sete positivos. O emprego desse novo procedimento trouxe mais um subsídio para compreensão do assunto, mas avanço mais concreto dependerá de documentação com soros de pessoas infectadas ou não pelo Trypanosoma cruzi conforme comprovação parasitológica.When indirect hemagglutination, indirect immunofluorescence and enzyme-linked immunosorbent assay are used together for serologically diagnosing Chagas disease, results that are considered discordant sometimes occur because there is disagreement between what these tests indicate. The availability of the chemiluminescent ELISA method enabled tests on 200 serum samples that had previously produced discordant results from the three abovementioned methods. CL-ELISA revealed that 193 of these samples were negative and seven were positive. The use of this new procedure provides further support for understanding this subject, but more concrete advances will depend on documentation with blood analyses from people previously demonstrated to be unquestionably infected or uninfected with Trypanosoma cruzi.

Development of ELISA-based methods to measure the anti-malarial drug chloroquine in plasma and in pharmaceutical formulations

Directory of Open Access Journals (Sweden)

Ronn Anita

2011-08-01

Full Text Available Abstract Background In Central and South America and Eastern and Southern Africa, Plasmodium vivax infections accounts for 71-81% and 5% of malaria cases, respectively. In these areas, chloroquine (CQ remains the treatment of choice for P. vivax malaria. In addition, CQ has recently proven to be an effective HIV-1 therapeutic agent. There is a dire need to continue monitoring quality of CQ as there is a major influx of substandard and fake formulations into malaria-endemic countries. The use of fake/substandard drugs will result in sub-therapeutic levels endangering the patient and possibly select for parasite resistance. The aim of this study was to develop an inexpensive, simple antibody-based ELISA to measure CQ concentrations in tablets and in plasma. Methods A monoclonal antibody (MAb that reacts with the N-side chain of the CQ molecule was prepared by use of a CQ analogue. A specific and reliable ELISA for detection of CQ was developed. The developed assay was validated by measuring CQ in tablets sold in Denmark, India and Sudan. Furthermore, kinetics of CQ concentrations in plasma of four volunteers, who ingested two tablets of Malarex® containing, 250 mg CQ base, were measured before drug intake, three hours later and thereafter at days 1, 3, 7, 14, 21 and 28. The same plasma samples were simultaneously measured by high performance liquid chromatography (HPLC. Results The ELISA proved an easy-to-handle and very sensitive tool for the detection of CQ with a lower limit of detection at 3.9 ng/ml. ELISA levels of CQ in plasma showed high agreement with the levels obtained by HPLC (r = 0.98. The specificity in the negative control group was 100%. Conclusion The developed ELISA can be used for quality screening of CQ in pharmaceutical formulations and for drug monitoring in malaria and in other infectious diseases, such as HIV, where CQ proved to be an effective therapeutic agent. The methodology has been exploited to develop monoclonal

Performance of the multitarget Mikrogen Chlamydia trachomatis IgG ELISA in the prediction of tubal factor infertility (TFI) in subfertile women : Comparison with the Medac MOMP IgG ELISA plus

NARCIS (Netherlands)

van Ess, Eleanne F.; Ouburg, Sander; Spaargaren, Joke; Land, Jolande A.; Morre, Servaas A.

2017-01-01

There is a need for more accurate Chlamydia trachomatis (CT) IgG antibody tests for tubal factor infertility (TFI) diagnostics. We evaluated the predictive value for TFI of Medac ELISA plus (MOMP) and multitarget Mikrogen ELISA (MOMP-CPAF-TARP). Based on Medac ELISA plus results, 183 subfertile

Specificity of Toxocara ELISA in tropical populations.

Science.gov (United States)

Lynch, N R; Wilkes, L K; Hodgen, A N; Turner, K J

1988-05-01

The diagnosis of human infection by Toxocara canis relies heavily upon serological tests, the specificity of which can be inadequate in regions of endemic helminthiasis. When different population groups of tropical Venezuela were evaluated using ELISA based upon Toxocara excretory-secretory antigen (TcESA), solid-phase adsorption of the sera with extracts of a wide variety of non-homologous parasites revealed the existence of significant cross-reactivity. This was effectively and conveniently overcome when the test sera were incubated in the presence of the soluble parasite extracts in a competitive inhibition ELISA. The mean reduction of ELISA values caused by pre-adsorption of the sera tested was 32.2%, and that caused by competitive inhibition was 42.3%, the effects of these two procedures being strongly correlated (r = 0.83). The magnitude of the reduction was inversely proportional to the actual ELISA value (r = -0.55), and ranged from a mean of 68.0% in sera from apparently healthy individuals of medium-high socio-economic level, down to 28.1% in heavily parasitized Amazon indians. Ascaris showed the greatest degree of cross-reactivity in these tests, although under conditions of competitive inhibition even sera with high levels of antibody against this parasite could be negative in Toxocara ELISA. Western blotting revealed a major 81,400 D component that was shared between Ascaris and TcESA. Our results indicate that the competitive inhibition of cross-reactivity by soluble non-homologous parasite extracts provides a convenient and economical means of increasing the specificity of ELISA for the determination of the seroprevalence of toxocariasis in tropical populations.

EVALUATION OF AN O-ANTIGEN ELISA FOR SCREENING CATTLE HERDS FOR SALMONELLA-TYPHIMURIUM

DEFF Research Database (Denmark)

Hoorfar, Jeffrey; Bitsch, V.

1995-01-01

A total of 2585 serum samples from 62 dairy herds located in four different regions of Denmark were tested in an O-antigen (0:1,4,5,12)-based ELISA for the detection of antibodies against Salmonella typhimurium. Ten closed herds from an island with no reported occurrence of salmonellosis for seve......A total of 2585 serum samples from 62 dairy herds located in four different regions of Denmark were tested in an O-antigen (0:1,4,5,12)-based ELISA for the detection of antibodies against Salmonella typhimurium. Ten closed herds from an island with no reported occurrence of salmonellosis...... for several years, and 12 herds from a salmonella enzootic area which had had clinical outbreaks of S typhimurium were used to define a herd ELISA cut-off value. When herds with at least 5 per cent of the serum samples having an optical density of >0.5 were considered ELISA-positive, all 10 herds from...... the salmonellosis-free island were ELISA-negative, and all but one of the 12 S typhimurium-infected herds were ELISA-positive, which resulted in a herd test sensitivity of 0.92 and herd test specificity of 1.0. Eleven of the 12 S typhimurium-infected herds were negative in a blocking ELISA based on a monoclonal...

The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis.

Science.gov (United States)

Davaasuren, Batdorj; Amgalanbaatar, Tovuu; Musinguzi, Simon Peter; Suganuma, Keisuke; Otgonsuren, Davaajav; Mossaad, Ehab; Narantsatsral, Sandagdorj; Battur, Banzragch; Battsetseg, Badgar; Xuan, Xuenan; Inoue, Noboru

2017-09-15

Trypanosoma equiperdum, which is the etiological agent of dourine, spreads through sexual intercourse in equines. Dourine (T. equiperdum) has been reported in Mongolia, where it is considered an economically important disease of horses. T. evansi has also been reported in Mongolian domestic animals. The objective of this study was to evaluate the potential application of recombinant T. evansi GM6 (rTeGM6-4r)-based diagnostic methods on a farm with an outbreak of non-tsetse transmitted horse trypanosomosis. Ninety-seven percent homology was found between the amino acid sequences of T. equiperdum GM6 and the GM6 of another Trypanozoon, which also shared the same cellular localization. This finding suggests the utility of rTeGM6-4r-based serodiagnostic methods for epidemiological studies and the diagnosis of both surra and dourine in Equidae. Fifty blood samples were examined from a herd of horses. The diagnostic value of an rTeGM6-4r-based ELISA and an rTeGM6-4r-based immunochromatographic test (ICT) were measured in comparison to a T. evansi crude antigen-based ELISA, which is a diagnostic method recommended by the OIE. However, this is not a perfect diagnostic method for trypanosomosis. Positive serum samples were detected in 46%, 42% and 28% of the tested horses using an rTeGM6-4r-based ELISA, crude antigen-based ELISA and rTeGM6-4r-based ICT, respectively. The sensitivity of rTeGM6-based ELISA was 81%, the specificity was 79%, and the agreement was moderate. We conclude that rTeGM6-4r-based ELISA and ICT represent alternative options for baseline epidemiological studies and the on-site diagnosis of horse trypanosomoses in the field, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy.

Science.gov (United States)

Liu, Y; Mundt, E; Mundt, A; Sylte, M; Suarez, D L; Swayne, D E; García, M

2010-03-01

An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus, purified, recombinant N1 protein from A/chicken/Indonesia/PA7/2003 (H5N1) virus. The N1-ELISA showed high selectivity for detection of N1 antibodies, with no cross-reactivity with other neuraminidase subtypes, and broad reactivity with sera to N1 subtype isolates from North American and Eurasian lineages. Sensitivity of the N1-ELISA to detect N1 antibodies in turkey sera, collected 3 wk after H1N1 vaccination, was comparable to detection of avian influenza antibodies by the commercial, indirect ELISAs ProFLOK AIV Plus ELISA Kit (Synbiotics, Kansas City, MO) and Avian Influenza Virus Antibody Test Kit (IDEXX, Westbrook, ME). However, 6 wk after vaccination, the Synbiotics ELISA kit performed better than the N1-ELISA and the IDEXX ELISA kit. An evaluation was made of the ability of the N1-ELISA to discriminate vaccinated chickens from subsequently challenged chickens. Two experiments were conducted, chickens were vaccinated with inactivated H5N2 and H5N9 viruses and challenged with highly pathogenic H5N1 virus, and chickens were vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected at 14 days postchallenge and tested by hemagglutination inhibition (HI), quantitative neuraminidase inhibition (NI), and N1-ELISA. At 2 days postchallenge, oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. The N1-ELISA was in fair agreement with VI and HI results. Although the N1-ELISA showed a lower sensitivity than the NI assay, it was demonstrated that detection of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus in vaccinated chickens. Therefore, N1-ELISA can facilitate a vaccination strategy with differentiation of infected from vaccinated animals using a neuraminidase heterologous approach.

Evaluation of antigen and antibody ELISA's for epidemiological surveys of tsetse-transmitted trypanosomosis in cattle

International Nuclear Information System (INIS)

Eisler, M.C.; Hopkins, J.S.; Machila, N.; Bossche, P. van den; Peregrine, A.S.; Luckins, A.G.

2000-01-01

Sensitivity and specificity of the FAO/IAEA antigen-detection enzyme-linked immunosorbent assay (ELISA) kits for diagnosis of bovine trypanosomosis were investigated using sera from experimental cattle infected by tsetse challenge with cloned populations of Trypanosoma congolense (3 populations) or T. vivax (1 population). The kits are based on monoclonal antibodies that recognise internal antigens of tsetse-transmitted trypanosomes. Ten cattle were infected with each trypanosome population for at least 60 days, and in combination with uninfected cohorts (n=16) were used in a double-blind study design. Sensitivity and specificity of the tests depended on the choice of positive-negative thresholds expressed as percent positivity with respect to the median OD of 4 replicates of the strong positive reference serum provided with the kit. In general, while overall specificities were high, sensitivities of the antigen-ELISA's were poor. For example, at a cut-off of 5% positivity, the sensitivities of the antigen-ELISA's were 11% for samples (n=1162) from T. congolense infected cattle (n=30), and 24% for samples (n=283) from T. vivax infected cattle (n=10). The corresponding specificity values were 95% and 79%, respectively. There were no values of the positive-negative threshold at which both sensitivity and specificity were satisfactory. Trypanosome species-specificities of the antigen-ELISA's were also poor. Sensitivity and species-specificity of the antigen-ELISA for T. brucei infections were not investigated. The indirect ELISA for the detection of anti-trypanosomal antibodies in bovine serum was adapted for use with dried blood spots on filter paper, and standardised using a strong positive reference serum and the percent positivity system of data expression. The antibody-ELISA was evaluated in Zambia for use in epidemiological surveys of the prevalence of tsetse-transmitted bovine trypanosomosis. Known negative samples (sera, n=209; blood spots, n=466) were

Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

International Nuclear Information System (INIS)

Liang, Yi; Huang, Xiaolin; Yu, Ruijin; Zhou, Yaofeng; Xiong, Yonghua

2016-01-01

The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H_2O_2) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H_2O_2 or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL"−"1 to 625 pg mL"−"1 with a limit of detection of 2.2 pg mL"−"1, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. - Highlights: • A novel fluorescence ELISA was first developed for the detection of OTA by using GOx-mediated fluorescence quenching of QDs. • The pH- and H_2O_2-sensitive MPA-capped CdTe QDs were used as a fluorescent signal output to improve the detection sensitivity. • This novel method open up a different vision to detect other mycotoxins and biomolecules.

Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.

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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.

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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.

A chromogranin A ELISA absent of an apparent high-dose hook effect observed in other chromogranin A ELISAs.

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Erickson, J Alan; Grenache, David G

2016-01-15

Routine testing for chromogranin A (CgA) using an established commercial ELISA revealed an apparent high-dose hook effect in approximately 15% of specimens. Investigations found the same effect in two additional ELISAs. We hypothesized that a CgA derived peptide(s) at high concentrations was responsible but experiments were inconclusive. Here we describe the analytical performance characteristics of the Chromoa™ CgA ELISA that did not display the apparent high-dose hook effect. Performance characteristics of the Chromoa ELISA were assessed. The reference interval was established utilizing healthy volunteers. Specimens producing the apparent high-dose hook effect in other assays were evaluated using the Chromoa ELISA. The limit of detection was 8ng/ml. Linearity was acceptable (slope=1.04, intercept=18.1 and r(2)=0.997). CVs were ≤4.6 and ≤9.3% for repeatability and within-laboratory imprecision, respectively. CgA was stable at ambient and refrigerated temperatures for a minimum of two and 14days, respectively. An upper reference interval limit of 95ng/ml was established. Specimens demonstrating the apparent high-dose hook effect in other ELISAs did not exhibit the phenomenon using the Chromoa ELISA. The Chromoa ELISA demonstrates acceptable performance for quantifying serum CgA. The apparent high-dose hook effect exhibited in other ELISAs was absent using the Chromoa assay. Copyright © 2015 Elsevier B.V. All rights reserved.

Elisa for the diagnosis and epidemiology of Brucella abortus infection in cattle in Chile

International Nuclear Information System (INIS)

Rojas, X.; Alonso, O.

1998-01-01

A serum bank of 1251 adult cows sera was prepared. The sera originated from animals of three different epidemiological groups: 1) 244 from infected cows, strain 19 vaccinated when calves; 2) 507 from herds free of infection but all cows were strain 19 vaccinated when calves and 3) the last group, 500 sera from cows free of infection and non-vaccinated. All the sera where tested with the routine Rose Bengal (RB) Rivanol (RIV) and Complement Fixation (CF) tests and additionally three enzyme immunoassays were performed. They included two indirect Elisa both using the kit from the Joint FAO/IAEA Division, Vienna, Austria. One assay used a polyclonal conjugated antibody (I-ELISAp) and the other a monoclonal conjugated antibody (I-ELISAm). The third assay was a competitive ELISA (C-ELISA) performed with sLPS, plus monoclonal antibody, M84, and goat anti-mouse antibody-HRPO. Using the CFT as 'gold standard' the sensitivities of all the methods were: RB 87.1%, RIV 87.1%, I-ELISAp 100% I-ELISAm 100%. The calculated specificity was: RB 100%, RIV 100%, I-ELISAp 96.4% and I-ELISAm 100%. In the group of infected animals (244) the following results were obtained: RB 13.5%, RIV 11.9%, CF 12.7%, I-ELISAp 50.8% and I-ELISAm 22.9%. Results for the non-vaccinated group were: RB 0.2%, RIV 0%, CFT 0.2%, I-ELISAp 6.9% and I-ELISAm 2.9%. The C-ELISA was performed on samples from the positive group or with positivity values close to the cut-off value in the I-ELISAm. In the infected group 28 out of 63 animals were detected as infected and from the non-vaccinated herds none of 15 I-ELISAm positive samples were detected as infected in the C-ELISA. (author)

Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

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Liang, Yi [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047 (China); Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Zhou, Yaofeng [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047 (China)

2016-09-14

The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H{sub 2}O{sub 2}) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H{sub 2}O{sub 2} or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL{sup −1} to 625 pg mL{sup −1} with a limit of detection of 2.2 pg mL{sup −1}, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. - Highlights: • A novel fluorescence ELISA was first developed for the detection of OTA by using GOx-mediated fluorescence quenching of QDs. • The pH- and H{sub 2}O{sub 2}-sensitive MPA-capped CdTe QDs were used as a fluorescent signal output to improve the detection sensitivity. • This novel method open up a different vision to detect other mycotoxins and biomolecules.

Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment.

Science.gov (United States)

Herremans, Tineke; Hogema, Boris M; Nabuurs, Marrigje; Peeters, Marcel; Wegdam-Blans, Marjolijn; Schneeberger, Peter; Nijhuis, Carla; Notermans, Daan W; Galama, Joep; Horrevorts, Anton; van Loo, Inge H M; Vlaminckx, Bart; Zaaijer, Hans L; Koopmans, Marion P; Berkhout, Hanneke; Socolovschi, Cristina; Raoult, Didier; Stenos, John; Nicholson, William; Bijlmer, Henk

2013-01-01

The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever. Copyright © 2013 Elsevier Inc. All rights reserved.

Development of a Monoclonal Antibody-Based Sandwich ELISA for Peanut Allergen Ara h 1 in Food

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Chuanlai Xu

2013-07-01

Full Text Available We have established a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA based on two monoclonal antibodies (mAb to measure the content of the major peanut allergen Ara h 1 in foods. Two mAbs were selected out of 12 murine hybridoma cells secreting Ara h 1-specific antibody. Using mAb 6 as the capture antibody and HRP-labelled mAb 4 as the detection antibody, the limit of detection (LOD the assay was 0.34 ng/mL. Cross-reaction analysis showed that this method was strongly specific and had no cross-reactions with Ara h 2, pea protein or soy protein. Sample analysis showed that this ELISA was a useful tool to monitor peanut allergens in food products by measuring Ara h 1 content.

Indirect three-dimensional printing of synthetic polymer scaffold based on thermal molding process

International Nuclear Information System (INIS)

Park, Jeong Hun; Jung, Jin Woo; Cho, Dong-Woo; Kang, Hyun-Wook

2014-01-01

One of the major issues in tissue engineering has been the development of three-dimensional (3D) scaffolds, which serve as a structural template for cell growth and extracellular matrix formation. In scaffold-based tissue engineering, 3D printing (3DP) technology has been successfully applied for the fabrication of complex 3D scaffolds by using both direct and indirect techniques. In principle, direct 3DP techniques rely on the straightforward utilization of the final scaffold materials during the actual scaffold fabrication process. In contrast, indirect 3DP techniques use a negative mold based on a scaffold design, to which the desired biomaterial is cast and then sacrificed to obtain the final scaffold. Such indirect 3DP techniques generally impose a solvent-based process for scaffold fabrication, resulting in a considerable increase in the fabrication time and poor mechanical properties. In addition, the internal architecture of the resulting scaffold is affected by the properties of the biomaterial solution. In this study, we propose an advanced indirect 3DP technique using projection-based micro-stereolithography and an injection molding system (IMS) in order to address these challenges. The scaffold was fabricated by a thermal molding process using IMS to overcome the limitation of the solvent-based molding process in indirect 3DP techniques. The results indicate that the thermal molding process using an IMS has achieved a substantial reduction in scaffold fabrication time and has also provided the scaffold with higher mechanical modulus and strength. In addition, cell adhesion and proliferation studies have indicated no significant difference in cell activity between the scaffolds prepared by solvent-based and thermal molding processes. (paper)

Diagnostic Value of ELISA Tests for the Detection of Specific Antibodies in Cats and Rabbits with Dermatophytosis

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Marinka Drobnič-Košorok

2002-01-01

Full Text Available Two indirect ELISA tests developed for the detection of specific IgG in cats and rabbits, infected with M. canis and T. mentagrophytes, respectively, were evaluated and compared. The levels of specific antibodies were determined in sera of 20 cats and 25 rabbits naturally infected with M. canis and T. mentagrophytes, respectively. Infection was confirmed by the results of fungal culture. Blood samples from 12 cats and 17 rabbits, previously unexposed to dermatophytes, served as negative controls. A significant increase in the level of specific antibodies in groups of infected animals was demonstrated. Sensitivity, specificity and predictive values of a positive and a negative test were determined to evaluate the diagnostic potential. ELISA for the detection of specific antibodies in cats infected with M. canis (ELISA-cats test exhibited 75.0 % of sensitivity at 91.7 % of specificity, whereas the test for the detection of specific antibodies in rabbits, infected with T. mentagrophytes (ELISA-rabbits test is highly sensitive (96.0 % and highly specific (94.1 %, confirming its encouraging diagnostic potential. The cross-reactivity of fungal antigens was tested by performing the assays with antigens M. canis, T. mentagrophytes, M. pachydermatis and A. fumigatus. There were no significant indications of cross-reactions in the test T. mentagrophytes-rabbits, whereas strong cross-reaction between dermatophyte antigens was observed in the test M. canis-cats.

Detection of toxoplasma-specific immunoglobulin G in human sera: performance comparison of in house Dot-ELISA with ECLIA and ELISA.

Science.gov (United States)

Teimouri, Aref; Modarressi, Mohammad Hossein; Shojaee, Saeedeh; Mohebali, Mehdi; Zouei, Nima; Rezaian, Mostafa; Keshavarz, Hossein

2018-05-08

In the current study, performance of electrochemiluminescence immunoassay (ECLIA) in detection of anti-toxoplasma IgG in human sera was compared with that of enzyme-linked immunosorbent assay (ELISA). Furthermore, performance of an in house Dot-ELISA in detection of anti-toxoplasma IgG was compared with that of ECLIA and ELISA. In total, 219 human sera were tested to detect anti-toxoplasma IgG using Dynex DS2® and Roche Cobas® e411 Automated Analyzers. Discordant results rechecked using immunofluorescence assay (IFA). Then, sera were used in an in house Dot-ELISA to assess toxoplasma-specific IgG. Of the 219 samples, two samples were found undetermined using ECLIA but reactive using ELISA. Using IFA, the two sera were reported unreactive. Furthermore, two samples were found reactive using ECLIA and unreactive using ELISA. These samples were reported reactive using IFA. The overall agreement for the two former methods was 98% (rZ0.98.1; P house Dot-ELISA included sensitivity of 79.5, specificity of 78.2, and accuracy of 78.9%, compared to ECLIA and ELISA. Positive and negative predictive values included 82.9 and 74.2%, respectively. A 100% sensitivity was found in in house Dot-ELISA for highly reactive sera in ECLIA and ELISA. ECLIA is appropriate for the first-line serological screening tests and can replace ELISA due to high speed, sensitivity, and specificity, particularly in large laboratories. Dot-ELISA is a rapid, sensitive, specific, cost-effective, user-friendly, and field-portable technique and hence can be used for screening toxoplasmosis, especially in rural fields or less equipped laboratories.

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

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Elisabetta Casarin

Full Text Available Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR disease. The avidin-nucleic-acid-nanoassembly (ANANAS is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

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Casarin, Elisabetta; Lucchese, Laura; Grazioli, Santina; Facchin, Sonia; Realdon, Nicola; Brocchi, Emiliana; Morpurgo, Margherita; Nardelli, Stefano

2016-01-01

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits

[Use of new immunoglobulin isotype-specific ELISA-systems to detect Salmonella infections in pigs].

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Ehlers, Joachim; Alt, Michael; Trepnau, Daniela; Lehmann, Jörg

2006-01-01

In Germany, the program for controlling salmonella infections in pigs is based on tests detecting salmonella-lipopolysaccharide (LPS) induced antibodies in meat-juice or blood. These conventional tests which are based on the technology of enzyme-linked immunosorbent assay (ELISA) detect exclusively or mainly immunoglobulin(lg)G antibodies. Meanwhile, novel ELISA systems (WCE-ELISA, 3-Isotype-Screening-ELISA) have been developed, which additionally detect the antibody classes IgM and IgA.This fact enables the registration of fresh salmonella infections (starting with day 5 p.i.) and thus, the distinction between early and older infections. The results show that animals with early salmonella infections appear significantly more often in herds with a high than with a low prevalence. With the newly developed tests this group of animals can be detected much more efficiently and precisely than with the tests used so far. Due to their clearly improved sensitivity the application of the WCE-ELISA and the 3-Isotype-Screening-ELISA in terms of the QS-Salmonella-Monitoring program can therefore significantly improve the selection of farms with potential salmonella excretors. Additionally, the WCE-ELISA can be applied very suitable for the examination of individual animals.

Undergraduate Laboratory Module for Implementing ELISA on the High Performance Microfluidic Platform

Science.gov (United States)

Giri, Basant; Peesara, Ravichander R.; Yanagisawa, Naoki; Dutta, Debashis

2015-01-01

Implementing enzyme-linked immunosorbent assays (ELISA) in microchannels offers several advantages over its traditional microtiter plate-based format, including a reduced sample volume requirement, shorter incubation period, and greater sensitivity. Moreover, microfluidic ELISA platforms are inexpensive to fabricate and allow integration of…

An Evidence-Based Approach to Plum Pox Virus Detection by DASI-ELISA and RT-PCR in Dormant Period

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Antonio Olmos

2008-01-01

Full Text Available An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASIELISA and RT-PCR.

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.

Validation of an indirect ELISA to detect antibodies against BoHV-1 in bovine and guinea-pig serum samples using ISO/IEC 17025 standards.

Science.gov (United States)

Parreño, Viviana; Romera, S Alejandra; Makek, Lucia; Rodriguez, Daniela; Malacari, Darío; Maidana, Silvina; Compaired, Diego; Combessies, Gustavo; Vena, María Marta; Garaicoechea, Lorena; Wigdorovitz, Andrés; Marangunich, Laura; Fernandez, Fernando

2010-10-01

Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections. Copyright (c) 2010 Elsevier B.V. All rights reserved.

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.

Grantee Spotlight: Elisa Rodriguez, Ph.D., M.S.

Science.gov (United States)

Dr. Elisa M. Rodriguez tests the feasibility of community-based participatory research approaches to engaging Hispanics, African Americans, and the medically underserved in the Buffalo, NY area in biospecimen donation for cancer research.

Application of 60Co γ-ray irradiated polystyrene microplate for anti-HCV ELISA

International Nuclear Information System (INIS)

Feng Bo; Zeng Hongyan; Tang Yufang; Wang Lu

2005-01-01

In order to explore the effect of 60 Co γ-ray irradiation on minor polypeptides absorption of polystyrene microplate, an indirect ELISA detection of anti-HCV was established, 60 Co γ-ray irradiated polystyrene microplates and the controls (without irradiation or UV-irradiated) were applied to absorb recombinant HCV antigens respectively. Cooperated with Bovine antihuman IgG labelled HRP, their related indices of sensitivity, specificity, homogeneity and stability were determinate. The results indicated that, optimum dose of the γ-ray irradiation is 8 kGy, and compared with the controls, detection sensitivity and homogeneity of the polystyrene microplate irradiated to 8 kGy could be improved markedly. (authors)

Influence of a peracetic acid-based immersion on indirect composite resin.

Science.gov (United States)

Samuel, Susana Maria Werner; Fracaro, Gisele Baggio; Collares, Fabrício Mezzomo; Leitune, Vicente Castelo Branco; Campregher, Ulisses Bastos

2011-06-01

The aim of this study was to evaluate the influence of immersion in a 0.2% peracetic acid-based disinfectant on the three-point flexural strength, water sorption and water solubility of an indirect composite resin. Specimens were produced according to ISO 4049:2000 specifications and were divided in two groups: Control group, with no disinfection and Disinfected group, with three 10 min immersions in the peracetic acid intercalated with 10 min immersions in sterile distilled water. All evaluations were conducted in compliance with ISO specifications. Three-point flexural strength, water sorption and solubility of indirect composite resin before and after immersion showed no statistical significant differences (p > 0.05) and met ISO standard requirements. Immersion in peracetic acid solution showed no influence in indirect composite resin tested properties.

Toxoplasma gondii antibodies sheep in Lages, Santa Catarina, Brazil, and comparison using IFA and ELISA Anticorpos toxoplásmicos em ovinos de Lages, Santa Catarina, Brasil, e comparação utilizando RIFI e ELISA

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Francine Bragagnolo Liz Stefen Sakata

2012-09-01

Full Text Available Toxoplasmosis in sheep is a disease of great importance in veterinary medicine, which causes economic losses in livestock and has a great impact on human health, since consumption of infected meat facilitates transmission of zoonotic infections. Blood samples from sheep (n = 360 were collected from 13 farm properties in the municipality of Lages, Santa Catarina, to estimate the prevalence of toxoplasmosis and identify risk factors associated with Toxoplasma gondii infection. T. gondii, antibodies were investigated by means of the indirect immunofluorescence assay (IFA and enzyme-linked immunosorbent assay (ELISA. Animals infected with T. gondii were found on 100% of the farms. IFA detected 56.9% (205/360 and ELISA 42.5% of the infected sheep. Breed was the only risk factor associated with the presence of T. gondii antibodies. ELISA showed sensitivity of 61%, specificity of 82% and kappa of 0.41, which was considered moderate. This allows use of ELISA as an alternative technique for diagnosing T. gondii in sheep.A toxoplasmose ovina é uma doença parasitária de elevada importância em medicina veterinária e em saúde pública, acarretando prejuízos na produção animal, gerados pelas perdas reprodutivas e econômicas, além de sua implicação na saúde humana, já que o consumo de carne infectada facilita a transmissão zoonótica. Para determinar a prevalência e identificar fatores de risco para a infecção por T. gondii em ovinos de Lages, Santa Catarina, amostras de sangue (n = 360 foram coletadas em 13 propriedades. Cada criador respondeu a um questionário para permitir a identificação dos fatores de risco da infecção. A pesquisa de anticorpos foi realizada por meio da Reação de Imunofluorescência Indireta (RIFI > 64 e do Ensaio Imunoenzimático Indireto (ELISA. Em 100% das propriedades foram encontrados animais positivos. Pela RIFI, 205 (56,94% ovinos apresentaram anticorpos contra T. gondii e pelo ELISA, 153 (42

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.

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.

Improved detection of equine antibodies against Sarcocystis neurona using polyvalent ELISAs based on the parasite SnSAG surface antigens.

Science.gov (United States)

Yeargan, Michelle R; Howe, Daniel K

2011-02-28

Equine protozoal myeloencephalitis (EPM) is a common neurologic disease of horses that is caused by the apicomplexan pathogen Sarcocystis neurona. To help improve serologic diagnosis of S. neurona infection, we have modified existing enzyme-linked immunosorbent assays (ELISAs) based on the immunogenic parasite surface antigens SnSAG2, SnSAG3, and SnSAG4 to make the assays polyvalent, thereby circumventing difficulties associated with parasite antigenic variants and diversity in equine immune responses. Two approaches were utilized to achieve polyvalence: (1) mixtures of the individual recombinant SnSAGs (rSnSAGs) were included in single ELISAs; (2) a collection of unique SnSAG chimeras that fused protein domains from different SnSAG surface antigens into a single recombinant protein were generated for use in the ELISAs. These new assays were assessed using a defined sample set of equine sera and cerebrospinal fluids (CSFs) that had been characterized by Western blot and/or were from confirmed EPM horses. While all of the polyvalent ELISAs performed relatively well, the highest sensitivity and specificity (100%/100%) were achieved with assays containing the rSnSAG4/2 chimera (Domain 1 of SnSAG4 fused to SnSAG2) or using a mixture of rSnSAG3 and rSnSAG4. The rSnSAG4 antigen alone and the rSnSAG4/3 chimera (Domain 1 of SnSAG4 fused to Domain 2 of SnSAG3) exhibited the next best accuracy at 95.2% sensitivity and 100% specificity. Binding ratios and percent positivity (PP) ratios, determined by comparing the mean values for positive versus negative samples, showed that the most advantageous signal to noise ratios were provided by rSnSAG4 and the rSnSAG4/3 chimera. Collectively, our results imply that a polyvalent ELISA based on SnSAG4 and SnSAG3, whether as a cocktail of two proteins or as a single chimeric protein, can give optimal results in serologic testing of serum or CSF for the presence of antibodies against S. neurona. The use of polyvalent SnSAG ELISAs will

Hyperglucagonaemia analysed by glucagon sandwich ELISA

DEFF Research Database (Denmark)

Albrechtsen, Nicolai Jacob Wewer; Hartmann, Bolette; Veedfald, Simon

2014-01-01

the extent to which the hyperglucagonaemia measured in clinical samples was caused by authentic glucagon. METHODS: We examined the performance of three commercial glucagon 'sandwich' ELISAs. The ELISA with the best overall performance was selected to compare glucagon measurements in clinical samples...... sensitivity for glucagon in plasma (>10-20 pmol/l). Thus, only the third assay was suitable for measuring glucagon concentrations in clinical samples. The ELISA and RIA measured similar glucagon levels in healthy individuals. Measurements of samples from individuals with abnormally high (type 2 diabetes...... or obese) or very elevated (post vagotomy with pyloroplasty, post-RYGB) glucagon levels were also similar in both assays. However, glucagon levels in participants with ESRD were much lower when measured by ELISA than by RIA, indicating that the apparent hyperglucagonaemia is not caused by fully processed...

Comparison of the Hemagglutination Inhibition Test and IgG ELISA in Categorizing Primary and Secondary Dengue Infections Based on the Plaque Reduction Neutralization Test

Directory of Open Access Journals (Sweden)

Nurhayati Lukman

2016-01-01

Full Text Available Secondary dengue infection by heterotypic serotypes is associated with severe manifestations of disease, that is, dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS. The World Health Organization (WHO has recommended criteria based on the hemagglutination inhibition (HI test to distinguish between primary and secondary dengue infections. Since the HI test has practical limitations and disadvantages, we evaluated the accuracy of WHO HI criteria and compared it with criteria based on an IgG enzyme-linked immunosorbent assay (ELISA using a plaque reduction neutralization test (PRNT as the gold standard. Both WHO HI criteria and IgG ELISA criteria performed strongly (16/16 in determining primary infection. However, to determine secondary infection, the IgG ELISA criteria performed better (72/73 compared to the WHO HI criteria (23/73.

Use of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma hyosynoviae distributions in pig herds with or without clinical arthritis

DEFF Research Database (Denmark)

Nielsen, Elisabeth Okholm; Lauritsen, Klara Tølbøll; Friis, Niels Filskov

2005-01-01

on porcine serum following experimental Mycoplasma infections as well as by analysis of samples from one Danish herd known to be free of M. hyosynoviae and samples from two Norwegian herds without clinical suspicion of M. hyosynoviae infections since their establishment. The epidemiology of M. hyosynoviae...... determined by culture of cross-sectional samples of whole-blood (n = 238) and tonsil scrapings (n = 322), respectively. Levels of serum antibodies (n = 396) were measured by the novel indirect ELISA test. There was no significant difference in the ELISA results between the MhA and the MhC herds. Pigs...

Indication of viruses and virus-specific antibodies by ELISA using conjugates based on β-lactamase obtained by genetic engineering

International Nuclear Information System (INIS)

Kharitonenkov, I.G.; Kordym, V.A.; Khristova, M.L.; Leonov, S.V.; Kirillova, V.S.; Chernykh, S.I.

1987-01-01

The method of enzyme-linked immunosorbent assay (ELISA), by means of which antigens and antibodies of different origin can be detected with high sensitivity and specificity, is an immunoenzymatic technique based on the use of conjugates, or macromolecular complexes formed by covalent attachment of enzyme molecules to antigen or antibody molecules. Conjugates based on peroxidase, alkaline phosphatase, and beta-galactosidase are most frequently used to construct immunoenzymatic test systems. The use of these enzymes in ELISA, however, is complicated by the fact that they are often present in free or bound form in the biological material under study, and that their substrates either possess low stability, are difficult to synthesize, or are toxic. In this paper, in order to avoid these shortcomings, the authors develop a method for the biosynthesis of lactamase conjugates which is based on genetic engineering, and demonstrate the viability and stability of these conjugates in radioimmunoenzymatic assay of viruses

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

Directory of Open Access Journals (Sweden)

Luiz Tadeu M. Figueiredo

1988-06-01

Full Text Available A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380. Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.A caracterização antigênica do vírus Jatobal (BeAn 423380 foi efetuada utilizando uma técnica de ELISA para deteccão de anticorpos que utiliza culturas celulares infectadas como antígeno e um micro teste de neutralização para vírus que utiliza o método imunoenzimático como auxiliar para a leitura dos resultados (NT-EIA. O vírus Jatobal foi caracterizado como um Bunyaviridae, gênero Bunyavirus, pertencente ao sorogrupo Simbu. A técnica de ELISA, utilizando culturas celulares infectadas como antígeno, trata-se de método sensível e confiável na identificação de agentes virais, possuindo muitas vantagens sobre ELISA convencionais e outros testes: elimina a preparação laboriosa de antígenos para o revestimento em fase sólida; permite que se teste de forma mais rápida e fácil que por CF, HAI e neutralização por redução de plaques um grande número de antisoros de vírus. ELISA e NT-EIA podem ser utilizados para a classificação de vírus que não produzem

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.

Introduction and use of ELISA-based technologies for the diagnosis and monitoring of foot-and-mouth disease in Hong Kong

International Nuclear Information System (INIS)

Sims, L.D.; Dyrting, K.C.; Lo, W.C.; Wong, K.W.

2000-01-01

ELISA-based tests were introduced to assist in the diagnosis and control of foot-and-mouth disease (FMD) in Hong Kong. The tests were used to identify and type FMD viruses in clinical samples, to provide an assessment of the efficacy of vaccination programmes as practised, to train staff in ELISA technology and to strengthen quality assurance for foot-and-mouth disease and other diagnostic tests. These tests have provided the tools needed to understand why foot-and-mouth disease occurs in the face of vaccination - an essential step towards control of this disease in Hung Kong. (author)

Constraining stellar binary black hole formation scenarios with eLISA eccentricity measurements

Science.gov (United States)

Nishizawa, Atsushi; Sesana, Alberto; Berti, Emanuele; Klein, Antoine

2017-03-01

A space-based interferometer such as the evolved Laser Interferometer Space Antenna (eLISA) could observe a few to a few thousands of progenitors of black hole binaries (BHBs) similar to those recently detected by Advanced LIGO. Gravitational radiation circularizes the orbit during inspiral, but some BHBs retain a measurable eccentricity at the low frequencies where eLISA is the most sensitive. The eccentricity of a BHB carries precious information about its formation channel: BHBs formed in the field, in globular clusters, or close to a massive black hole (MBH) have distinct eccentricity distributions in the eLISA band. We generate mock eLISA observations, folding in measurement errors, and using a Bayesian model selection, we study whether eLISA measurements can identify the BHB formation channel. We find that a handful of observations would suffice to tell whether BHBs were formed in the gravitational field of an MBH. Conversely, several tens of observations are needed to tell apart field formation from globular cluster formation. A 5-yr eLISA mission with the longest possible armlength is desirable to shed light on BHB formation scenarios.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples

Science.gov (United States)

Nekouei, Omid; Durocher, Jean; Keefe, Greg

2016-01-01

This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. PMID:27429469

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.

Science.gov (United States)

Liu, Xing; Tang, Zongwen; Duan, Zhenhua; He, Zhenyun; Shu, Mei; Wang, Xianxian; Gee, Shirley J; Hammock, Bruce D; Xu, Yang

2017-03-01

A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC 50 of 0.64ng/mL and a linear range (IC 20 -IC 80 ) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of Candida albicans Sap2 in cancer patient serum samples by an indirect competitive enzyme-linked immunosorbent assay for the diagnosis of candidiasis.

Science.gov (United States)

Wang, Yicun; Gao, Xiang; Zhi Gang, J U; Liu, Jingyuan; Dong, Shuai; Wang, Li

2013-01-01

The secreted aspartyl proteinases 2 (Sap2) of Candida albicans (C. albicans) is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1:1200 in an indirect ELISA, and detected Sap2 concentration up to 1 ng/mL. Of the 286 cancer serum samples tested, 16.8% were found as candidiasis. The test was simple and economical to perform and had a level of sensitivity for detection of low-titer positive sera; thus, it may be proven to be of value in epidemiological studies on candidiasis.

Interactions Between Indirect DC-Voltage Estimation and Circulating Current Controllers of MMC-Based HVDC Transmission Systems

DEFF Research Database (Denmark)

Wickramasinghe, Harith R.; Konstantinou, Georgios; Pou, Josep

2018-01-01

Estimation-based indirect dc-voltage control in MMCs interacts with circulating current control methods. This paper proposes an estimation-based indirect dc-voltage control method for MMC-HVDC systems and analyzes its performance compared to alternative estimations. The interactions between......-state and transient performance is demonstrated using a benchmark MMC-HVDC transmission system, implemented in a real-time digital simulator. The results verify the theoretical evaluations and illustrate the operation and performance of the proposed indirect dc-voltage control method....

Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA.

Science.gov (United States)

Arilla, M C; Ibarrola, I; Eraso, E; Aguirre, M; Martínez, A; Asturias, J A

2001-08-01

Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts. To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens. Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate. The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts. This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.

A Simple and Specific Noncompetitive ELISA Method for HT-2 Toxin Detection

Directory of Open Access Journals (Sweden)

Henri O. Arola

2017-04-01

Full Text Available We developed an HT-2 toxin-specific simple ELISA format with a positive read-out. The assay is based on an anti-immune complex (IC scFv antibody fragment, which is genetically fused with alkaline phosphatase (AP. The anti-IC antibody specifically recognizes the IC between a primary anti-HT-2 toxin Fab fragment and an HT-2 toxin molecule. In the IC ELISA format, the sample is added together with the scFv-AP antibody to the ELISA plate coated with the primary antibody. After 15 min of incubation and a washing step, the ELISA response is read. A competitive ELISA including only the primary antibody recognizes both HT-2 and T-2 toxins. The anti-IC antibody makes the assay specific for HT-2 toxin, and the IC ELISA is over 10 times more sensitive compared to the competitive assay. Three different naturally contaminated matrices: wheat, barley and oats, were used to evaluate the assay performance with real samples. The corresponding limits of detection were 0.3 ng/mL (13 µg/kg, 0.1 ng/mL (4 µg/kg and 0.3 ng/mL (16 µg/kg, respectively. The IC ELISA can be used for screening HT-2 toxin specifically and in relevant concentration ranges from all three tested grain matrices.

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.

A noise simulator for eLISA: Migrating LISA Pathfinder knowledge to the eLISA mission

OpenAIRE

Armano, M.; Audley, H.; Auger, G.; Baird, J.; Binetruy, P.; Born, Michael; Bortoluzzi, D.; Brandt, N.; Bursi, A.; Caleno, M.; Cavalleri, A.; Cesarini, A.; Cruise, M.; Danzmann, Karsten; Diepholz, I.

2015-01-01

We present a new technical simulator for the eLISA mission, based on state space modeling techniques and developed in MATLAB. This simulator computes the coordinate and velocity over time of each body involved in the constellation, i.e. the spacecraft and its test masses, taking into account the different disturbances and actuations. This allows studying the contribution of instrumental noises and system imperfections on the residual acceleration applied on the TMs, the latter reflecting the ...

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

ELISA for Detection of Soya Proteins in Meat Products

Directory of Open Access Journals (Sweden)

Eva Renčová

2009-01-01

Full Text Available Indirect competitive ELISA method for the detection of soya proteins in meat products was developed. The detection limit of the method is 0.5% of the weight of added soya protein. A total of 131 meat product samples such as salamis or sausages from the Czech Republic market were investigated for the presence of soya proteins. Soya proteins were detected in 84% of the investigated samples without any declaration on the package of the product. The use of vegetable additives, namely soya in meat products in the market of the Czech Republic is very frequent and the restriction of its usage by legislation relates only to some kinds of durable products and ham (Act 264/2003 Coll.. The need for sensitive inspecting methods for soya protein detection is not only associated with the economic aspect (adulteration, but mainly with consumer health protection in case of allergy to soya proteins.

Evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect ELISAs using latent class analysis

DEFF Research Database (Denmark)

Toftaker, Ingrid; Toft, Nils; Stokstad, Maria

2018-01-01

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) are responsible for respiratory disease and diarrhea in cattle worldwide. The Norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. The objective of this study...... was to estimate sensitivity and specificity across different cut-off values for the MVD-Enferplex BCV/BRSV multiplex, by comparing them to a commercially available ELISA, the SVANOVIR® BCV-Ab and SVANOVIR® BRSV-Ab, respectively. We analyzed bulk tank milk samples from 360 herds in a low- and 360 herds in a high...

Hapten design and indirect competitive immunoassay for parathion determination: Correlation with molecular modeling and principal component analysis

Energy Technology Data Exchange (ETDEWEB)

Liu Yihua [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Jin Maojun [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Gui Wenjun [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Cheng Jingli [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Guo Yirong [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Zhu Guonian [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China)]. E-mail: zhugn@zju.edu.cn

2007-05-22

A novel procedure for parathion hapten design is described. The optimal antigen for parathion was selected after molecular modeling studies of six types of potentially immunizing haptens with the aim to identify the best mimicking target analyte. Heterologous competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed after screening a battery of competitors as coating antigens. The relationship between the heterology degree of the competitor and the resulting immunoassay detectability was investigated according to the electronic similarities of the competitor haptens and the target analyte. Molecular modeling and principal component analysis were performed to understand the electronic distribution and steric parameters of the haptens at their minimum energetic levels. The results suggested that the competitors should have a high heterology to produce assays with good detectability values. An indirect competitive ELISA was finally selected for further investigation. The immunoassay had an IC{sub 50} value of 4.79 ng mL{sup -1} and a limit of detection of 0.31 ng mL{sup -1}. There was little or no cross-reactivity to similar compounds tested except for the insecticide parathion-methyl, which showed a cross-reactivity of 7.8%.

[Effect evaluation of three ELISA kits in detection of fasciolasis].

Science.gov (United States)

Ai, Lin; Chen, Mu-Xin; Chen, Shao-Hong; Chu, Yan-Hong; Cai, Yu-Chun; Zhou, Xiao-Nong; Chen, Jia-Xu

2013-04-01

To evaluate the effect of 3 ELISA kits on detection of human fasciolasis. Twenty-six serum samples from patients with fasciolasis, 180 serum samples from patients with other parasitic diseases as well as 26 serum samples from healthy people were detected by ELISA kits which using soluble antigen of Fasciola gigantica, Fasciola hepatica (Fg-ELISA and Fh-ELISA) as well as IgG antigen ELISA detection kits made by DRG company in Germany. The effects of the 3 kits were evaluated. The sensitivities of Fg-ELISA, Fh-ELISA, and DRG-ELISA were 100.0%, 80.8% (95% CI: 65.7%-95.9%) and 100.0%, respectively; the specificities of the three were 87.9% (95% CI: 83.5%-92.4%), 85.0%(95% CI: 80.1%-89.9%) and 83.5% (95% CI: 78.4%-88.6%), respectively, and Youden indexes of them were 0.88, 0.66 and 0.84, respectively. The detection rate of Fg-ELISA (100%) was significantly higher than that of Fh-ELISA (80.8%) (P DRG-ELISA for clinical sample tests as well as massive screening in fasciolasis endemic areas in southwest China.

E.L.I.S.A. en coccidioidomicosis humana E.L.I.S.A. in human coccidioidomycosis

Directory of Open Access Journals (Sweden)

Iris Nora Tiraboschi

1991-08-01

Full Text Available Se realizó E.L.I.S.A. con exoantígeno de Coccidioides immitis para la detectión de anticuerpos, en 67 sueros humanos diluidos 1/1000, 1/2000, 1/4000 y 1/8000. De los 18 sueros de enfermos de coccidioidomicosis comprobada por examen directo, cultivo y/o histología, 5 fueron negativos, en otros 13 fueron positivos en una o varias diluciones. 3/26 sueros de personas sanas, coccidioidino positivas, fueron positivos en títulos de 1/1000 y el resto no tuvo anticuerpos detectables. No presentaron reacciones positivas ninguno de los sueros controles de personas sanas, pero sí lo hicieron 4/8 pacientes con otras micosis. Se concluye que E.L.I.S.A. es útil para la detección de mínimas cantidades de anticuerpos o en sueros que no pueden ser procesados por fijación de complemento. No es recomendable el uso de la técnica en forma aislada por al presencia de reacciones cruzadas.An E.L.I.S.A. test for antibody detection, with an exo-antigen of Coccidioides immitis was standardized in 67 humans sera diluited in 1/1000, 1/2000, 1/4000 and 1/8000. Eightheen sera from mycologically proved cases of coccidioidomycosis were studied: 5 were negative and 13 were positive in some dilutions. 3/26 sera of healthy persons who presented positive skin tests with coccidioidin were positive and the other 23 sera did not have positive reactions. None of the 15 sera of healthy human exhibited positive E.L.I.S.A. Serum samples of 8 patients suffering other deep mycosis were studied, 4 of them presented cross-reactions in E.L.I.S.A. tests. E.L.I.S.A. test seems to be a useful Serologic technique for antibody detection in anticomplementary serum samples or when a low concentration of antibodies should be detected. As it is very sensitive, cross-reactions with other mycoses are frequent, thus the use other more specific serologic technique together E.L.I.S.A. is recommended.

A sandwich ELISA for measurement of the primary glucagon-like peptide-1 metabolite

DEFF Research Database (Denmark)

Wewer Albrechtsen, Nicolai J; Asmar, Ali; Jensen, Frederik

2017-01-01

developed a sandwich ELISA recognizing both GLP-1 9-36NH2 and nonamidated GLP-1 9-37. The ELISA was validated using analytical assay validation guidelines and by comparing it to a subtraction-based method, hitherto employed for estimation of GLP-1 9-36NH2 Its accuracy was evaluated from measurements...

Ultrasensitive microfluidic solid-phase ELISA using an actuatable microwell-patterned PDMS chip.

Science.gov (United States)

Wang, Tanyu; Zhang, Mohan; Dreher, Dakota D; Zeng, Yong

2013-11-07

Quantitative detection of low abundance proteins is of significant interest for biological and clinical applications. Here we report an integrated microfluidic solid-phase ELISA platform for rapid and ultrasensitive detection of proteins with a wide dynamic range. Compared to the existing microfluidic devices that perform affinity capture and enzyme-based optical detection in a constant channel volume, the key novelty of our design is two-fold. First, our system integrates a microwell-patterned assay chamber that can be pneumatically actuated to significantly reduce the volume of chemifluorescent reaction, markedly improving the sensitivity and speed of ELISA. Second, monolithic integration of on-chip pumps and the actuatable assay chamber allow programmable fluid delivery and effective mixing for rapid and sensitive immunoassays. Ultrasensitive microfluidic ELISA was demonstrated for insulin-like growth factor 1 receptor (IGF-1R) across at least five orders of magnitude with an extremely low detection limit of 21.8 aM. The microwell-based solid-phase ELISA strategy provides an expandable platform for developing the next-generation microfluidic immunoassay systems that integrate and automate digital and analog measurements to further improve the sensitivity, dynamic ranges, and reproducibility of proteomic analysis.

Detection of Candida albicans Sap2 in cancer patient serum samples by an indirect competitive enzyme-linked immunosorbent assay for the diagnosis of candidiasis

Directory of Open Access Journals (Sweden)

Yicun Wang

2013-01-01

Full Text Available Background: The secreted aspartyl proteinases 2 (Sap2 of Candida albicans (C. albicans is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. Aim: In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA. Materials and Methods: Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1:1200 in an indirect ELISA, and detected Sap2 concentration up to 1 ng/mL. Results: Of the 286 cancer serum samples tested, 16.8% were found as candidiasis. The test was simple and economical to perform and had a level of sensitivity for detection of low-titer positive sera; thus, it may be proven to be of value in epidemiological studies on candidiasis.

Diagnostic accuracy study of a factor VIII ELISA for detection of factor VIII antibodies in congenital and acquired haemophilia A.

Science.gov (United States)

Batty, Paul; Moore, Gary W; Platton, Sean; Maloney, James C; Palmer, Ben; Bowles, Louise; Pasi, K John; Rangarajan, Savita; Hart, Daniel P

2015-10-01

Antibody formation to factor VIII (FVIII) remains the greatest clinical and diagnostic challenge to the haemophilia-treating physician. Current guidance for testing for inhibitory FVIII antibodies (inhibitors) recommends the functional Nijmegen-Bethesda assay (NBA). A FVIII ELISA offers a complementary, immunological approach for FVIII antibody testing. It was the aim of this study to retrospectively evaluate the performance of a FVIII ELISA (index) for detection of FVIII antibodies, compared with the NBA (reference). All samples sent for routine FVIII antibody testing at two haemophilia Comprehensive Care Centres, were tested in parallel using the NBA and a solid-phase, indirect FVIII ELISA kit (Immucor). A total of 497 samples from 239 patients (severe haemophilia A=140, non-severe haemophilia A=85, acquired haemophilia A=14) were available for analysis. Sixty-three samples tested positive by the NBA (prevalence 12.7%, 95% confidence interval [CI], 9.9-15.9 %), with a median inhibitor titre of 1.2 BU/ml (range 0.7-978.0). The FVIII ELISA demonstrated a specificity of 94.0% (95%CI, 91.3-96.0), sensitivity of 77.8% (95%CI, 65.5-87.3), negative predictive value of 96.7% (95%CI, 94.5-98.2), positive predictive value 65.3% (95%CI, 53.5-76.0), negative likelihood ratio 0.2 (95%CI, 0.1-0.4), positive likelihood ratio 13.0 (95%CI, 8.7-19.3) and a diagnostic odds ratio of 54.9 (95%CI, 27.0-112.0). Strong positive correlation (r=0.77, pNBA (log adjusted) and FVIII ELISA optical density. In conclusion, FVIII ELISA offers a simple, specific, surveillance method enabling batch testing of non-urgent samples for the presence of FVIII antibodies.

Synthetic positive controls for ELISA test kits for detection of IgA and IgM antibodies to Chlamydia trachomatis

Directory of Open Access Journals (Sweden)

O. Y. Galkin

2015-01-01

Full Text Available The enzyme-linked immunosorbent assay (ELISA is the most informative and versatile method of serological diagnostics. The possibility of detecting by ELISA specific antibodies of different classes allow to differentiate primary infectious process and its remission, exacerbation and chronic disease (holding of differential diagnosis. This approach is implemented in the methodology for evaluation of patients for presence of humoral immune response against the causative agent of urogenital chlamydiosis. As with other infections immediately after Chlamydia trachomatis infection the specific IgM antibodies are formed, and subsequently basic projective antibodies of IgG class are synthesized. However, at exacerbation of chronic urogenital chlamydiosis specific IgA antibodies can be synthesized. That is why comprehensive evaluation of patients for presence of humoral immune response to Ch. trachomatis involves plasma testing of specific antibodies of all three classes. The essential problem in the production of ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood plasma containing specific antibodies. But specific IgM- and IgA-positive sera are deficit raw materials. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested methodological approach to use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it’s possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethylcyclohexane-1-carboxylate and reductive amination-mediated conjugation (by sodium periodate. It was found that synthetic positive controls obtained by different methods are characterized by higher titer compared to IgM- and IgA-positive high

Evaluation of CSFV Antibody ELISAs for the differentiation of infected from vacci-nated animals

DEFF Research Database (Denmark)

Schroeder, Sabine; Blome, Sandra; Koenen, Frank

countries and out-breaks occurred recently e.g. in Germany, France, Hungary, Romania, Bulgaria, and the Slovak Republic. Preventive vaccination is prohibited within the EU, but emergency vaccination can be part of the strategy in case of a contingency. Using conventional vaccines, differentiation...... of vaccinated from infected animals (DIVA) is not possible. Newly developed modified live marker vaccines allow a DIVA strategy based on the use of enzyme linked immunosorbent assay (ELISA) tests. The aim of this study was to evaluate CSF virus (CSFV) Antibody ELISAs, com-mercially available in Europe......, for their diagnostic sensitivity as well as for their potential in differentiating between infected and marker vaccinated animals. Two newly available ELISAs were included into the tests, the Priocheck® CSFV Erns ELISA, a special DIVA test, and the LDL Pigtype® CSFV Antibody ELISA. An inter-laboratory comparison test...

Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1.

Science.gov (United States)

Zhu, Min; Gong, Xue; Hu, Yonghong; Ou, Weijun; Wan, Yakun

2014-12-20

Influenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, high specificity and stability. In this study, a novel Nanobody-based bioassay was developed for rapid, low-cost and sensitive detection of the influenza H5N1 virus. Nanobodies specific to H5N1 virus were selected from a VHH library by phage display technology. In this system, the biotinylated Nanobody was directionally captured by streptavidin coated on ELISA plate, which can specifically capture the H5N1 virus. Another Nanobody conjugated with HRP was used as a detector. A novel directional enzyme-linked immunosorbent assay for H5N1 using specific Nanobodies was established and compared to the conventional undirected ELISA assay. We have successfully constructed a high quality phage display Nanobody library and isolated two Nanobodies against H5N1 with high affinity and specificity. These two Nanobodies were further used to prepare the biosensor detection system. This streptavidin-biotin-based directional double Nanobodies sandwich ELISA for H5N1 detection showed superiority over the commonly undirectional ELISA protocol. The linear range of detection for standards in this immunoassay was approximately 50-1000 ng/mL and the detection limit was 14.1 ng/mL. The average recoveries of H5N1 virus from human serum samples were in the range from 94.58% to 114.51%, with a coefficient of variation less than 6.5%. Collectively, these results demonstrated that the proposed detection system is an alternative diagnostic tool that enables a rapid, inexpensive, sensitive and specific detection of the

Quality control using a multilevel logistic model for the Danish pig Salmonella surveillance antibody-ELISA programme

DEFF Research Database (Denmark)

Bak, Hanne; Ekeroth, Lars; Houe, Hans

2007-01-01

In Denmark, the level of Salmonella infection in pig herds is monitored with a surveillance programme using an indirect antibody ELISA. Our purpose with the present study was to determine whether sample results from the programme were useful in the quality control of this ELISA. Test results from...... throughout. Analysis of the test results from the wells with test samples gave good information on systematic errors across the microtitre plates, and severe errors appeared significant even when data from short time periods were used....... the year 2003, in which the laboratory experienced a technical problem with an automatic microtitre-plate washing machine, were examined statistically. We chose 3 months for the analysis: January, where the problem was moderate, June with the problem more serious, and November, where the problem had been...... solved. A logistic analysis was carried out with outcome 0 for a negative test result and I for a positive test result. Row and column on the microtitre plates, multiprobe robot, and their interactions were included as fixed effects, and date, plate, and slaughterhouse were included as random effects...

Chemiluminescence ELISA for the detection of oxidative DNA base damage using anti-8-hydroxy-2'-deoxyguanosine antibody. Application to the detection of irradiated foods

International Nuclear Information System (INIS)

Kikuchi, Masahiro; Funayama, Tomoo; Sakashita, Tetsuya; Satoh, Katsuya; Narumi, Issay; Kobayashi, Yashihiko; Gunawardane, Chaminda R.; Alam, Md. Khorshed; Dzomir, A. Zainuri Mohd.; Pitipanaarachchi, Ramya C.; Hamada, Nobuyuki; Wada, Seiichi

2007-01-01

Since ionizing radiation is used for sterilizing or lowering the microbial content of foods as a means of reducing food losses and securing food safety, the development of versatile detection methods of irradiated foods is necessary for appropriate management. In an effort to distinguish between irradiated and non-irradiated food, a method based on the detection of oxidative DNA base damage using the chemiluminescence enzyme-linked immunosorbent assay (ELISA) with anti-8-hydroxy-2'-deoxyguanosine antibody was developed. In the course of optimizing the reaction conditions for the ELISA, a 30-mer synthetic oligonucleotide containing 8-hydroxyguanine (8-oxoG) was used. Under the optimized conditions, the correlation between chemiluminescence intensity and 8-oxoG content in oligonucleotides was obtained. It was shown that this chemiluminescence ELISA method could be applied to chicken, beef and pork that were irradiated with over 3 kGy. Twenty milligrams of a loaf of meat was sufficient to distinguish between irradiated and non-irradiated meat by this method. (author)

Elisa Studio showroom = Elisa Studio showroom / Jan Joonas Graps ; kommenteerinud Tiina Kuusisto

Index Scriptorium Estoniae

Graps, Jan Joonas, 1972-

2015-01-01

Elisa Studio showroom Helsingis Lasiplatsi, Mannerheimintie 22-24. Sisekujunduse autorid Jan Joonas Graps, Ken-Kristjan Ruut, Anne Määrmann (JanKen Wisespace). Lühidalt loovstuudiost JanKen Wisespace

Elisa technology consolidation study overview

Science.gov (United States)

Fitzsimons, E. D.; Brandt, N.; Johann, U.; Kemble, S.; Schulte, H.-R.; Weise, D.; Ziegler, T.

2017-11-01

The eLISA (evolved Laser Interferometer Space Antenna) mission is an ESA L3 concept mission intended to detect and characterise gravitational radiation emitted from astrophysical sources [1]. Current designs for eLISA [2] are based on the ESA study conducted in 2011 to reformulate the original ESA/NASA LISA concept [3] into an ESA-only L1 candidate named NGO (New Gravitational Observatory) [4]. During this brief reformulation period, a number of significant changes were made to the baseline LISA design in order to create a more costeffective mission. Some of the key changes implemented during this reformulation were: • A reduction in the inter satellite distance (the arm length) from 5 Gm to 1 Gm. • A reduction in the diameter of the telescope from 40 cm to 20 cm. • A reduction in the required laser power by approximately 40%. • Implementation of only 2 laser arms instead of 3. Many further simplifications were then enabled by these main design changes including the elimination of payload items in the two spacecraft (S/C) with no laser-link between them (the daughter S/C), a reduction in the size and complexity of the optical bench and the elimination of the Point Ahead Angle Mechanism (PAAM), which corrects for variations in the pointing direction to the far S/C caused by orbital dynamics [4] [5]. In the run-up to an L3 mission definition phase later in the decade, it is desirable to review these design choices and analyse the inter-dependencies and scaling between the key mission parameters with the goal of better understanding the parameter space and ensuring that in the final selection of the eLISA mission parameters the optimal balance between cost, complexity and science return can be achieved.

Field evaluation of a new commercially available ELISA based on a recombinant antigen for diagnosing Chlamydophila abortus (Chlamydia psittaci serotype 1) infection.

Science.gov (United States)

Buendía, A J; Cuello, F; Del Rio, L; Gallego, M C; Caro, M R; Salinas, J

2001-02-12

A new commercially available ELISA (ELISAr-Chlamydia) for detecting antibodies against Chlamydophila abortus has been evaluated using sheep field serum samples. The ELISA is based on a recombinant antigen which expresses part of a protein from the 80-90kDa family that is specific to C. abortus. Sera (105) from six flocks with confirmed ovine chlamydial abortion (OEA) outbreaks were used in this study, as well as sera (258) from 18 flocks which had suffered no OEA in the last lambing. The ELISAr-Chlamydia was compared with the complement fixation test (CFT) and with an ELISA using purified C. abortus elementary bodies (ELISA-EB), employing as reference technique a comparative microimmunofluorescence test that differentiates C. abortus infection from Chlamydophila pecorum infection. The results showed that the sensitivity of ELISAr-Chlamydia was 90.9% with a specificity of 85.9%, the sensitivity of CFT was 71.0% with a specificity of 83.6%, while the sensitivity of ELISA-EB was 95.2% and the specificity was 54.2%. Furthermore, ELISAr-Chlamydia was the test with fewer false positives resulting from positive reactivity to C. pecorum, although 15% of the sera positive for C. pecorum but negative for C. abortus antibodies reacted positively. This study demonstrated with field material that ELISAr-Chlamydia provides the most balanced results between sensitivity and specificity, especially in flocks with no clinical OEA but reactivity to C. abortus.

Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays.

Science.gov (United States)

Gornowicz-Porowska, Justyna; Seraszek-Jaros, Agnieszka; Bowszyc-Dmochowska, Monika; Kaczmarek, Elżbieta; Pietkiewicz, Paweł; Bartkiewicz, Paweł; Dmochowski, Marian

2017-02-01

Pemphigus and bullous pemphigoid (BP) are identified by autoantibodies (abs) against desmoglein 1, 3 (DSG1/3) and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF) using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection) and modified (IgG4 detection) mosaic for indirect immunofluorescence (IIFc - IIF commercial, IIFm - IIF modified) and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP) molecular diagnostics. To compare diagnostic accuracy of commercial (IgG detection) and modified (IgG4 detection) mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher's exact test. There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1)/desmoglein3 (DSG3)/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm.

Galactic binaries with eLISA

OpenAIRE

Nelemans, G.

2013-01-01

I review what eLISA will see from Galactic binaries -- double stars with orbital periods less than a few hours and white dwarf (or neutron star/black hole) components. I discuss the currently known binaries that are guaranteed (or verification) sources and explain why the expected total number of eLISA Galactic binaries is several thousand, even though there are large uncertainties in our knowledge of this population, in particular that of the interacting AM CVn systems. I very briefly sketch...

Bayesian estimation of sensitivity and specificity of Coxiella burnetii antibody ELISA tests in bovine blood and milk

DEFF Research Database (Denmark)

Paul, Suman; Toft, Nils; Agerholm, Jørgen S.

2013-01-01

Serological tests for Coxiella burnetii (the causative agent of Q fever) antibodies are usually based on enzyme linked immunosorbent assay (ELISA) although this method is not thoroughly evaluated. The objective of this study was to determine the sensitivity and specificity of an ELISA for detection...... lactating cows is relatively easy, non-invasive and inexpensive and hence milk ELISA may be a better option for screening lactating cows. But, blood ELISA is an option for screening non-lactating cattle....

Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1.

Science.gov (United States)

Duffort, O; Calabozo, B; González, R; Carpizo, J A; Barber, D; Polo, F

2004-12-01

Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content. 2004 S. Karger AG, Basel.

PREPARATION OF CONJUGATE FOR USE IN AN ELISA FOR HUMORAL IMMUNE RESPONSE AGAINST EGG DROP SYNDROME VIRUS IN LAYER CHICKS

Directory of Open Access Journals (Sweden)

M. K. Mansoor, S. U. Rahman, I. Hussain, M. H. Rasool and M. A. Zahoor

2004-10-01

Full Text Available An indirect enzyme-linked immunosorbent assay (ELISA was performed for the detection of antibodies against Egg Drop Syndrome (EDS virus. Virus identification was done through haemaggluti- nation inhibition (HI test using known antisera. Antichicken immunoglobulins were raised in goats and purified by ammonium sulphate precipitation technique. These goat-antichicken immunoglobulins were conjugated with horseradish peroxidase. Twenty-seven serum samples were collected from a layers flock vaccinated against EDS and specific antibodies were determined by using a horseradish conjugate.

The association between reality-based beliefs and indirectly experienced traumatization.

Science.gov (United States)

Shiri, Shimon; Wexler, Isaiah D; Schwartz, Isabella; Kadari, Michal; Kreitler, Shulamith

2010-12-01

The purpose of the study was to examine the association between belief types and the magnitude of indirect traumatization. Specific types of beliefs were defined in terms of the cognitive orientation theory, which is a cognitive-motivational approach to the understanding, predicting, and changing of behaviors. Belief types that were analyzed included beliefs about self, general beliefs, beliefs about norms, and goal beliefs as they relate to personal growth. Study participants included 38 rescuers (body handlers), 37 nurses, and 31 rehabilitation workers who treated injured civilians that had been exposed to politically motivated violence. The Cognitive Orientation for Posttraumatic Growth Scale was used to assess beliefs about personal growth. The Revised Posttraumatic Stress Disorder Inventory was administered to evaluate indirect traumatization. The results indicate that three of the four belief types related to personal growth were associated with the level of indirect traumatization. Optimistic and positive beliefs about self and general beliefs were associated with a lower level of indirect traumatization symptomatology, suggesting that these types of beliefs may counteract indirect traumatization. On the other hand, stronger goal beliefs were associated with greater indirect traumatization. The negative association between positive goal beliefs and indirect trauma may be related to the gap the individual perceives between the hoped-for ideals and the trauma-stricken reality. These results indicate the importance of cognitive beliefs and their possible role in determining the response to indirect traumatization.

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)

Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

International Nuclear Information System (INIS)

Maria Lina R; Andi Yasmon

2011-01-01

There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32 P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

Development, standardisation and validation of ELISA methods to improve the control of trypanosomosis

International Nuclear Information System (INIS)

Rebeski, D.E.; Winger, E.M.; Robinson, M.M.; Dwinger, R.H.; Crowther, J.R.

2000-01-01

During the period from 1995 to 2000, comprehensive laboratory and field studies on enzyme-linked immunosorbent assay (ELISA) methods for detection of trypanosomal antibodies and antigens were undertaken to improve the proficiency of diagnostic laboratories involved in control of trypanosomosis in the tropics. The work was initiated by the FAO/IAEA through the Coordinated Research Programme D3.20.13 and undertaken in close collaboration with Research Agreement Holders and Research Contract Holders. Initially, the CRP facilitated the field evaluation of three direct sandwich antigen detection ELISAs based on monoclonal antibodies. Diagnostic laboratories were supported with ELISA equipment, disposables, and training. ELISA reagents were produced in sufficient quantities and distributed in a standardised kit format. As a result of the laboratory and field evaluation studies, the assays were found unreliable for trypanosomosis control and rejected for routine use in diagnostic laboratories. At that time, no standardised ELISA system was available for trypanosomosis that was considered suitable for distribution and use under tropical conditions. Through the CRP, a new generation of standardised antibody ELISAs were developed and established using a novel approach, namely the use of antigen-precoated ELISA plates. In addition, the potential of native and denatured trypanosomal antigens as diagnostic candidates was examined. In-house and field evaluation studies in the tropics demonstrated that a reasonable robustness with an acceptable diagnostic assay proficiency was achieved by means of utilising plates precoated with denatured antigens. Moreover, a data charting method for continuous monitoring of the operational performance of the ELISAs was developed and established. It was routinely used as remote control and follow up tool saving the need for costly expert missions to the diagnostic laboratories during the assay validation period. In parallel, preliminary studies

ELISA based techniques for the identification of foot-and-mouth disease virus and vaccine evaluation in Bangladesh

International Nuclear Information System (INIS)

Sil, B.K.; Taimur, M.J.F.A.

2000-01-01

Epidemiology of FMD infection was studied at farm and field levels. The rate of outbreaks increases following the monsoons and remained throughout the winter (until March). Cattle were found to be more susceptible (96.43%) than buffalo (1.01%), goat (2.27%) and sheep (0.27%). Exotic and their cross-bred animals were more susceptible than local breeds of animals (69.89% and 30.11% respectively). Dynamic FMD infection was also studied at the farm level and it was found that infection was directly related with herd immunity, season of the year and climatic conditions. Outbreaks of FMD at rural area were found to be associated with the introduction of new animals from the market in a herd (75.21%) or transportation of infected animals by road or boats. A total of 956 FMD suspected samples from 257 different field outbreaks were collected during the last five years (1995-1999). At the same time, 367 convalescent sera were collected for the analysis FMD antibodies and the virus involved in infection. FMD suspected epithelial samples were tested using indirect ELISA and 875 samples (85.36%) were found positive either against O, A or Asia I. Throughout the last five years, FMD virus type O predominated (54.07%) over FMD virus types Asia I (19%), and virus type A (16.8%) and 6.48% were found to be negative. None of the epithelial samples was found positive against FMD type C. However, five convalescence sera collected from the northern part of Bangladesh showed very strong reaction (>1:240) against FMD virus type C in LPB-ELISA. Vaccination failure was found one of the major constraints towards the control of FMD using vaccine and factors like lack of potent vaccine, inadequate vaccination coverage, poor cold chain, lack of vaccine evaluation and poor health conditions played an important role in this area. Three FMD vaccine candidates were tested using LPB-ELISA both at laboratory and field conditions. Locally produced bi-valent vaccine (O and A) developed a satisfactory

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)

Constraining stellar binary black hole formation scenarios with eLISA eccentricity measurements

OpenAIRE

Nishizawa, Atsushi; Sesana, Alberto; Berti, Emanuele; Klein, Antoine

2016-01-01

A space-based interferometer such as eLISA could observe few to few thousands progenitors of black hole binaries (BHBs) similar to those recently detected by Advanced LIGO. Gravitational radiation circularizes the orbit during inspiral, but some BHBs retain a measurable eccentricity at the low frequencies where eLISA is most sensitive. The eccentricity of a BHB carries precious information about its formation channel: BHBs formed in the field, in globular clusters, or close to a massive black...

Performance of microscopy and ELISA for diagnosing Giardia duodenalis infection in different pediatric groups.

Science.gov (United States)

Silva, Renata K N R; Pacheco, Flávia T F; Martins, Adson S; Menezes, Joelma F; Costa-Ribeiro, Hugo; Ribeiro, Tereza C M; Mattos, Ângela P; Oliveira, Ricardo R; Soares, Neci M; Teixeira, Márcia C A

2016-12-01

Techniques for Giardia diagnosis based on microscopy are usually applied as routine laboratory testing; however, they typically exhibit low sensitivity. This study aimed to evaluate Giardia duodenalis and other intestinal parasitic infections in different pediatric groups, with an emphasis on the comparison of Giardia diagnostic techniques. Feces from 824 children from different groups (diarrheic, malnourished, with cancer and from day care) were examined by microscopy and ELISA for Giardia, Cryptosporidium sp. and Entamoeba histolytica coproantigen detection. Giardia-positive samples from day-care children, identified by either microscopy or ELISA, were further tested by PCR targeting of the β-giardin and Gdh genes. Statistically significant differences (Psp. in diarrheic and malnourished groups; infections by Entamoeba histolytica were found only in children with diarrhea. Considering positivity for Giardia by at least one method, ELISA was found to be more sensitive than microscopy (97% versus 55%). To examine discrepancies among the diagnostic methods, 71 Giardia-positive stool samples from day-care children were tested by PCR; of these, DNA was amplified from 51 samples (77.4%). Concordance of positivity between microscopy and ELISA was found for 48 samples, with 43 confirmed by PCR. Parasite DNA was amplified from eleven of the 20 Giardia samples (55%) identified only by ELISA. This study shows the higher sensitivity of ELISA over microscopy for Giardia diagnosis when a single sample is analyzed and emphasizes the need for methods based on coproantigen detection to identify this parasite in diarrheic fecal samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A Simple ELISA Exercise for Undergraduate Biology.

Science.gov (United States)

Baker, William P.; Moore, Cathy R.

Understanding of immunological techniques such as the Enzyme Linked Immuno Sorbent Assay (ELISA) is an important part of instructional units in human health, developmental biology, microbiology, and biotechnology. This paper describes a simple ELISA exercise for undergraduate biology that effectively simulates the technique using a paper model.…

Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays

Directory of Open Access Journals (Sweden)

Justyna Gornowicz-Porowska

2017-02-01

Full Text Available Introduction : Pemphigus and bullous pemphigoid (BP are identified by autoantibodies (abs against desmoglein 1, 3 (DSG1/3 and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection and modified (IgG4 detection mosaic for indirect immunofluorescence (IIFc – IIF commercial, IIFm – IIF modified and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP molecular diagnostics. Aim : To compare diagnostic accuracy of commercial (IgG detection and modified (IgG4 detection mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Material and methods : Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher’s exact test. Results: There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1/desmoglein3 (DSG3/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. Conclusions : The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm.

Does size matter? Study of performance of pseudo-ELISAs based on molecularly imprinted polymer nanoparticles prepared for analytes of different sizes.

Science.gov (United States)

Cáceres, C; Canfarotta, F; Chianella, I; Pereira, E; Moczko, E; Esen, C; Guerreiro, A; Piletska, E; Whitcombe, M J; Piletsky, S A

2016-02-21

The aim of this work is to evaluate whether the size of the analyte used as template for the synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) can affect their performance in pseudo-enzyme linked immunosorbent assays (pseudo-ELISAs). Successful demonstration of a nanoMIPs-based pseudo-ELISA for vancomycin (1449.3 g mol(-1)) was demonstrated earlier. In the present investigation, the following analytes were selected: horseradish peroxidase (HRP, 44 kDa), cytochrome C (Cyt C, 12 kDa) biotin (244.31 g mol(-1)) and melamine (126.12 g mol(-1)). NanoMIPs with a similar composition for all analytes were synthesised by persulfate-initiated polymerisation in water. In addition, core-shell nanoMIPs coated with polyethylene glycol (PEG) and imprinted for melamine were produced in organics and tested. The polymerisation of the nanoparticles was done using a solid-phase approach with the correspondent template immobilised on glass beads. The performance of the nanoMIPs used as replacement for antibodies in direct pseudo-ELISA (for the enzymes) and competitive pseudo-ELISA for the smaller analytes was investigated. For the competitive mode we rely on competition for the binding to the nanoparticles between free analyte and corresponding analyte-HRP conjugate. The results revealed that the best performances were obtained for nanoMIPs synthesised in aqueous media for the larger analytes. In addition, this approach was successful for biotin but completely failed for the smallest template melamine. This problem was solved using nanoMIP prepared by UV polymerisation in an organic media with a PEG shell. This study demonstrates that the preparation of nanoMIP by solid-phase approach can produce material with high affinity and potential to replace antibodies in ELISA tests for both large and small analytes. This makes this technology versatile and applicable to practically any target analyte and diagnostic field.

Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65.

Science.gov (United States)

Liu, Maojun; DU, Gaimei; Zhang, Yue; Wu, Yuzi; Wang, Haiyan; Li, Bin; Bai, Yun; Feng, Zhixin; Xiong, Qiyan; Bai, Fangfang; Browning, Glenn F; Shao, Guoqing

2016-09-01

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection.

Short communication: ELISA system for screening of bovine mastitis caused by Prototheca zopfii.

Science.gov (United States)

Kano, Rui; Sato, Ayano; Sobukawa, Hideto; Sato, Yuko; Ito, Takaaki; Suzuki, Kazuyuki; Hasegawa, Atsuhiko; Kamata, Hiroshi

2016-08-01

Prototheca zopfii is an achlorophyllic alga that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. This study evaluated the use of an ELISA system for distinguishing cows with mastitis due to P. zopfii genotype 2 from healthy cows and cows with chronic candidal mastitis. We also investigated the transitional changes of specific antibody titers in healthy cows injected with inactivated P. zopfii genotype 2 cells. The ELISA system exhibited the highest sensitivity (94%) and specificity (100%) for chronic protothecal mastitis when the positive cutoff value was set at 43.4 ELISA units. Anti-protothecal IgG titers were positive in all cows after they were inoculated with inactivated P. zopfii genotype 2 cells. These results indicated that ELISA detection of anti-protothecal IgG in serum provided specificity and sensitivity sufficient for diagnosing protothecal mastitis. Thus, an ELISA system incorporating this specific antiserum is expected to be valuable for definitive field-based diagnosis of bovine mastitis due to P. zopfii genotype 2. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Follow-up of neurocysticercosis patients after treatment using an antigen detection ELISA

Directory of Open Access Journals (Sweden)

Nguekam

2003-03-01

Full Text Available Seven patients with active neurocysticercosis (NCC received an eight days treatment with albendazole and were followed up using computed tomography (CT-scan and a monoclonal antibody based ELISA for the detection of circulating antigen (Ag-ELISA. Only three patients were cured as was shown by CT-scan and by the disappearance of circulating antigens one month after treatment. After a second course of albendazole therapy, two other patients became seronegative. CT-scan showed the disappearance of viable cysts in all persons who became seronegative whereas patients who were not cured remained seropositive. These preliminary results show that this Ag-ELISA is a promising technique for monitoring the success of treatment of NCC patients because of the excellent correlation between the presence of circulating antigens and of viable brain cysts.

Analysis of fenbendazole residues in bovine milk by ELISA.

Science.gov (United States)

Brandon, David L; Bates, Anne H; Binder, Ronald G; Montague, William C; Whitehand, Linda C; Barker, Steven A

2002-10-09

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

OpenAIRE

Richardson, M D; Stubbins, J M; Warnock, D W

1982-01-01

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen ...

Seroprevalence of Toxoplasma gondii in southern districts of Tamil Nadu using IgG-ELISA.

Science.gov (United States)

Sucilathangam, G; Palaniappan, N; Sreekumar, C; Anna, T

2012-10-01

The present study was conducted to assess the seroprevalence of Toxoplasma gondii in and around Tirunelveli by in-house IgG assay using ELISA. Serum samples from 175 immunodeficient and 175 immunocompetent patients were collected at Tirunelveli district, Tamil Nadu from May 2006 to October 2007. They were subjected into in-house IgG assay using enzyme-linked immune sorbent assay (ELISA) in which tachyzoite soluble antigen derived from solubilised whole organisms was used. Out of 350 patients tested by IgG ELISA, 46 patients (13.14%) had antibodies for toxoplasmosis with mean OD value of 0.2 ± 0.073 and the OD value ranged from 0.144 to 0.444. Among the immunocompetent group of 175 patients, 19 patients (10.86%) had antibodies to toxoplasmosis whereas, in immunodeficient group of 175 patients, 27 patients (15.43%) had antibodies for toxoplasmosis. There was no statistical difference (P > 0.05) between the immunocompetent and immunodeficient group. The sensitivity and specificity of IgG ELISA in detecting toxoplasmosis was 90 and 100%, respectively. The overall seroprevalence of toxoplasmosis in and around Tirunelveli district of Tamil Nadu was 13.14% based on IgG ELISA. The study has proved ELISA to be a sensitive and specific procedure for the serodiagnosis of toxoplasmosis.

Quantitative sandwich ELISA for the determination of fish in foods.

Science.gov (United States)

Faeste, Christiane K; Plassen, Christin

2008-01-01

Allergy to fish represents one of the most prevalent causes for severe food-allergic reactions. Therefore, food authorities in different countries have implemented mandatory labeling of fish in pre-packed foods. Detection of fish proteins in food has previously been based on the use of patient serum. In the present study, a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of fish in food matrixes has been developed and validated, using a polyclonal rabbit anti-cod parvalbumin antibody for capture and a biotinylated conjugate of the same antibody for detection. By employing the ubiquitous muscle protein parvalbumin as target the method succeeds to detect a variety of fish. However, the ELISA is specific for fish and does not cross-react with other species. Recoveries ranged from 68-138% in typical food matrixes, while the intra- and inter-assay precisions were parvalbumin ELISA with a limit of detection of 0.01 mg parvalbumin/kg food, about 5 mg fish/kg food, seems sufficient to detect fish protein traces in foods at levels low enough to minimize the risk for fish allergic consumers.

Science with the space-based interferometer eLISA. III: probing the expansion of the universe using gravitational wave standard sirens

Energy Technology Data Exchange (ETDEWEB)

Tamanini, Nicola; Caprini, Chiara [Institut de Physique Théorique, CEA-Saclay, CNRS UMR 3681, Université Paris-Saclay, F-91191 Gif-sur-Yvette (France); Barausse, Enrico [Sorbonne Universités, UPMC Université Paris 6, UMR 7095, Institut d' Astrophysique de Paris, 98 bis Bd Arago, 75014 Paris (France); Sesana, Alberto [School of Physics and Astronomy, The University of Birmingham, Edgbaston, Birmingham B15 2TT (United Kingdom); Klein, Antoine [Department of Physics and Astronomy, The University of Mississippi, University, MS 38677 (United States); Petiteau, Antoine, E-mail: nicola.tamanini@cea.fr, E-mail: chiara.caprini@cea.fr, E-mail: barausse@iap.fr, E-mail: asesana@star.sr.bham.ac.uk, E-mail: aklein@physics.montana.edu, E-mail: antoine.petiteau@apc.univ-paris7.fr [APC, Université Paris Diderot, Observatoire de Paris, Sorbonne Paris Cité, 10 rue Alice Domon et Léonie Duquet, 75205 Paris Cedex 13 (France)

2016-04-01

We investigate the capability of various configurations of the space interferometer eLISA to probe the late-time background expansion of the universe using gravitational wave standard sirens. We simulate catalogues of standard sirens composed by massive black hole binaries whose gravitational radiation is detectable by eLISA, and which are likely to produce an electromagnetic counterpart observable by future surveys. The main issue for the identification of a counterpart resides in the capability of obtaining an accurate enough sky localisation with eLISA. This seriously challenges the capability of four-link (2 arm) configurations to successfully constrain the cosmological parameters. Conversely, six-link (3 arm) configurations have the potential to provide a test of the expansion of the universe up to z ∼ 8 which is complementary to other cosmological probes based on electromagnetic observations only. In particular, in the most favourable scenarios, they can provide a significant constraint on H{sub 0} at the level of 0.5%. Furthermore, (Ω{sub M}, Ω{sub Λ}) can be constrained to a level competitive with present SNIa results. On the other hand, the lack of massive black hole binary standard sirens at low redshift allows to constrain dark energy only at the level of few percent.

Effect of processing on the detectability of peanut protein by ELISA.

Science.gov (United States)

Iqbal, Amjad; Ateeq, Nadia

2013-12-01

Chicken IgY was used for the detection and quantification of peanut proteins by indirect competitive ELISA. The method was optimized by using a checker board approach to determine the optimal concentration of coating antigen, primary antibody and secondary antibody. Peanut protein could be detected in foods down to levels of 10 ppm. The effect of physical (heat treatment at 80 °C and 100 °C) and chemical (acid, alkali and reducing sugar) treatments on the IgY binding of peanut proteins was investigated. The optimized assay was relatively sensitive for the roasted peanut proteins. However, the binding ability of chicken IgYs to peanut proteins was found to be significantly altered by denaturation and hydrolysis of proteins. It was also observed that the effect of Millard chemistry on the detectability of peanut protein was less pronounced at high temperatures than at low temperatures. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of an ELISA kit in the serological diagnosis of Babesia bovis for epidemiological studies

International Nuclear Information System (INIS)

Martins, J.R.; Correa, B.L.; Cereser, V.H.; Artiles, J.M.; Alves-Branco, F.P.J.

1998-01-01

An enzyme linked immunosorbent assay (ELISA) kit for detect antibodies to Babesia bovis, an intraerytrocitic bovine parasite was evaluated using known negative and positive samples and the results were compared with an indirect immunofluorescent antibody test (IFAT). Results obtained with field samples were used to estimate seroprevalence of B. bovis in an endemic area to the cattle tick (Boophilus microplus) vector of bovine babesiosis. Percentage of positivity (PP) values (optical density of tested serum/mean optical density of positive control) on 274 negative samples, had major values ranged in the frequency of 4.0 to 7.0 PP. Comparison between ELISA and IFAT showed an agreement of 93.3% on field sera samples, collected in areas of low, good and high soil fertility in the region of Bage (31 degree 20 min 13 sec S, 54 degree 06 min 21 sec W), RS, Brazil. From 5,082 tested sera, 3,751 (73%) were positive for B. bovis antibodies. No significant difference (P>0.05) was observed between results from calves living in areas of low and good soil fertility (80 and 82% of seroprevalence, respectively). However, calves living in soil of high fertility showed a minor inoculation rate for B. bovis (63% of seroprevalence), indicating needs of measures to prevent losses due to babesiosis. (author)

Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

International Nuclear Information System (INIS)

Tahara, Yusuke; Huang, Zhe; Kiritoshi, Tetsuro; Onodera, Takeshi; Toko, Kiyoshi

2014-01-01

The monitoring of salivary cortisol as a key biomarker of an individual’s stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt–100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

Energy Technology Data Exchange (ETDEWEB)

Tahara, Yusuke; Huang, Zhe; Kiritoshi, Tetsuro [Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka (Japan); Onodera, Takeshi [Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka (Japan); Toko, Kiyoshi, E-mail: toko@ed.kyushu-u.ac.jp [Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka (Japan); Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka (Japan)

2014-05-30

The monitoring of salivary cortisol as a key biomarker of an individual’s stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt–100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

European Union funded project on the development of a whole complement deficiency screening ELISA

DEFF Research Database (Denmark)

Würzner, Reinhard; Tedesco, Francesco; Garred, Peter

2015-01-01

A whole complement ELISA-based assay kit, primarily designed to screen for deficiencies in components of the complement system was developed during a European Union grant involving more than a dozen European scientists and a small-medium enterprise company (Wieslab, which later merged into Eurodi......A whole complement ELISA-based assay kit, primarily designed to screen for deficiencies in components of the complement system was developed during a European Union grant involving more than a dozen European scientists and a small-medium enterprise company (Wieslab, which later merged...

Evaluation of SD BIOLINE H. pylori Ag rapid test against double ELISA with SD H. pylori Ag ELISA and EZ-STEP H. pylori Ag ELISA tests.

Science.gov (United States)

Negash, Markos; Kassu, Afework; Amare, Bemnet; Yismaw, Gizachew; Moges, Beyene

2018-01-01

Helicobacter pylori antibody titters fall very slowly even after successful treatment. Therefore, tests detecting H. pylori antibody lack specificity and sensitivity. On the other hand, H. pylori stool antigen tests are reported as an alternative assay because of their reliability and simplicity. However, the comparative performance of H. pylori stool antigen tests for detecting the presence of the bacterium in clinical specimens in the study area is not assessed. Therefore, in this study we evaluated the performance of SD BIOLINE H. pylori Ag rapid test with reference to the commercially available EZ- STEP ELISA and SD BIOLINE H. pylori Ag ELISA tests. Stool samples were collected to analyse the diagnostic performance of SD BIOLINE H. pylori Ag rapid test kit using SD H. pylori Ag ELISA kit and EZ- STEP ELISA tests as a gold standard. Serum samples were also collected from each patient to test for the presence of H. pylori antibodies using dBest H. pylori Test Disk. Sensitivity, specificity, predictive values and kappa value are assessed. P values H. pylori Ag rapid test were: 95.6% (95% CI, 88.8-98.8), 92.5% (95%CI, 89-94.1%), 86.7% (95% CI, 80.5-89.6), and 97.6% (95% CI, 993.9-99.3) respectively. The performance of SD BIOLINE H. pylori Ag rapid test was better than the currently available antibody test in study area. Therefore, the SD BIOLINE Ag rapid stool test could replace and be used to diagnose active H. pylori infection before the commencement of therapy among dyspeptic patients.

Evaluation of two commercially available ELISA kits for the determination of melatonin concentrations in amniotic fluid throughout pregnancy.

Science.gov (United States)

Bagci, Soyhan; Altuntas, Özlem; Katzer, David; Berg, Christoph; Willruth, Arne; Reutter, Heiko; Bartmann, Peter; Müller, Andreas; Zur, Berndt

2017-01-01

Background The aim of the present study is to evaluate the utility of extraction versus non-extraction-based commercial melatonin ELISA kits for determining the melatonin concentration in amniotic fluid obtained in early and late pregnancy. Methods Pregnancy duration less than 28 weeks was defined as early and from 28 weeks until delivery as late gestation. Nine samples were obtained in early and 18 in late pregnancy. Two commercially available melatonin ELISA kits (melatonin ELISA RE54021, including methanol-based extraction and direct saliva melatonin ELISA RE 54041, not including an extraction step, both from IBL-International, Germany) were used to determine melatonin concentrations in amniotic fluid. Results The mean melatonin concentration in ELISAs assayed by the non-extraction was significantly lower than those assayed after extraction. Subgroup analysis showed that there was no significant difference between melatonin concentration measured by non-extraction versus extraction ELISA in early pregnancy (11.2 ± 7.4 vs. 12.2 ± 7.7, respectively, P = 0.463) but that the mean melatonin concentration in late pregnancy was significantly lower when assayed by non-extraction ELISA than when assayed by extraction ELISA (14.8 ± 9.3 vs. 145.1 ± 179.3, respectively; P pregnancy was rather poor (r 2  = 0.271, P = 0.022), as opposed to the good correlation found in early pregnancy (r 2  = 0.929, P melatonin assay without an extraction step, such as direct saliva ELISA, does not seem to be a valid method to determine the melatonin concentration of amniotic fluid, especially in late gestation.

DETERMINATION OF HISTAMINE IN FISH USING ELISA TECHNIQUE

NARCIS (Netherlands)

KRUGER, C; SEWING, U; STENGEL, G; KEMA, [No Value; WESTERMANN, J; MANZ, B

1995-01-01

The analysis of histamine in fish and fish products via competitive ELISA is described. The advantages of this method are easy sample preparation and handling, screening capabilities, and low costs. Automation enables the performance of the assay with higher series of samples. The Histamine-ELISA is

Development of a serodiagnostic IgM-ELISA for tick-borne encephalitis virus using subviral particles with strep-tag.

Science.gov (United States)

Nakayasu, Miki; Hirano, Minato; Muto, Memi; Kobayashi, Shintaro; Kariwa, Hiroaki; Yoshii, Kentaro

2018-06-23

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. IgM antibody detection is useful for the serological diagnosis of TBEV infection, because IgM has high specificity for each flavivirus and indicates a recent infection. Commercial IgM-ELISA kits are somewhat expensive and difficulties in their sensitivity have been suggested due to their format and formalin-inactivated antigens. Therefore, the development of an inexpensive IgM-ELISA with high specificity and sensitivity is needed. In this study, a μ-capture ELISA was developed to detect TBEV-specific IgM antibodies using subviral particles (SPs) with strep-tag (strep-SP-IgM-ELISA). The results of our strep-SP-IgM-ELISA were highly correlated with diagnoses made by the neutralization test (sensitivity: 94.1%), and our strep-SP-IgM-ELISA could detect anti-TBEV IgM antibodies in patients who could not be diagnosed with the neutralization test. Besides, 51 of 52 positive samples by a commercial IgM-ELISA were also diagnosed as positive by our strep-SP-IgM-ELISA (98.1%), and our strep-SP-IgM-ELISA could detect anti-TBEV IgM antibodies in all samples that were inconclusive based on the commercial IgM-ELISA. Our strep-SP-IgM-ELISA will be useful for diagnoses in TBE-endemic areas. Copyright © 2018 Elsevier GmbH. All rights reserved.

An Indirect Route for Ethanol Production

Energy Technology Data Exchange (ETDEWEB)

Eggeman, T.; Verser, D.; Weber, E.

2005-04-29

The ZeaChem indirect method is a radically new approach to producing fuel ethanol from renewable resources. Sugar and syngas processing platforms are combined in a novel way that allows all fractions of biomass feedstocks (e.g. carbohydrates, lignins, etc.) to contribute their energy directly into the ethanol product via fermentation and hydrogen based chemical process technologies. The goals of this project were: (1) Collect engineering data necessary for scale-up of the indirect route for ethanol production, and (2) Produce process and economic models to guide the development effort. Both goals were successfully accomplished. The projected economics of the Base Case developed in this work are comparable to today's corn based ethanol technology. Sensitivity analysis shows that significant improvements in economics for the indirect route would result if a biomass feedstock rather that starch hydrolyzate were used as the carbohydrate source. The energy ratio, defined as the ratio of green energy produced divided by the amount of fossil energy consumed, is projected to be 3.11 to 12.32 for the indirect route depending upon the details of implementation. Conventional technology has an energy ratio of 1.34, thus the indirect route will have a significant environmental advantage over today's technology. Energy savings of 7.48 trillion Btu/yr will result when 100 MMgal/yr (neat) of ethanol capacity via the indirect route is placed on-line by the year 2010.

A simplified indirect bonding technique

Directory of Open Access Journals (Sweden)

Radha Katiyar

2014-01-01

Full Text Available With the advent of lingual orthodontics, indirect bonding technique has become an integral part of practice. It involves placement of brackets initially on the models and then their transfer to teeth with the help of transfer trays. Problems encountered with current indirect bonding techniques used are (1 the possibility of adhesive flash remaining around the base of the brackets which requires removal (2 longer time required for the adhesive to gain enough bond strength for secure tray removal. The new simplified indirect bonding technique presented here overcomes both these problems.

Odour-based context reinstatement effects with indirect measures of memory: the curious case of rosemary.

Science.gov (United States)

Ball, Linden J; Shoker, Jaswinder; Miles, Jeremy N V

2010-11-01

Previous studies examining environmental context-dependent memory (ECDM) effects using indirect measures of memory have produced inconsistent findings. We report three experiments that examined ECDM in an indirect memory paradigm (word-fragment completion) using ambient odours as environmental contexts. Expt 1 manipulated the odour present at learning and testing (rosemary or lemon) to produce reinstated-context or switched-context conditions. Reinstating rosemary led to a striking ECDM effect, indicating that indirect memory testing can be sensitive to ECDM manipulations. Odour ratings also indicated that rosemary induced a more unpleasant mood in participants than lemon. Expt 2 assessed the influence on indirect retrieval of odour-based mood induction as well as odour distinctiveness, and indicated that rosemary's capacity to promote ECDM effects appears to arise from an additive combination of its unpleasantness-inducing properties and its distinctiveness. Expt 3 partially supported these proposals. Overall, our findings indicate that some odours are capable of producing ECDM effects using indirect testing procedures. Moreover, it appears that it is the inherent proprieties of odours on dimensions such as unpleasantness and distinctiveness that mediate the emergence of ECDM effects, thereby explaining the particular potency of rosemary's mnemonic influence when it is reinstated.

Development of hen antihepatitis B antigen IgY-based conjugate for ELISA assay

Directory of Open Access Journals (Sweden)

Najat Muayed Nafea

2015-01-01

Conclusions: This study showed that laying hens can be used as an alternative source for production of polyclonal antibodies against HBsAg and anti-HBs IgY could be labeled with HRP enzyme and could subsequently be used successfully as secondary antibody in ELISA for detection of HBsAg in the patients sera.

Case study on human α1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

DEFF Research Database (Denmark)

Hansen, Henning Gram; Kildegaard, Helene Faustrup; Min Lee, Gyun

2016-01-01

Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme-linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available...... ELISA kits. We observed that estimations of recombinant human ø1-antitrypsin (rø1AT) titer by Coomassie-stained SDS-PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based...... on biolayer interferometry and reversed-phase high-performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off-the-shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers...

AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

Science.gov (United States)

An ELISA assay for heme oxygenase (HO-l ) Abstract A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

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.

INDIRECT LABOR COSTS AND IMPLICATIONS FOR OVERHEAD ALLOCATION

OpenAIRE

Bea Chiang

2013-01-01

Cost accounting typically allocates indirect labor cost to cost object based on direct labor hours. The allocation process implicitly assumes that indirect labor costs vary proportionally with direct labor hours. The assumption of a linear relationship between indirect and direct labor is particularly suspicious at low production volume levels because there tends to be a fixed component in indirect labor. The linearity assumption is also challenged by recent increasing complexity of indirect ...

Standardization of the indirect enzyme-linked immunosorbent assay for detection of antibodies against Newcastle disease virus in chickens

International Nuclear Information System (INIS)

Della Porta, A.J.; Young, J.; Hansson, E.; Spencer, T.

1994-01-01

Newcastle disease is the major viral disease of poultry causing significant economic losses in most countries except Australia and New Zealand. Serological monitoring of poultry has traditionally been carried out using the haemagglutinin-inhibition (HI) test. More recently, ELISA has been used for the same purpose. This paper described the use of an indirect ELISA for assay of antibodies in chickens against Newcastle disease viruses and compares some of the parameters for this test. The sucrose density gradient purified, inactivated, antigen enabled performance of the test without the addition of any blocking agents other than the usual Tween 20. A range of plates was compared and the most suitable plate was found to be a polystyrene haemagglutination plate giving an excellent positive to negative ratio of 33.2, compared with some expensive ELISA plates which gave very low +ve/-ve ratios. Various incubation conditions for the steps in the ELISA were compared and incubation with shaking at room temperature (24 to 28 deg. C) gave adequate reactivity whilst simplifying incubation conditions and speeding up the test. The negative cut-off value was determined by testing 1632 HI negative specific pathogen free sera from birds of a wide age range. The reactivity of sera in the ELISA was standardized using a standard curve on every plate and converting the readings to ELISA units (EUs) in the range of 16 to 512 EUs. The EU values of these sera were not normally distributed and so the 95% cut-off was determined by ranking the values in descending order and retaining only the top 5% of the values as false positives. This resulted in a cut-off value of 33.6 EUs, with few of HI positive sera having values lower than this cut-off. The use of a standard curve on each plate is recommended in order to standardize the assay and to determine the ELISA units for the test sera. (author). 14 refs, 2 figs, 1 tab

PM1-Alpha ELISA: the assay of choice for the detection of anti-PM/Scl autoantibodies?

Science.gov (United States)

Mahler, Michael; Fritzler, Marvin J

2009-03-01

A characteristic serological feature of patients suffering from the overlap polymyositis and scleroderma (PM/Scl) syndrome are antibodies to the human counterpart of the yeast exosome referred to as the PM/Scl complex. Historically, the detection of anti-PM/Scl antibodies was laborious and relied largely on indirect immunofluorescence and immunodiffusion techniques. In 1992 the major autoantigen PM/Scl-100 was identified and cloned. Subsequently, the major epitopes were mapped and one of these, termed PM1-Alpha, became the antigen for a novel ELISA exhibiting high sensitivity and specificity for the detection of anti-PM/Scl antibodies. Comparative studies with other methods using other PM/Scl autoantigens have shown that the PM1-Alpha ELISA has higher sensitivity and specificity than assays that employed recombinant PM/Scl-75c and PM/Scl-100. Anti-PM1-Alpha antibodies were identified in 55.0% of sera from PM/Scl overlap syndrome patients, but were also seen in 7.9% of SSc and in 7.5% of PM patients. The frequency in other systemic autoimmune diseases and in infectious diseases was significant lower. In summary, the data derived from individual studies suggest that PM1-Alpha may become the "gold standard" for the detection of anti-PM/Scl antibodies.

Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples.

Science.gov (United States)

Meza-Lucas, Antonio; Pérez-Villagómez, María-Fernanda; Martínez-López, José-Patricio; García-Rodea, Ricardo; Martínez-Castelán, María-Guadalupe; Escobar-Gutiérrez, Alejandro; de-la-Rosa-Arana, Jorge-Luis; Villanueva-Zamudio, Altagracia

2016-09-01

A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100 % was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples.

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.

Nested-PCR and a new ELISA-based NovaLisa test kit for malaria diagnosis in an endemic area of Thailand.

Science.gov (United States)

Thongdee, Pimwan; Chaijaroenkul, Wanna; Kuesap, Jiraporn; Na-Bangchang, Kesara

2014-08-01

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.

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.

Cornelius Elisa Bertus Bremekamp

NARCIS (Netherlands)

Lanjouw, J.

1969-01-01

Cornelis Elisa Bertus Bremekamp was born at Dordrecht on February 7th 1888. He is therefore now just past eighty, and he has been a member of the Koninklijke Nederlandse Botanische Vereniging for sixty years. He studied at the Utrecht State University and, like many of his contemporaries, was

Outstanding insecurities concerning the use of an Ov16-based ELISA in the Amazonia onchocerciasis focus

Directory of Open Access Journals (Sweden)

Sérgio Luiz Bessa Luz

2014-07-01

Full Text Available In a recent issue of Memórias do Instituto Oswaldo Cruz, published in Rio de Janeiro in February 2014 (109: 87-92, Adami et al. have published a survey reporting Mansonella parasite prevalence in the Amazon Region. This report makes a useful contribution to the existing knowledge of filarial parasite distribution within the Amazon area, parasite prevalence rates in relation to age and occupation and provides observations on the possible clinical impact of Mansonella ozzardi. Their publication also provides an account of what appears to be a novel ELISA that has recently been used in the Simuliidae and Onchocerciasis Laboratory of the Oswaldo Cruz Institute, Rio de Janeiro, Brazil. We are concerned that the publication of this ELISA may have created an excessively positive impression of the effectiveness of the onchocerciasis recrudescence serological surveillance tools that are presently available for use in the Amazonia onchocerciasis focus. In this letter we have, thus, sought to highlight some of the limitations of this ELISA and suggest how continuing insecurities concerning the detection of antibodies to Onchocerca volvulus within the Amazonia onchocerciasis focus might be minimised.

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

Optimisation of sandwich ELISA based on monoclonal antibodies for the specific measurement of pregnancy-associated plasma protein (PAPP-A) in acute coronary syndrome

DEFF Research Database (Denmark)

Rossen, Marie; Iversen, Kasper; Teisner, Ane

2007-01-01

OBJECTIVES: PAPP-A has become the principal biochemical serum marker in first trimester screening for Down syndrome, the original data being based on results of a radioimmunoassay (RIA). Recent observations using sandwich ELISA technology have proposed PAPP-A as a potential marker in patients wit...

Multimeric recombinant M2e protein-based ELISA: a significant improvement in differentiating avian influenza infected chickens from vaccinated ones.

Directory of Open Access Journals (Sweden)

Farshid Hadifar

Full Text Available Killed avian influenza virus (AIV vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006 was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.

Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA

DEFF Research Database (Denmark)

Bosteen, Markus Høybye; Dahlbäck, Björn; Nielsen, Lars Bo

2015-01-01

that specifically recognizes human apoM in plasma using commercially available reagents. Commercial apoM antibodies were screened for compatibility in a sandwich ELISA-based assay. One optimal pair of antibodies was chosen, and sample preparation, buffers, and incubation times were optimized to generate a simple...... and reproducible method. Validation and comparison to a previously described ELISA for apoM confirmed that the assay displays a high degree of sensitivity, specificity, and precision. Our results show that commercially available antibodies can be used to accurately measure human plasma apoM. This method can...

Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA).

Science.gov (United States)

Zeng, Weiwei; Yao, Wei; Wang, Yingying; Li, Yingying; Bermann, Sven M; Ren, Yan; Shi, Cunbin; Song, Xinjian; Huang, Qiwen; Zheng, Shuchen; Wang, Qing

2017-05-01

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture. Copyright © 2017 Elsevier B.V. All rights reserved.

Multi-Laboratory Validation of Estrone (E1) ELISA Methods

Science.gov (United States)

This project is a round-robin evaluation of commercially available Enzyme-Linked Immunosorbent Assay (ELISA) technology to quantitatively or qualitatively measure the hormone estrone (E1) in combined animal feeding operation (CAFO) receiving streams. ELISA is meant to be a simpl...

Anti-signal recognition particle autoantibody ELISA validation and clinical associations.

Science.gov (United States)

Aggarwal, Rohit; Oddis, Chester V; Goudeau, Danielle; Fertig, Noreen; Metes, Ilinca; Stephens, Chad; Qi, Zengbiao; Koontz, Diane; Levesque, Marc C

2015-07-01

The aim of this study was to develop and validate a quantitative anti-signal recognition particle (SRP) autoantibody serum ELISA in patients with myositis and longitudinal association with myositis disease activity. We developed a serum ELISA using recombinant purified full-length human SRP coated on ELISA plates and a secondary antibody that bound human IgG to detect anti-SRP binding. Protein immunoprecipitation was used as the gold standard for the presence of anti-SRP. Serum samples from three groups were analysed: SRP(+) myositis subjects by immunoprecipitation, SRP(-) myositis subjects by immunoprecipitation and non-myositis controls. The ELISA's sensitivity, specificity, positive predictive value and negative predictive value were evaluated. Percentage agreement and test-retest reliability were assessed. Serial samples from seven SRP immunoprecipitation-positive subjects were also tested, along with serum muscle enzymes and manual muscle testing. Using immunoprecipitation, we identified 26 SRP(+) myositis patients and 77 SRP(-) controls (including 38 patients with necrotizing myopathy). Non-myositis control patients included SLE (n = 4) and SSc (n = 7) patients. Anti-SRP positivity by ELISA showed strong agreement (97.1%) with immunoprecipitation (κ = 0.94). The sensitivity, specificity, positive predictive value, and negative predictive value of the anti-SRP ELISA were 88, 100, 100 and 96, respectively. The area under the curve was 0.94, and test-retest reliability was strong (r = 0.91, P < 0.001). Serial samples showed that anti-SRP levels paralleled changes in muscle enzymes and manual muscle testing. We developed a quantitative ELISA for detecting serum anti-SRP autoantibodies and validated the assay in myositis. Longitudinal assessment of SRP levels by ELISA may be a useful biomarker for disease activity. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions

Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA

Directory of Open Access Journals (Sweden)

Birkelund Svend

2004-02-01

Full Text Available Abstract Background Serology is often used for the diagnosis of Mycoplasma pneumoniae. It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M. pneumoniae. The two proteins, P116 and P1, are found to be immunogenic. By using these in ELISA it is possible to identify an immune response against M. pneumoniae in serum samples. Results A recombinant protein derived from the P116 protein and one from the P1 protein were used in two ELISA tests, rP116-ELISA and rP1-ELISA. Human serum samples from patients with atypical pneumonia were tested and compared to the results of the complement fixation test. There was a good agreement between the two tests but the rP1-ELISA showed the best discrimination between positive and negative samples. Conclusion Two ELISA tests based on recombinant proteins have been analysed and compared to the complement fixation test results. The two ELISA tests were found suitable for use in serodiagnostics of M. pneumoniae infections. The use of specific antigens eliminates the risk of cross reaction to an immune response against other bacteria.

Novel sensitive monoclonal antibody based competitive enzyme-linked immunosorbent assay for the detection of raw and processed bovine beta-casein.

Science.gov (United States)

Castillo, Daniela S; Cassola, Alejandro

2017-01-01

Cow milk protein allergy (CMPA) is the most common childhood food allergy, which can sometimes persist or can newly develop in adulthood with severe symptoms. CMPA's treatment is complete dietary avoidance of milk proteins. To achieve this task, patients have to be aware of milk proteins found as "hidden allergens" in food commodities. In regard to milk proteins, it has been reported that allergenicity of caseins remains unaffected upon heat treatment. For these reasons, we aimed to obtain monoclonal antibodies (mAbs) against native and denatured β-casein, one of the most abundant and antigenic caseins, in order to develop an indirect competitive ELISA (icELISA) to detect and quantify traces of this milk allergen in raw and processed foodstuffs. We developed two specific hybridoma clones, 1H3 and 6A12, which recognized β-casein in its denatured and native conformations by indirect ELISA (iELISA). Cross-reaction analysis by Western blot and iELISA indicated that these mAbs specifically recognized β-casein from bovine and goat milk extracts, while they did not cross-react with proteins present in other food matrixes. These highly specific mAbs enabled the development of sensitive, reliable and reproducible icELISAs to detect and quantify this milk protein allergen in food commodities. The extraction of β-casein from foodstuff was efficiently carried out at 60°C for 15 minutes, using an extraction buffer containing 1% SDS. The present study establishes a valid 1H3 based-icELISA, which allows the detection and quantification -0.29 ppm and 0.80 ppm, respectively- of small amounts of β-casein in raw and processed foods. Furthermore, we were able to detect milk contamination in incurred food samples with the same sensitivity as a commercial sandwich ELISA thus showing that this icELISA constitutes a reliable analytical method for control strategies in food industry and allergy prevention.

Radiolinja, Vodafone või Elisa

Index Scriptorium Estoniae

2004-01-01

Jüri Kaljundi, Tarvo Ulejev ning Anne Mere vastavad küsimusele, kas Radiolinja peaks jätma alles olemasoleva nime, võtma nimeks Soomes peagi Radiolinjat asendama hakkava Elisa või rahvusvaheliselt tuntud kaubamärgi Vodafone

Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays

DEFF Research Database (Denmark)

Møller, H J; Møller-Pedersen, T; Damsgaard, T E

1995-01-01

The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA...... cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate...... correlated well (r = 0.90) with the concentrations obtained with a traditional KS-ELISA that uses purified aggrecan as standard and coating antigen, and KS in both serum and synovial fluid could be measured with sufficient linearity....

Unreliability of three commercial Coxiella burnetii phase II IgM ELISA kits for the seroscreening of acute Q fever in human cases.

Science.gov (United States)

Stephen, Selvaraj; Ambroise, Stanley; Pradeep, Jothimani; Gunasekaran, Dhandapany; Sangeetha, Balakrishnan; Sarangapani, Kengamuthu

2017-09-01

Seroprevalence of Q fever (QF) caused by Coxiella burnetii has been reported from different parts of India. Usually serological/molecular tests are employed for detection of infection. The present study was undertaken to verify the validity of three different QF phase II IgM ELISA kits for acute QF diagnosis by comparing with the gold standard indirect fluorescent antibody assay (IFA). Fifty eight serum samples collected from 42 patients (26 patients provided acute sample only and 16 both acute and convalescent samples) which were examined by all three commercial kits, were cross-checked with QF Phase II IgM IFA for confirmation. Eleven patients were positive for C. burnetii antibodies by IFA in acute and/or convalescent serum samples. Taking IFA as a reference, percentages of sensitivity, specificity, positive predictive value and negative predictive value for Virion-Serion/Vircell/NovaTec were 36.36, 61.29, 25.00, 73.08; 81.82, 35.48, 31.03, 84.62 and 100, 25.81, 32.35, 100 per cent, respectively. The three different ELISA kits exhibited poor agreement amongst them and unacceptable level of false positivity. IFA remains to be the only option for diagnosing acute QF. Discrepancy between the clinical findings and IFA/ELISA results needs confirmation by C. burnetii DNA detection in real-time polymerase chain reaction.

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

DEFF Research Database (Denmark)

Klausen, Joan; Huda, A.; Ekeroth, Lars

2003-01-01

concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP. A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture......-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six...... culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80-96% at the cut-off values of 4...

A multi-purpose ultrasonic streaming mixer for integrated magnetic bead ELISAs

International Nuclear Information System (INIS)

Brandhoff, Lukas; Lang, Walter; Vellekoop, Michael J; Zirath, Helene; Peham, Johannes; Wiesinger-Mayr, Herbert; Salas, Mariugenia; Haller, Anna; Spittler, Andreas; Schnetz, Guntram

2015-01-01

We present an ultrasonic streaming mixer for disposable and on-chip magnetic bead ELISAs. The ultrasonic transducer is placed at system-level to keep cost per chip as low as possible, and is coupled to the chip by means of a solid ultrasonic horn. The system provides mixing of liquids, as well as dispersion of the superparamagnetic beads in the ELISA. Additionally it can be used clean the chamber surface from nonspecifically bound proteins during the washing steps in the ELISA protocol. Using our system the time for the ELISA protocol has been greatly reduced down to 30 min. (paper)

Acoustic waves and the detectability of first-order phase transitions by eLISA

Science.gov (United States)

Weir, David J.

2017-05-01

In various extensions of the Standard Model it is possible that the electroweak phase transition was first order. This would have been a violent process, involving the formation of bubbles and associated shock waves. Not only would the collision of these bubbles and shock waves be a detectable source of gravitational waves, but persistent acoustic waves could enhance the signal and improve prospects of detection by eLISA. I summarise the results of a recent campaign to model such a phase transition based on large-scale hydrodynamical simulations, and its implications for the eLISA mission.

ELISA, a demonstrator environment for information systems architecture design

Science.gov (United States)

Panem, Chantal

1994-01-01

This paper describes an approach of reusability of software engineering technology in the area of ground space system design. System engineers have lots of needs similar to software developers: sharing of a common data base, capitalization of knowledge, definition of a common design process, communication between different technical domains. Moreover system designers need to simulate dynamically their system as early as possible. Software development environments, methods and tools now become operational and widely used. Their architecture is based on a unique object base, a set of common management services and they host a family of tools for each life cycle activity. In late '92, CNES decided to develop a demonstrative software environment supporting some system activities. The design of ground space data processing systems was chosen as the application domain. ELISA (Integrated Software Environment for Architectures Specification) was specified as a 'demonstrator', i.e. a sufficient basis for demonstrations, evaluation and future operational enhancements. A process with three phases was implemented: system requirements definition, design of system architectures models, and selection of physical architectures. Each phase is composed of several activities that can be performed in parallel, with the provision of Commercial Off the Shelves Tools. ELISA has been delivered to CNES in January 94, currently used for demonstrations and evaluations on real projects (e.g. SPOT4 Satellite Control Center). It is on the way of new evolutions.

Refining the LPS-Antigen in Salmonella Antibody Elisa for Poultry Enhanced Specificity without Impairing Sensitivity

DEFF Research Database (Denmark)

Lauritsen, Klara Tølbøl; Lind, Peter; Klausen, Joan

2014-01-01

In the Danish serological surveillance for Salmonella in poultry (serum and egg yolk) a mix-ELISA is used, based on S. typhimurium and S. enteritidis antigens (Feld et al., 2000). When we evaluated results of the test retrospectively, over the years an unacceptably large fraction of seropositive...... findings could not be confirmed by the subsequent confirmatory bacteriological sampling in the herd. Therefore we tried to enhance specificity of the ELISA, without losing sensitivity, by refining the antigens used....

Comparação entre diversos antígenos para o diagnóstico de Anaplasma marginale por ELISA Comparison between several antigens for diagnosis of Anaplasma marginale by ELISA

Directory of Open Access Journals (Sweden)

Carlos A.N. Ramos

2010-01-01

immunosorbent assays (ELISAs, are the most used due to its versatility and practice. However, due to the high number of antigens currently available, an evaluation becomes necessary to define which antigens present the better performance in the diagnosis of anaplasmosis. Sera from cattle positive or negative to A. marginale by PCR, and sera from cattle proceeding from Brazil and Costa Rica, were tested by ELISAs based in recombinant MSP1a, MSP2, and MSP5, a pool of the three recombinant proteins, and initial body lisate antigen (CI. Using sera from A. marginale positive cattle by PCR, the highest sensitivity was shown by CI ELISA. Nevertheless, the highest specificity, with sera from negative cattle by PCR, was shown by recombinants ELISAs. The percentiles of positive cattle from Brazil and Costa Rica were higher with CI ELISA. Reasons for such differences were discussed.

The logic of indirect speech

Science.gov (United States)

Pinker, Steven; Nowak, Martin A.; Lee, James J.

2008-01-01

When people speak, they often insinuate their intent indirectly rather than stating it as a bald proposition. Examples include sexual come-ons, veiled threats, polite requests, and concealed bribes. We propose a three-part theory of indirect speech, based on the idea that human communication involves a mixture of cooperation and conflict. First, indirect requests allow for plausible deniability, in which a cooperative listener can accept the request, but an uncooperative one cannot react adversarially to it. This intuition is supported by a game-theoretic model that predicts the costs and benefits to a speaker of direct and indirect requests. Second, language has two functions: to convey information and to negotiate the type of relationship holding between speaker and hearer (in particular, dominance, communality, or reciprocity). The emotional costs of a mismatch in the assumed relationship type can create a need for plausible deniability and, thereby, select for indirectness even when there are no tangible costs. Third, people perceive language as a digital medium, which allows a sentence to generate common knowledge, to propagate a message with high fidelity, and to serve as a reference point in coordination games. This feature makes an indirect request qualitatively different from a direct one even when the speaker and listener can infer each other's intentions with high confidence. PMID:18199841

Homologous ELISA for detection of oligomeric human TNF: properties of the assay.

Science.gov (United States)

Petyovka, N; Lyach, L; Voitenok, N N

1995-10-26

In order to quantify oligomeric human tumor necrosis factor-alpha (TNF), we have developed a sensitive homologous enzyme-linked immunosorbent assay (Hm-ELISA) using the same monoclonal antibody (MoAb) for both solid and liquid phase. Different anti-TNF MoAb have been compared in terms of their efficacy in the Hm-ELISA, affinity, neutralization capacity and epitope specificity. The data suggest, that effectiveness in the Hm-ELISA may represent a novel characteristic of MoAb. Of the MoAbs tested, 5 N was capable of recognizing oligomeric TNF in the Hm-ELISA with a detection limit of 15 pg/ml. Furthermore, using Hm-ELISA against human TNF, interleukin-8 (IL-8) and lymphotoxin, we have demonstrated that these cytokines are oligomeric in physiological solutions, but are converted into monomeric forms in the presence of the non-ionic detergent Tween 20. High salt buffer was employed to abrogate a nonspecific false positive reaction in the Hm-ELISA found in nearly half of the plasma samples obtained from healthy subjects. Finally, a good correlation between the Hm-ELISA and the L929 bioassay was observed for natural and recombinant TNF measured in human plasma.

ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

DEFF Research Database (Denmark)

de Witte, H; Pappot, H; Brünner, N

1997-01-01

A sandwich-type ELISA has been developed for the assessment of complexes between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti-uPA antibodies and a biotinylated mouse...... anti-uPAR monoclonal antibody (MAb). The detection limit of the assay is approximately 0.5 fmol/ml. A linear dose-response is obtained with up to 40 fmol/ml of uPA:uPAR complexes, while uPA and uPAR separately do not cause any response in the ELISA. A buffer which has been used previously for optimal...... extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti-uPA or anti-uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross-linked uPA:uPAR complexes added to tumor...

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

Directory of Open Access Journals (Sweden)

Pace Umberto

2006-05-01

Full Text Available Abstract Background We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i products of the proteolytic (e.g. tryptic digestion of the protein to be identified and (ii unique to the target protein, as far as one can know from the published sequences. Results In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. Conclusion The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

Development of an ELISA-based kit for the on-farm determination of progesterone in milk

Czech Academy of Sciences Publication Activity Database

Simerský, Radim; Swaczynová, Jana; Morris, David; Fránek, M.; Strnad, Miroslav

2007-01-01

Roč. 52, č. 1 (2007), s. 19-28 ISSN 0375-8427 Institutional research plan: CEZ:AV0Z50380511 Keywords : bovine milk * ELISA * oestrus Subject RIV: CE - Biochemistry Impact factor: 0.645, year: 2007 http://old.vri.cz/docs/vetmed/52-1-19.pdf

The use of enzyme-linked immunosorbent assay (ELISA) for the diagnosis and monitoring of foot-and-mouth disease in the Philippines

International Nuclear Information System (INIS)

Verin, B.C.; Arvesu, R.M.

2000-01-01

The establishment and use of the indirect sandwich ELISA for the detection of foot-and-mouth disease (FMD) virus antigen sero-types O, A and C and the liquid phase blocking ELISA (LPB-ELISA) for antibody levels against similar FMD sero-types has been adopted for routine diagnosis at the FMD diagnostic laboratory, PAHC. A total of 552 epithelial samples and 4401 serum samples were tested starting 1995 to 1998. Out of 552, 84 (17.9%) were found negative and 468 (84.78%) diagnosed as positive for sero-types O and C (42% of the total positives). Within 4 years, 62 representative samples were sent to WRL for FMD for confirmation diagnosis. From 62 samples sent 54 (87%) were diagnosed as positive and 8 (12.9%) were negative. Serum samples received were either for diagnosis (71 samples), surveillance (3002 serum), post vaccination titre (1303 serum) and for the FAO/IAEA external quality assurance programme (25 samples) by the FAO/IAEA Co-ordinated Research Project on FMD. The assay has been a useful tool in the fast diagnosis and confirmation of FMD suspect cases and in the measurement of antibodies against FMDV in serum samples from all animals either vaccinated or infected. In the future, the assay will be use for potency testing of imported vaccines and for monitoring and surveillance purposes to show freedom from disease for the support documentation from OIE. (author)

Indirect estimators in US federal programs

CERN Document Server

1996-01-01

In 1991, a subcommittee of the Federal Committee on Statistical Methodology met to document the use of indirect estimators - that is, estimators which use data drawn from a domain or time different from the domain or time for which an estimate is required. This volume comprises the eight reports which describe the use of indirect estimators and they are based on case studies from a variety of federal programs. As a result, many researchers will find this book provides a valuable survey of how indirect estimators are used in practice and which addresses some of the pitfalls of these methods.

Development of an ELISA for evaluation of swab recovery efficiencies of bovine serum albumin.

Directory of Open Access Journals (Sweden)

Nadja Sparding

Full Text Available After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin with a detection range of 1.32-80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0-60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.

Gyro Drift Correction for An Indirect Kalman Filter Based Sensor Fusion Driver

Directory of Open Access Journals (Sweden)

Chan-Gun Lee

2016-06-01

Full Text Available Sensor fusion techniques have made a significant contribution to the success of the recently emerging mobile applications era because a variety of mobile applications operate based on multi-sensing information from the surrounding environment, such as navigation systems, fitness trackers, interactive virtual reality games, etc. For these applications, the accuracy of sensing information plays an important role to improve the user experience (UX quality, especially with gyroscopes and accelerometers. Therefore, in this paper, we proposed a novel mechanism to resolve the gyro drift problem, which negatively affects the accuracy of orientation computations in the indirect Kalman filter based sensor fusion. Our mechanism focuses on addressing the issues of external feedback loops and non-gyro error elements contained in the state vectors of an indirect Kalman filter. Moreover, the mechanism is implemented in the device-driver layer, providing lower process latency and transparency capabilities for the upper applications. These advances are relevant to millions of legacy applications since utilizing our mechanism does not require the existing applications to be re-programmed. The experimental results show that the root mean square errors (RMSE before and after applying our mechanism are significantly reduced from 6.3 × 10−1 to 5.3 × 10−7, respectively.

A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

Directory of Open Access Journals (Sweden)

Anju Mohan

2016-07-01

Full Text Available Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT, microtiter plate agglutination test (MAT, indirect hemagglutination assay (IHA, and indirect enzyme-linked immunosorbent assay (iELISA as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001. The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005. The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002. However, the difference in mean iELISA titers of infected cattle (1.3678±0.014 and healthy vaccinated cattle (1.367±0.014 was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

Modification the ELISA Kit for diagnosis of “Pseudomonas aeruginosa and comparing it with ordinary ELISA kit”

Directory of Open Access Journals (Sweden)

Taghreed A. Mohammad

2017-07-01

Full Text Available The first aim of the present study was to diagnosis Pseudomonas  aeruginosa by many tests. This study consisted "200 patients " who suffered from burn wound and compare with 100 health individuals (male and female as a control group, Vitek test was used to diagnose  118 (87 "local isolate  ATCC 15692" with 31 other isolate of Pseudomonas  aeruginosa  ((ATCC 15690, ATCC 15688  from 200 samples which were taken from burn patients. This result was similar to Analytical profile index ( API   test (118 isolates of  P. aeruginosa with 82 isolations of  other bacteria. Then the detection P. aeruginosa isolate  ATCC 15692 by new ELISA Technique and comparing its with modify the ordinary ELISA kit.

Indirect Reciprocity; A Field Experiment.

Science.gov (United States)

van Apeldoorn, Jacobien; Schram, Arthur

2016-01-01

Indirect reciprocity involves cooperative acts towards strangers, either in response to their kindness to third parties (downstream) or after receiving kindness from others oneself (upstream). It is considered to be important for the evolution of cooperative behavior amongst humans. Though it has been widely studied theoretically, the empirical evidence of indirect reciprocity has thus far been limited and based solely on behavior in laboratory experiments. We provide evidence from an online environment where members can repeatedly ask and offer services to each other, free of charge. For the purpose of this study we created several new member profiles, which differ only in terms of their serving history. We then sent out a large number of service requests to different members from all over the world. We observe that a service request is more likely to be rewarded for those with a profile history of offering the service (to third parties) in the past. This provides clear evidence of (downstream) indirect reciprocity. We find no support for upstream indirect reciprocity (in this case, rewarding the service request after having previously received the service from third parties), however. Our evidence of downstream indirect reciprocity cannot be attributed to reputational effects concerning one's trustworthiness as a service user.

SENSIBILIDAD DE INMUNOIMPRESIÓN-ELISA Y DAS-ELISA EN EL DIAGNÓSTICO Y MUESTREO DEL VIRUS DE LA TRISTEZA DE LOS CÍTRICOS EN HUERTOS COMERCIALES DE TAMAULIPAS, MEXICO

Directory of Open Access Journals (Sweden)

N. Ruiz-García

2009-01-01

Full Text Available El Virus de la tristeza de los cítricos (VTC causa una enfermedad de interés regulatorio para la citricultura mexicana. El diagnóstico y un muestreo oportuno y confiable es esencial para aplicar estrategias de manejo ante el avance en territorio nacional del pulgón café, su principal vector. Con el fin de contar con un método eficaz y eficiente para el muestreo y detección del VTC, se evaluó el desempeño del método de inmunoimpresión-ELISA con respecto a DAS-ELISA, el método oficial de diagnóstico, debido a su economía, facilidad y rapidez. Con este propósito se evaluaron 7,421 árboles, considerando la edad del brote y de infección, provenientes de 11 huertos comerciales de Tamaulipas. El método de inmunoimpresión-ELISA superó en sensibilidad y capacidad de pronóstico de positivos a DAS-ELISA en el diagnóstico de árboles con infección reciente o desconocida del VTC (P¿0.028. La disposición de brotes positivos en el dosel fue heterogénea siendo descrita por la función beta binomial (P¿0.16-0.23. Con base en esta función y la reproducción de los resultados de impresiones dobles por pecíolo (93.2 % se sugiere un tamaño óptimo de muestra de 10 pecíolos por árbol en impresiones simples. Este método fue 54.9 % más económico que DAS-ELISA y los diagnósticos se realizaron en una sexta parte del tiempo requerido en esta última prueba. Sin embargo, por el requerimiento de la norma oficial mexicana vigente (NOM- 031-FITO-2000, se recomienda el método de inmunoimpresión-ELISA como un método rápido para discriminar árboles positivos en muestreos de campo que deben posteriormente ser verificados por el método oficial de diagnóstico.

ELISA technique standardisation for human toxocariasis diagnosis

International Nuclear Information System (INIS)

Espinoza, Y.; Suarez, R.; Huiza, A.

2003-01-01

To standardise ELISA technique for toxocara canis human infection diagnosis by using excreted-secreted antigen prepared in our country. T. canis eggs were collected by incubation with formalin (2%) at 28 o C in order to obtain third stage larvae that were freed and incubated in RPMI at 37 o C for 7 days; the medium was replaced by a similar one and stored at -20 o C. Antigen was concentrated and protein dosage was made. Sera from patients with toxocariasis and newborns were used as positive and negative controls by ELISA technique, dilutions 1/4 to 1/1024. Polystyrene plates were sensitised with antigen in several concentrations and conjugated peroxidase with horseradish IgG, anti human IgG and substrate OPD were used. Absorbance was read with spectrophotometer (Multiskan plus labsystems) at 492 nm. Cut off point was determined by negative sera absorbencies arithmetic mean plus 3 standard deviations. Antigen concentration was 50 ug/mL, sera dilution 1/128, conjugate dilution 1/1000 with optical density above 0,241. ELISA technique for serologic diagnosis of human infection by toxocara canis could be used in epidemiological studies in our country. Its efficacy will be determined in future studies

Two distinct neural mechanisms underlying indirect reciprocity.

Science.gov (United States)

Watanabe, Takamitsu; Takezawa, Masanori; Nakawake, Yo; Kunimatsu, Akira; Yamasue, Hidenori; Nakamura, Mitsuhiro; Miyashita, Yasushi; Masuda, Naoki

2014-03-18

Cooperation is a hallmark of human society. Humans often cooperate with strangers even if they will not meet each other again. This so-called indirect reciprocity enables large-scale cooperation among nonkin and can occur based on a reputation mechanism or as a succession of pay-it-forward behavior. Here, we provide the functional and anatomical neural evidence for two distinct mechanisms governing the two types of indirect reciprocity. Cooperation occurring as reputation-based reciprocity specifically recruited the precuneus, a region associated with self-centered cognition. During such cooperative behavior, the precuneus was functionally connected with the caudate, a region linking rewards to behavior. Furthermore, the precuneus of a cooperative subject had a strong resting-state functional connectivity (rsFC) with the caudate and a large gray matter volume. In contrast, pay-it-forward reciprocity recruited the anterior insula (AI), a brain region associated with affective empathy. The AI was functionally connected with the caudate during cooperation occurring as pay-it-forward reciprocity, and its gray matter volume and rsFC with the caudate predicted the tendency of such cooperation. The revealed difference is consistent with the existing results of evolutionary game theory: although reputation-based indirect reciprocity robustly evolves as a self-interested behavior in theory, pay-it-forward indirect reciprocity does not on its own. The present study provides neural mechanisms underlying indirect reciprocity and suggests that pay-it-forward reciprocity may not occur as myopic profit maximization but elicit emotional rewards.

Evaluation of an Anti-rPA IgG ELISA for Measuring the Antibody Response in Mice

National Research Council Canada - National Science Library

Little, S

2004-01-01

A recombinant protective antigen (rPA)-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the serological response of female A/J mice after inoculation with the new rPA-based anthrax vaccine...

LISA Pathfinder and eLISA news

Science.gov (United States)

Thorpe, James Ira; Mueller, Guido

2014-01-01

Two important gatherings of the space-based gravitational-wave detector community were held in Zurich, Switzerland this past March. The first was a meeting of the Science Working Team for LISA Pathfinder (LPF), a dedicated technology demonstrator mission for a future LISA-like gravitational wave observatory. LPF is entering an extremely exciting phase with launch less than 15 months away. All flight components for both the European science payload, known as the LISA Technology Package (LTP), and the NASA science payload, known as the Space Technology 7 Disturbance Reduction System (ST7-DRS), have been delivered and are undergoing integration. The final flight component for the spacecraft bus, a cold-gas thruster based on the successful GAIA design, will be delivered later this year. Current focus is on completing integration of the science payload (see Figures 1 and 2) and preparation for operations and data analysis. After a launch in Summer 2015, LPF will take approximately 90 days to reach its operational orbit around the Earth-Sun Lagrange point (L1), where it will begin science operations. After 90 days of LTP operations followed by 90 days of DRS operations, LPF will have completed its prime mission of paving the way for a space-based observatory of gravitational waves in the milliHertz band. Immediately following the meeting of the LPF team, the eLISA consortium held its third progress meeting. The consortium (www.elisascience.org) is the organizing body of the European space-based gravitational-wave community, and it was responsible for the "The Gravitational Universe" whitepaper that resulted in the November 2013 election of a gravitational-wave science theme for ESA's Cosmic Visions L3 opportunity. In preparation for an L3 mission concept call, which is expected later this decade, and for launch in the mid 2030s, the eLISA consortium members are coordinating technology development and mission study activities which will build on the LPF results. The final

Acute hospital, community, and indirect costs of stroke associated with atrial fibrillation: population-based study.

LENUS (Irish Health Repository)

Hannon, Niamh

2014-10-30

No economic data from population-based studies exist on acute or late hospital, community, and indirect costs of stroke associated with atrial fibrillation (AF-stroke). Such data are essential for policy development, service planning, and cost-effectiveness analysis of new therapeutic agents.

The use of ELISAs for monitoring exposure of pig herds to Brachyspira hyodysenteriae

Directory of Open Access Journals (Sweden)

Song Yong

2012-01-01

Full Text Available Abstract Background Swine dysentery (SD, a mucohaemorrhagic diarrhoeal disease of pigs, results from infection of the large intestine with the spirochaete Brachyspira hyodysenteriae. ELISA systems using whole spirochaete cells (WC and the B. hyodysenteriae outer membrane lipoprotein Bhlp29.7 previously have been established as potential diagnostic tools for SD. However, their true value in identifying infected herds remains unclear. The present study aimed to compare the performance of whole-cell and Bhlp29.7 based ELISAs in detecting specific immunoglobulin class IgG and IgM to B. hyodysenteriae in growing pigs, and additionally evaluated whether meat juice could serve as a source of specific antibodies. Results Levels of circulating IgG and IgM reacting with WC spirochaete preparations and recombinant Bhlp29.7 peaked 4-6 weeks post-infection in the experimentally challenged pigs, and remained elevated in the present study. In a cohort of pigs on an infected farm levels of antibody directed against both antigens showed a progressive increase with time. However, other than for the level of IgG against WC antigen, a significant increase in antibody levels also was observed in a cohort of pigs on a non-infected farm. In addition, assays using meat juice had 100% specificity and equivalent sensitivity to those based on serum, and likewise the best performance was achieved using the WC IgG ELISA. Conclusions IgG ELISAs using either WC or Bhlp29.7 as plate-coating antigens were shown to be useful for monitoring the dynamics of B. hyodysenteriae infection in grower pigs. Of the two antigens, the WC preparation tended to give better discrimination between pigs from infected and non-infected farms. Testing of meat juice was shown to have potential for identifying infected herds.

Expression of the Tpanxb1 gene from Taenia pisiformis and its potential diagnostic value by dot-ELISA.

Science.gov (United States)

Yang, Deying; Chen, Lin; Wu, Xuhang; Zhou, Xuan; Li, Mei; Chen, Zuqin; Nong, Xiang; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2014-04-01

Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in rabbits that results in economic losses. To date, there has been limited information available on the early detection of infection by this parasite. This study describes a dot-ELISA method based on an autologous antigen annexin B1 (Tpanxb1). Its potential for serodiagnosis of rabbit cysticercosis was also evaluated. Western blot analysis revealed that the recombinant Tpanxb1 (rTpanxb1) protein could be specifically recognized by rabbit anti-sera. In serum trials, the antibodies could be detected by dot-ELISA using rTpanxb1 at 14 days post-infection. The positive response was present for up to 49 days post-infection. Based on the necropsy results of 169 rabbit samples, the relative sensitivity and specificity of the dot-ELISA were 94.55% and 92.86%, respectively. This study provides a foundation for studying the immunological function of annexin and its application to control Taenia cestodes.

Applicability of Commercially Available ELISA Kits for the Quantification of Faecal Immunoreactive Corticosterone Metabolites in Mice

DEFF Research Database (Denmark)

Abelson, Klas S P; Kalliokoski, Otto; Teilmann, Anne Charlotte

2016-01-01

Background: Commercially available ELISA kits are popular among investigators that quantify faecal corticosterone or cortisol metabolites (FCM) for stress assessment in animals. However, in faeces, these assays mainly detect immunoreactive glucocorticoid metabolites. Since different assays contain......: The present study was designed to investigate corticosterone (CORT) in serum and FCM levels in faeces of laboratory mice, as quantified in four different ELISA kits (DRG EIA-4164, Demeditec DEV9922, Enzo ADI-900-097 and Cayman EIA kit 500655). Assay kits were chosen based on the origin of the antibody...... assays, in both groups of mice. In faecal samples, there was no consistent positive correlation between the levels detected in the four assays and the measured concentration of FCM also differed between assays. Conclusion: Whereas commercially available CORT ELISAs are frequently successfully used...

An ELISA-inhibition test using monoclonal antibody for the serology of leprosy

NARCIS (Netherlands)

Klatser, P. R.; de Wit, M. Y.; Kolk, A. H.

1985-01-01

In this study a mouse monoclonal antibody (47-9) is described, which recognized an epitope on the 36 kD protein antigen of M. leprae. The monoclonal antibody showed specificity for M. leprae. An ELISA-inhibition test based on the competitive inhibition by antibodies from human test sera of the

A new ELISA for determination of potency in snake antivenoms.

Science.gov (United States)

Rial, A; Morais, V; Rossi, S; Massaldi, H

2006-09-15

A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.

Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes

DEFF Research Database (Denmark)

Mucci, Juan; Carmona, Santiago J.; Volcovich, Romina

2017-01-01

Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up....... cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic...... method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects). The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96...

EMT nurjas Rate.ee ostuga Elisa kõnekaardiplaani / Gert D. Hankewitz

Index Scriptorium Estoniae

Hankewitz, Gert D.

2006-01-01

Ilmunud ka: Delovõje Vedomosti 12. apr. lk. 2. Elisa juhatuse esimehe Sami Seppäneni sõnul ostis EMT suhtlusportaali Rate.ee, kuna sai teada, et Elisa ning portaal plaanivad koos uue kõnekaardi turule tuua

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...

Development of ELISA kit for rat albumin

International Nuclear Information System (INIS)

Yuan Zhigang; Han Shiquan; Liu Yibing; Xu Wenge; Jia Juanjuan

2009-01-01

The Anti-rat albumin serum was prepared by immunized the sheep with rat albumin. A ELISA method was established for rat albumin. The measurement range of the assay was 1-50 mg/L, sensitivity of the assay was 0.42 mg/L, recovery rate was 85.0%-106.0%. Intra-and inter-assay variation coefficients were <8.9% and <12.8% respectively. The correlation coefficients between measured and expected values were 0.999 after serial dilution of the urine samples with high concentrations of rat albumin. A good correlation was observed between the ELISA and RIA methods, and the kit for rat albumin might provide a convenience in exploitation of renal drugs and experimental injury of the kidney. (authors)

Elisa ja EMT kemplevad, kumb pakub paremat levi / Toivo Tänavsuu

Index Scriptorium Estoniae

Tänavsuu, Toivo

2007-01-01

Mobiilifirma Elisa väitel on Tartus kõige kvaliteetsem leviala Elisal. EMT võrgujuhi Margus Malvi hinnangul on EMT ja Elisa võrgud Tartus samaväärsed, ainus erinevus on kliendi kaugus tugijaamast. Lisa: Mobiililevi kvaliteet

ELISA reader does not interfere by mobile phone radiofrequency radiation

Science.gov (United States)

Mortazavi, Seyyed Mohammad Javad; Baradaran-Ghahfarokhi, Hamid Reza; Abdi, Mohammad Reza; Baradaran-Ghahfarokhi, Milad; Mostafavi, Nayyer Sadat; Mahmoudi, Golshan; Berenjkoub, Nafiseh; Akmali, Zahra; Hossein-Beigi, Fahimeh; Arsang, Vajiheh

2016-01-01

Background: The increasing number of mobile phones can physically cause electromagnetic interference (EMI) in medical environments; can also cause errors in immunoassays in laboratories. The ELISA readers are widely used as a useful diagnostic tool for Enzymun colorimetric assay in medicine. The aim of this study was to investigate whether the ELISA reader could be interfered by the exposure to the 900 MHz cell phones in the laboratory. Materials and Methods: Human serum samples were collected from 14 healthy donors (9 women and 5 men) and each sample was divided into four aliquots and was placed into four batches for the in-vitro quantitative determination of human chorionic gonadotropin (hCG). During colorimetric reading of the first, second, and third batches, the ELISA reader (Stat Fax 2100, Awareness Technology, Inc., USA) was exposed to 0.5, 1.0, and 2.0 W exposure of 900 MHz radiation, respectively. For the forth batch (control group), no radiation was applied. All experiments were performed comparing ELISA read out results of the I, II, and III batches with the control batch, using the Wilcoxon test with criterion level of P = 0.050. Results: The final scores in the exposed batches I, II, and III were not statistically significant relative to the control batch (P > 0.05). The results showed that 900 MHz radiation exposure did not alter the ELISA measured levels of hCG hormone in I (P = 0.219), II (P = 0.909), and III (P = 0.056) batches compared to the control batch. Conclusion: This study showed that ELISA reader does not interfere by mobile phone RF radiation at a closed contact (less than 5 cm distance). However, we recommend that medical institutions discuss these issues in the context of their specific use of technologies and frame a policy that is clear and straightforward to guide staff, patients, and visitors. PMID:27376040

Pedal indirect lymphangiography

International Nuclear Information System (INIS)

Lee, Kil Woo; Hong, Myung Sun; Kim, In Jae

1994-01-01

Recently, indirect lymphangiography has been developed as a relatively good and noninvasive imaging modality of the lymphatic system at extremities. But the disadvantage of the indirect lymphangiography is a low contrast ratio between the surrounding tissues and the contrast media in lymphatic vessels, because dimeric nonionic contrast media is water soluble and diluted in the proximal leg lymphatic vessels. We could have relatively better image than previously published images for the leg lymphatic system, when we injected contrast media with adequate high pressure in intradermal space of the interdigital areas at the foot dorsum. So, we would like to report the results. We could study all 9 lymphedemas(primary: 6, secondary: 3) from April 1990 to May 1993 on outpatient base. They were diagnosed as lymphedema clinically and radiologically. Ten ml of dimeric nonionic aget, iotrolan(Isovist 300) was injected into intradermal space with five 30-gauge needles. The injection speed was more than 0.2 ml/min. We have done one side pedal lymphangiogram in 30 minutes. The evaluation of the anterior superficial lymphatics was according to the criteria of the Weissleder. The results were as follows: 1. All lymphatic vessels from foot to inguinal area could be visualized. 2. Two or three inferior inguinal lymph nodes could be visualized about 42%. 3. The most common abnormal finding of the lymphedma was the neovascularization of the lymphatics on indirect pedal lymphangiogram. If we use adequate technique relatively high pressure injection, correct intradermal needle insertion, adequate soft tissue exposure technique indirect lymphangiography is considered to be a safe and noninvasive imaging modality for the evaluation of the lymphedema of lower extremity lymphatics including inferior inguinal lymph nodes

Biomass Indirect Liquefaction Strategy Workshop Summary Report

Energy Technology Data Exchange (ETDEWEB)

none,

2014-07-01

This report is based on the proceedings of the U.S. Department of Energy Bioenergy Technologies Office Biomass Indirect Liquefaction Strategy Workshop. The workshop, held March 20–21, 2014, in Golden, Colorado, discussed and detailed the research and development needs for biomass indirect liquefaction. Discussions focused on pathways that convert biomass-based syngas (or any carbon monoxide, hydrogen gaseous stream) to liquid intermediates (alcohols or acids) and further synthesize those intermediates to liquid hydrocarbons that are compatible as either a refinery feed or neat fuel.

Application of Microwave Irradiation and Heat to Improve Gliadin Detection and Ricin ELISA Throughput with Food Samples

Directory of Open Access Journals (Sweden)

Eric A. E. Garber

2015-06-01

Full Text Available The utility of microwave irradiation to accelerate the onset of equilibrium and improve ELISA performance was examined using ELISAs for the detection of the plant toxin ricin and gliadin. The ricin ELISA normally requires several one hour incubations at 37 °C, a total assay time of approximately five hours, and employs a complex buffer containing PBS, Tween-20®, and non-fat milk. Different energy levels and pulse designs were compared to the use of abbreviated incubation times at 37 °C for the detection of ricin in food. The use of microwave irradiation had no significant advantage over the application of heat using an oven incubator and performed worse with some foods. In contrast, a gliadin ELISA that relied on 30 min incubation steps at room temperature and a salt-based buffer performed better upon irradiation but also displayed improvement upon incubating the microtiter plate at 37 °C. Whether microwave irradiation was advantageous compared to incubation in an oven was inconclusive. However, by abbreviating the incubation time of the ricin ELISA, it was possible to cut the assay time to less than 2 hours and still display LOD values < 10 ppb and recoveries of 78%–98%.

State and force observers based on multibody models and the indirect Kalman filter

Science.gov (United States)

Sanjurjo, Emilio; Dopico, Daniel; Luaces, Alberto; Naya, Miguel Ángel

2018-06-01

The aim of this work is to present two new methods to provide state observers by combining multibody simulations with indirect extended Kalman filters. One of the methods presented provides also input force estimation. The observers have been applied to two mechanism with four different sensor configurations, and compared to other multibody-based observers found in the literature to evaluate their behavior, namely, the unscented Kalman filter (UKF), and the indirect extended Kalman filter with simplified Jacobians (errorEKF). The new methods have some more computational cost than the errorEKF, but still much less than the UKF. Regarding their accuracy, both are better than the errorEKF. The method with input force estimation outperforms also the UKF, while the method without force estimation achieves results almost identical to those of the UKF. All the methods have been implemented as a reusable MATLAB® toolkit which has been released as Open Source in https://github.com/MBDS/mbde-matlab.

Indirect Fluorescent Antibody Technique based Prevalence of Surra in Equines

Directory of Open Access Journals (Sweden)

Ahsan Nadeem, Asim Aslam*, Zafar Iqbal Chaudhary, Kamran Ashraf1, Khalid Saeed1, Nisar Ahmad1, Ishtiaq Ahmed and Habib ur Rehman2

2011-04-01

Full Text Available This project was carried out to find the prevalence of trypanosomiasis in equine in District Gujranwala by using indirect fluorescent antibody technique and thin smear method. Blood samples were collected from a total of 200 horses and donkeys of different ages and either sex. Duplicate thin blood smears were prepared from each sample and remaining blood samples were centrifuged to separate the serum. Smears from each animal were processed for giemsa staining and indirect fluorescent antibody test (IFAT. Giemsa stained smears revealed Trypanosome infection in 4/200 (2.0% samples and IFAT in 12/200 (6.0% animals.

Development and evaluation of porcine cysticercosis QuickELISA in Triturus EIA analyzer.

Science.gov (United States)

Handali, Sukwan; Pattabhi, Sowmya; Lee, Yeuk-Mui; Silva-Ibanez, Maria; Kovalenko, Victor A; Levin, Andrew E; Gonzalez, Armando E; Roberts, Jacquelin M; Garcia, Hector H; Gilman, Robert H; Hancock, Kathy; Tsang, Victor C W

2010-01-01

We evaluated three diagnostic antigens (recombinant GP50, recombinant T24H, and synthetic Ts18var1) for cysticercosis and found that all three performed well in detecting cysticercosis in humans and pigs in several assay formats. These antigens were adapted to a new antibody detection format (QuickELISA). With one single incubation step which involves all reactants except the enzyme substrate, the QuickELISA is particularly suited for automation. We formatted the QuickELISA for the Triturus EIA analyzer for testing large numbers of samples. We found that in QuickELISA formats rGP50 and rT24H have better sensitivity and specificity than sTs18var1 for detecting porcine cysticercosis.

Indirect taxes on food in Southern Brazil

Directory of Open Access Journals (Sweden)

Denize Mirian da Silva

2013-12-01

Full Text Available The objective of this paper is to estimate the indirect tax burden on food for ten income classes, based on income and household total expenditure in southern Brazil. Thus it can be seen as indirect taxes on foods affect the monetary income and consumption pattern of households. To reach the objectives proposed, will be used the Pintos-Payeras (2008 model. The database iscomposed by microdata from the Household Budgeting Survey (POF 2008-2009 and the tax regulations of the country and the southern states of Brazil. The results show that indirect taxes on food in Southern Brazil is regressive when based on income and expenditure of household , ie , the poorest people pay proportionately more taxes and have their consumption pattern highest taxed ICMS (Brazilian value added tax is the tax that contributes most to the regressivity.

Direct and indirect effects for neighborhood-based clustered and longitudinal data

OpenAIRE

VanderWeele, T.J.

2010-01-01

Definitions of direct and indirect effects are given for settings in which individuals are clustered in groups or neighborhoods and in which treatments are administered at the group level. A particular intervention may affect individual outcomes both through its effect on the individual and by changing the group or neighborhood itself. Identification conditions are given for controlled direct effects and for natural direct and indirect effects. The interpretation of these identification condi...

Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

Science.gov (United States)

Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

2016-09-01

Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Establishment of a quantitative ELISA capable of determining peptide - MHC class I interaction

DEFF Research Database (Denmark)

Sylvester-Hvid, C; Kristensen, N; Blicher, T

2002-01-01

dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards...

Low-Frequency Gravitational-Wave Science with eLISA/ NGO

Science.gov (United States)

Amaro-Seoane, Pau; Aoudia, Sofiane; Babak, Stanislav; Binetruy, Pierre; Berti, Emanuele; Bohe, Alejandro; Caprini, Chiara; Colpi, Monica; Cornish, Neil J.; Danzmann, Karsten;

The evaluation of CLIA, RIA and MSP-ELISA for measurement of thyroid hormones

International Nuclear Information System (INIS)

Jing Hua; Li Dan; Chen Yiguang; Zhou Huiqin; Xu Liyan

2005-01-01

To compare the characteristics of chemiluminescent immunoassay (CLIA), RIA and magnetic solid phase enzyme-linked immnosorbent assay (MSP-ELISA) in measuring thyroid hormones, TT 3 , TT 4 , FT 3 , FT 4 and TSH were tested in 40 samples of human serum and in standard samples of thyroid hormones by CLIA, RIA and MSP-ELISA respectively. The linearity, relativiy, precision, recovery of these three met hods were compared. The result showed that there were no statistical differences between CLIA, RIA and MSP-ELISA in linearity and relativity. CLIA was better than RIA and MSP-ELISA in precision and accuracy. (authors)

Development and evaluation of an ELISA for human trefoil factor 3

DEFF Research Database (Denmark)

Vestergaard, Else Marie; Poulsen, Steen Seier; Grønbaek, Henning

2002-01-01

are warranted. METHODS: An ELISA was developed that uses two antibodies from rabbits immunized with recombinant human TFF3 and a calibrator (3-100 pmol/L) prepared from recombinant human TFF3. RESULTS: The ELISA had a detection limit of 3.0 pmol/L. The imprecision (CV) was 5-9% for mean concentrations of 13...... exacerbation of chronic inflammatory bowel disease restricted to the colon, normal concentrations and only minor variations during treatment and tapering were observed. CONCLUSIONS: The ELISA measures TFF3 in human serum and represents a specific and precise method for measurement of TFF3, which...

Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB in Buffer and Milk

Directory of Open Access Journals (Sweden)

Yacine Nia

2016-09-01

Full Text Available The aim of this work was to organize the first proficiency test (PT dedicated to staphylococcal enterotoxin B (SEB detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness.

Agricultural production - Phase 2. Indonesia. ELISA for epidemiology of brucellosis

International Nuclear Information System (INIS)

Sutherland, S.S.

1992-01-01

This document is a travel report of a three-week mission (from October 13 to November 1, 1991) to Indonesia within the framework of ''The implementation of ELISA technology for the sero-diagnosis of important livestock diseases''. The mission evaluated the implementation of ELISA technology at the Regional Laboratories and its role in the surveillance and control of bovine brucellosis

CONTRIBUTION OF INDIRECT TAXES

Directory of Open Access Journals (Sweden)

CHIRCULESCU MARIA FELICIA

2015-08-01

Full Text Available The work is based on the fact that at any time and in any society, taxation is regarded as undesirable for all taxpayers. The existence and it's manifestation is justified, because the operation of any company involves costs that must be covered by sufficient resources. Since ancient times, each state has adopted its own tax system, more or less perfected, as the state has experienced a greater or lesser economic and military power At the base of this work stays the fact that tax systems are a key factor influencing the overall efficiency of the economy. They determine the size tendency to save, invest and work, influencing the increase in production and employment, which is essential sights integral economic strategy, making tax reform an important component of economic reform. This paper aims to analyze the indirect taxes and their contribution to the public revenues in Romania, the purpose paper contains an analysis based on statistical series as indirect taxation is where tax harmonization was possible. Through analyzes, the paper aims to provide answers to the problem of the contradiction between the growing need for budgetary revenues, which entails a continuous amplification and diversification of taxation, on the one hand, and the need to stimulate economic development, on the other hand. The harmonization of indirect taxation had been achieved since this touches the free movement of goods and the freedom to supply services, not being able to say the same thing about direct taxation, which is why the European Community Treaty does not specify expressly the alignment of direct taxation, considering that direct taxation is a matter of Internal Policies that, for a country free option.

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

DEFF Research Database (Denmark)

Selman, L; Henriksen, M L; Brandt, J

2012-01-01

-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies....... The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined...... and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes....

Development of a species-specific coproantigen ELISA for human Taenia solium taeniasis.

Science.gov (United States)

Guezala, Maria-Claudia; Rodriguez, Silvia; Zamora, Humberto; Garcia, Hector H; Gonzalez, Armando E; Tembo, Alice; Allan, James C; Craig, Philip S

2009-09-01

Taenia solium causes human neurocysticercosis and is endemic in underdeveloped countries where backyard pig keeping is common. Microscopic fecal diagnostic methods for human T. solium taeniasis are not very sensitive, and Taenia saginata and Taenia solium eggs are indistinguishable under the light microscope. Coproantigen (CoAg) ELISA methods are very sensitive, but currently only genus (Taenia) specific. This paper describes the development of a highly species-specific coproantigen ELISA test to detect T. solium intestinal taeniasis. Sensitivity was maintained using a capture antibody of rabbit IgG against T. solium adult whole worm somatic extract, whereas species specificity was achieved by utilization of an enzyme-conjugated rabbit IgG against T. solium adult excretory-secretory (ES) antigen. A known panel of positive and negative human fecal samples was tested with this hybrid sandwich ELISA. The ELISA test gave 100% specificity and 96.4% sensitivity for T. solium tapeworm carriers (N = 28), with a J index of 0.96. This simple ELISA incorporating anti-adult somatic and anti-adult ES antibodies provides the first potentially species-specific coproantigen test for human T. solium taeniasis.

The Platelia Aspergillus ELISA in diagnosis of invasive pulmonary aspergilosis (IPA).

Science.gov (United States)

Siemann, M; Koch-Dörfler, M

2001-01-01

The sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was evaluated with 66 serum samples and 113 specimens of the respiratory tract obtained from 52 patients with pulmonary diseases. The patients were divided into five groups: proven invasive pulmonary aspergillosis (IPA) (five patients), probable IPA (seven patients), Aspergillus colonization (eight patients) or unlikely Aspergillus infection (27 patients). Another five patients with doubtful diagnostic test results are discussed in detail. The results of the Platelia Aspergillus ELISA (Sanofi Pasteur, Freiburg, Germany) in testing specimens of the respiratory tract were 90% sensitivity in proven (serum 38%), 60% in probable (serum 37%) and 71% in Aspergillus colonization (serum 0%). Furthermore, 85% of the Aspergillus spp. from positive cultures of specimens of the respiratory tract were also detected in the ELISA. A total of 57% of the culture negative specimens of patients with a least one positive culture or proven aspergillosis in a series of specimens were positive in the ELISA.

Generation of polyclonal antibody with high avidity to rosuvastatin and its use in development of highly sensitive ELISA for determination of rosuvastatin in plasma

Directory of Open Access Journals (Sweden)

Al-Malaq Hamoud A

2011-07-01

Full Text Available Abstract In this study, a polyclonal antibody with high avidity and specificity to the potent hypocholesterolaemic agent rosuvastatin (ROS has been prepared and used in the development of highly sensitive enzyme-linked immunosorbent assay (ELISA for determination of ROS in plasma. ROS was coupled to keyhole limpt hemocyanin (KLH and bovine serum albumin (BSA using carbodiimide reagent. ROS-KLH conjugate was used for immunization of female 8-weeks old New Zealand white rabbits. The immune response of the rabbits was monitored by direct ELISA using ROS-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and avidity to ROS was scarified and its sera were collected. The IgG fraction was isolated and purified by avidity chromatography on protein A column. The purified antibody showed high avidity to ROS; IC50 = 0.4 ng/ml. The specificity of the antibody for ROS was evaluated by indirect ELISA using various competitors from the ROS-structural analogues and the therapeutic agents used with ROS in a combination therapy. The proposed ELISA involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG and 3,3',5,5'-tetramethylbenzidine (TMB as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the color intensity in the assay wells. The assay enabled the determination of ROS in plasma at concentrations as low as 40 pg/ml.

Indirection and computer security.

Energy Technology Data Exchange (ETDEWEB)

Berg, Michael J.

2011-09-01

The discipline of computer science is built on indirection. David Wheeler famously said, 'All problems in computer science can be solved by another layer of indirection. But that usually will create another problem'. We propose that every computer security vulnerability is yet another problem created by the indirections in system designs and that focusing on the indirections involved is a better way to design, evaluate, and compare security solutions. We are not proposing that indirection be avoided when solving problems, but that understanding the relationships between indirections and vulnerabilities is key to securing computer systems. Using this perspective, we analyze common vulnerabilities that plague our computer systems, consider the effectiveness of currently available security solutions, and propose several new security solutions.

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.

Selectivity verification of cardiac troponin monoclonal antibodies for cardiac troponin detection by using conventional ELISA

Science.gov (United States)

Fathil, M. F. M.; Arshad, M. K. Md; Gopinath, Subash C. B.; Adzhri, R.; Ruslinda, A. R.; Hashim, U.

2017-03-01

This paper presents preparation and characterization of conventional enzyme-linked immunosorbent assay (ELISA) for cardiac troponin detection to determine the selectivity of the cardiac troponin monoclonal antibodies. Monoclonal antibodies, used to capture and bind the targets in this experiment, are cTnI monoclonal antibody (MAb-cTnI) and cTnT monoclonal antibody (MAb-cTnT), while both cardiac troponin I (cTnI) and T (cTnT) are used as targets. ELISA is performed inside two microtiter plates for MAb-cTnI and MAb-cTnT. For each plate, monoclonal antibodies are tested by various concentrations of cTnI and cTnT ranging from 0-6400 µg/l. The binding selectivity and level of detection between monoclonal antibodies and antigen are determined through visual observation based on the color change inside each well on the plate. ELISA reader is further used to quantitatively measured the optical density of the color changes, thus produced more accurate reading. The results from this experiment are utilized to justify the use of these monoclonal antibodies as bio-receptors for cardiac troponin detection by using field-effect transistor (FET)-based biosensors coupled with substrate-gate in the future.

Merozoite proteins from Babesia sp. BQ1 (Lintan) as potential antigens for serodiagnosis by ELISA.

Science.gov (United States)

Guan, G Q; Chauvin, A; Rogniaux, H; Luo, J X; Yin, H; Moreau, E

2010-05-01

Babesia sp. BQ1 (Lintan) is a Babesia isolated from sheep infested with Haemaphysalis qinghaiensis in China, and is closely related to B. motasi based on the 18S rRNA gene sequence. In the present study, an ELISA was developed with merozoite antigens of Babesia sp. BQ1 (Lintan) (BQMA) purified from in vitro culture. When the positive threshold was chosen as 30% of the antibodies rate, evaluated with 198 negative sera, the specificity was 95.5%. Except for Babesia sp. Tianzhu, there was no cross-reaction between BQMA and positive sera from Babesia sp. BQ1 (Ningxian)-, Babesia sp. Hebei-, Babesia sp. Xinjiang-, Theileria luwenshuni-, T. uilenbergi-, or Anaplasma ovis-infected sheep, which are the dominant haemoparasites of small ruminants in China. Specific antibodies against Babesia sp. BQ1 (Lintan) were produced 1 or 2 weeks post-infection and a high level of antibodies persisted for more than 8 months in experimentally infected sheep. This ELISA was tested on 974 sera collected from field-grazing sheep in 3 counties of Gansu province, northwestern China to evaluate the seroprevalence of Babesia sp. BQ1 (Lintan) infection and the average positive rate was 66.84%. The feasibility of increasing the specificity of this BQMA-based ELISA, by using some BQMA antigens for serodiagnosis is discussed.

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

Science.gov (United States)

Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

2016-01-01

Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

Science.gov (United States)

Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

2016-01-01

Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds

DEFF Research Database (Denmark)

Grøndahl-Hansen, Jan; Barfod, Kristen; Klausen, Joan

2003-01-01

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot...... of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes....... phenol-water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2...

Smoking Cessation Is Associated With Lower Indirect Costs.

Science.gov (United States)

Baker, Christine L; Bruno, Marianna; Emir, Birol; Li, Vicky W; Goren, Amir

2018-06-01

This study quantified differences in indirect costs due to decreased work productivity between current and former smokers. Former smokers were further categorized by number of years since quitting to assess corresponding differences. Data on employed individuals were obtained from the 2013 US National Health and Wellness Survey (NHWS; N = 75,000). Indirect costs were calculated for current smokers and former smokers from weekly wages based on age and sex. The annual total indirect costs for current smokers were $1327.53, $1560.18, and $1839.87 higher than for those who quit 0 to 4 years, 5 to 10 years, and more than or equal to 11 years prior, respectively. There were no significant differences in mean total indirect costs between the former smoker groups. Current smokers showed significantly higher total annual indirect costs compared with former smokers, independently of the number of years since quitting smoking.

Detection of Candida albicans Sap2 in cancer patient serum samples by an indirect competitive enzyme-linked immunosorbent assay for the diagnosis of candidiasis

OpenAIRE

Yicun Wang; Xiang Gao; J U Zhi gang; Jingyuan Liu; Shuai Dong; Li Wang

2013-01-01

Background: The secreted aspartyl proteinases 2 (Sap2) of Candida albicans (C. albicans) is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. Aim: In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA). Materials and Methods: Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1...

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Science.gov (United States)

Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

2014-07-01

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. © 2013 Wiley Periodicals, Inc.

Development and primary application of ELISA method for detecting residual calf serum protein

International Nuclear Information System (INIS)

Li Hua; Yu Jiankun; Hong Chao; Long Ruixiang; Zhai Yougang; Xu Qiongfang; Cui Pingfang; Ding Xuefeng; Xie Zhongping

2005-01-01

To develop a new DAS-ELISA (double antibody sandwich ELISA) kit for detecting residual calf serum protein (CSP) in vaccines, calf sera from different district were pooled and used to immunize rabbits and hens respectively. Then, the IgY from yolk and the anti-CSP IgG from rabbit were separated and purified. The purified IgY was used as the coating antibody, and purified rabbit anti-CSP IgG was labeled by HRP. The optimal concentration of IgY was 25-30 μg/mL. The coating buffer was 0.01mol/L PBS(pH7.4) containing 0.4% glutin. The optimal dilutions of HRP-IgG were from 1:2000 to 1:3000. The sensitivity of this ELISA method was higher (up to 2.5μg/mL) than that of current RPHA, the variation coefficient was about 6.3%-9.4%, and the recovery rate was 90.4%-112.8%. Furthermore, there was no cross-reaction with sera of pig, monkey and guinea pig. Twenty lots of vaccines with Al(OH) 3 or without Al(OH) 3 were tested by ELISA and RPHA respectively. The results proved that the adjuvant of Al(OH) 3 had fewer influence on ELISA than on RPHA, the variation of PRHA among different lots of vaccines was more significant than ELISA. The ELISA method is a highly sensitive and useful method to detect CSP in vaccines. (authors)

Indirect inference with time series observed with error

DEFF Research Database (Denmark)

Rossi, Eduardo; Santucci de Magistris, Paolo

estimation. We propose to solve this inconsistency by jointly estimating the nuisance and the structural parameters. Under standard assumptions, this estimator is consistent and asymptotically normal. A condition for the identification of ARMA plus noise is obtained. The proposed methodology is used......We analyze the properties of the indirect inference estimator when the observed series are contaminated by measurement error. We show that the indirect inference estimates are asymptotically biased when the nuisance parameters of the measurement error distribution are neglected in the indirect...... to estimate the parameters of continuous-time stochastic volatility models with auxiliary specifications based on realized volatility measures. Monte Carlo simulations shows the bias reduction of the indirect estimates obtained when the microstructure noise is explicitly modeled. Finally, an empirical...

Performance analysis of a bio-gasification based combined cycle power plant employing indirectly heated humid air turbine

Energy Technology Data Exchange (ETDEWEB)

Mukherjee, S., E-mail: sankha.deepp@gmail.com; Mondal, P., E-mail: mondal.pradip87@gmail.com; Ghosh, S., E-mail: sudipghosh.becollege@gmail.com [Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah – 711103, West Bengal (India)

2016-07-12

Rapid depletion of fossil fuel has forced mankind to look into alternative fuel resources. In this context, biomass based power generation employing gas turbine appears to be a popular choice. Bio-gasification based combined cycle provides a feasible solution as far as grid-independent power generation is concerned for rural electrification projects. Indirectly heated gas turbine cycles are promising alternatives as they avoid downstream gas cleaning systems. Advanced thermodynamic cycles have become an interesting area of study to improve plant efficiency. Water injected system is one of the most attractive options in this field of applications. This paper presents a theoretical model of a biomass gasification based combined cycle that employs an indirectly heated humid air turbine (HAT) in the topping cycle. Maximum overall electrical efficiency is found to be around 41%. Gas turbine specific air consumption by mass is minimum when pressure ratio is 6. The study reveals that, incorporation of the humidification process helps to improve the overall performance of the plant.

Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

Directory of Open Access Journals (Sweden)

Myllyharju Johanna

2010-06-01

Full Text Available Abstract Background We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H, the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation. Results We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase® verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield. Conclusions Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due

Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays

DEFF Research Database (Denmark)

Møller, H J; Møller-Pedersen, T; Damsgaard, T E

1995-01-01

The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA...... inhibition studies, immunoblotting and HPLC analyses confirmed the presence of substantial amounts of KS, probably as a large proteoglycan (> 120 kDa). Commercial and heterogenic glycosaminoglycan preparations therefore must be used with great caution in immunological analyses. On the other hand the shark...... cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate...

Prospects of eLISA for Detecting Galactic Binary Black Holes Similar to GW150914

OpenAIRE

Seto, Naoki

2016-01-01

We discuss the prospects of eLISA for detecting gravitational waves (GWs) from Galactic binary black holes (BBHs) similar to GW150914. For a comoving merger rate that is consistent with current observation, eLISA is likely to identify at least one BBH with a sufficient signal-to-noise ratio. In addition, eLISA has a potential to measure the eccentricity of the BBH as small as $e\\sim 0.02$, corresponding to the residual value $e\\sim 10^{-6}$ at 10Hz. Therefore, eLISA could provide us with a cr...

Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

Science.gov (United States)

Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A; González-Warleta, Marta; Mezo, Mercedes; Ubeira, Florencio M

2016-07-01

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1-10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.

Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens

Science.gov (United States)

Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; González-Warleta, Marta; Mezo, Mercedes; Ubeira, Florencio M.

2016-01-01

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1–10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis. PMID:27438470

The use of filter paper plasticized with polyvinyl alcohol-glutaraldehyde in ELISA

Directory of Open Access Journals (Sweden)

G.H.T.S. Barbosa

2000-07-01

Full Text Available F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3% w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.

A novel approach for osteocalcin detection by competitive ELISA using porous silicon as a substrate.

Science.gov (United States)

Rahimi, Fereshteh; Mohammadnejad Arough, Javad; Yaghoobi, Mona; Davoodi, Hadi; Sepehri, Fatemeh; Amirabadizadeh, Masood

2017-11-01

In this study, porous silicon (PSi) was utilized instead of prevalent polystyrene platforms, and its capability in biomolecule screening was examined. Here, two types of porous structure, macroporous silicon (Macro-PSi) and mesoporous silicon (Meso-PSi), were produced on silicon wafers by electrochemical etching using different electrolytes. Moreover, both kinds of fresh and oxidized PSi samples were investigated. Next, osteocalcin as a biomarker of the bone formation process was used as a model biomarker, and the colorimetric detection was performed by competitive enzyme-linked immunosorbent assay (ELISA). Both Macro-PSi and Meso-PSi substrates in the oxidized state, specifically the Meso-porous structure, were reported to have higher surface area to volume ratio, more capacitance of surface-antigen interaction, and more ability to capture antigen in comparison with the prevalent platforms. Moreover, the optical density signal of osteocalcin detected by the ELISA technique was notably higher than the common platforms. Based on the findings of this study, PSi can potentially be used in the ELISA to achieve better results and consequently more sensitivity. A further asset of incorporating such a nanometer structure in the ELISA technique is that the system response to analyte concentration could be maintained by consuming lower monoclonal antibody (or antigen) and consequently reduces the cost of the experiment. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

Science.gov (United States)

Liu, Shuangshuang; Li, Shasha; Zhang, Yang; Wang, Ye; Zhu, Yumeng; Wang, Bin; Chen, Zhi-Nan

2017-01-01

The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane-spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen-immunoaffinity chromatography purification. The purity of rabbit anti-CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti-CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti-hepatoma HAb18 monoclonal antibodies and purified rabbit anti-CD147 polyclonal antibodies. The present study demonstrated that antigen-immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs. PMID:28487989

Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance.

Science.gov (United States)

Estevez, M-Carmen; Belenguer, Jose; Gomez-Montes, Silvia; Miralles, Javier; Escuela, Alfonso M; Montoya, Angel; Lechuga, Laura M

2012-12-07

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 μg L(-1)) and an IC(50) of 3.2 nM (0.64 μg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.

An ELISA for the quantitation of von Willebrand Factor

DEFF Research Database (Denmark)

Vinholt, Pernille Just; Overgaard, Martin; Diederichsen, Axel Cosmus Pyndt

2013-01-01

for measurement of von Willebrand factor-osteoprotegerin complex (VWF:OPG) in human plasma. Furthermore, the significance of VWF:OPG complex as a marker of cardiovascular disease (CVD) was evaluated. PATIENTS/METHODS: A sandwich ELISA for quantification of VWF:OPG was developed using a polyclonal rabbit anti...... with and without documented coronary calcification (total n=118). RESULTS AND CONCLUSIONS: The assay detected VWF:OPG complexes in human plasma, while no significant signal was observed when testing solutions containing VWF or recombinant OPG alone. Importantly, the ELISA assay was able to detect in vitro formed...

Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA.

Science.gov (United States)

Fukushi, Shuetsu; Fukuma, Aiko; Kurosu, Takeshi; Watanabe, Shumpei; Shimojima, Masayuki; Shirato, Kazuya; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ohnishi, Kazuo; Ato, Manabu; Melaku, Simenew Keskes; Sentsui, Hiroshi; Saijo, Masayuki

2018-01-01

Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

International ring trial to detect anti-Trichinella IgG by ELISA on pig sera.

Science.gov (United States)

In the present study, the sensitivity and specificity of the ELISA assay determined by the CRLP validation was 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% an...

Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

Science.gov (United States)

Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

2015-01-01

Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

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.

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.

Assessment of Metabolic Flexibility of Old and Adult Mice Using Three Noninvasive, Indirect Calorimetry-Based Treatments

NARCIS (Netherlands)

Duivenvoorde, L.P.M.; Schothorst, van E.M.; Swarts, J.J.M.; Keijer, J.

2015-01-01

Indirect calorimetry (InCa) can potentially be used to noninvasively assess metabolic and age-related flexibility. To assess the use of InCa for this purpose, we tested the sensitivity and response stability over time of three InCa-based treatments in old versus adult mice. Diurnal patterns of

Cloning and expression of nucleocapsid protein of feline infectious peritonitis virus and establishment of an indirect ELISA for EIPV%猫传染性腹膜炎病毒核衣壳蛋白的表达及间接ELISA法的建立

Institute of Scientific and Technical Information of China (English)

熊炜; 林颖峥; 王艳; 魏晓锋; 王巧全; 黄忠荣; 胡建华; 李健

2014-01-01

本研究对猫传染性腹膜炎病毒（FIPV）N蛋白的编码基因进行克隆和原核表达，并在纯化重组N蛋白的基础上，建立了FIPV抗体间接ELISA检测方法。研究结果显示，该重组纯化的N蛋白具有良好的抗原反应性，可用于FIPV阳性血清的筛查，为我国出入境检疫部门监控FIPV疫情提供技术支撑。%In this study,the gene of nucleocapsid protein(N protein)of feline infectious peritonitis virus(FIPV) was cloned and expressed in Escherichia coli. And an indirect ELISA method was established with the purified recombi-nant N protein. The results showed this recombinant N protein had perfect antigen reactivity of FIPV and could be used to screen FIPV positive serum,and the study was helpful to monitor the FIPV infection status.

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables.

Science.gov (United States)

Moreno, María J; D'Arienzo, Pasquale; Manclús, Juan J; Montoya, Angel

2011-01-01

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I₅₀ value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

Utility of loop-mediated isothermal amplification assay, polymerase chain reaction, and elisa for diagnosis of leptospirosis in South Indian patients

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Mallika Sengupta

2017-01-01

Full Text Available Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines' criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis. Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05. Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings.

Calculation and decomposition of indirect carbon emissions from residential consumption in China based on the input–output model

International Nuclear Information System (INIS)

Zhu Qin; Peng Xizhe; Wu Kaiya

2012-01-01

Based on the input–output model and the comparable price input–output tables, the current paper investigates the indirect carbon emissions from residential consumption in China in 1992–2005, and examines the impacts on the emissions using the structural decomposition method. The results demonstrate that the rise of the residential consumption level played a dominant role in the growth of residential indirect emissions. The persistent decline of the carbon emission intensity of industrial sectors presented a significant negative effect on the emissions. The change in the intermediate demand of industrial sectors resulted in an overall positive effect, except in the initial years. The increase in population prompted the indirect emissions to a certain extent; however, population size is no longer the main reason for the growth of the emissions. The change in the consumption structure showed a weak positive effect, demonstrating the importance for China to control and slow down the increase in the emissions while in the process of optimizing the residential consumption structure. The results imply that the means for restructuring the economy and improving efficiency, rather than for lowering the consumption scale, should be adopted by China to achieve the targets of energy conservation and emission reduction. - Highlights: ► We build the input–output model of indirect carbon emissions from residential consumption. ► We calculate the indirect emissions using the comparable price input–output tables. ► We examine the impacts on the indirect emissions using the structural decomposition method. ► The change in the consumption structure showed a weak positive effect on the emissions. ► China's population size is no longer the main reason for the growth of the emissions.

Investigation of enrofloxacin residues in broiler tissues using ELISA and LC-MS/MS.

Science.gov (United States)

Panzenhagen, Pedro Henrique N; Aguiar, Waldemir S; Gouvêa, Raquel; de Oliveira, Andréa M G; Barreto, Fabiano; Pereira, Virgínia L A; Aquino, Maria Helena C

2016-01-01

This study investigated the efficiency of an enrofloxacin ELISA test kit to detect the presence of enrofloxacin residues in broiler tissues compared with LC-MS/MS. Broiler tissues from 72 samples consisting of 60 breast muscle, six pools of livers (500 g each) and six pools of kidneys (500 g each) were obtained from six different slaughterhouses. Breast muscle from 10 carcasses and pools of livers and kidneys from approximately 200 carcasses of the same flock were collected from each slaughterhouse. ELISA and HPLC were used to identify and quantify the contamination of the samples with enrofloxacin. A total of 72% of the analysed samples contained enrofloxacin residues detected by the ELISA and 22.2% were detected by LC-MS/MS. The mean values of enrofloxacin contamination found in chicken breast by ELISA and HPLC were 8.63 and 12.25 μg kg(-1), respectively. None of the samples exceeded the maximum limit of 100 μg kg(-1) by both methods set by the European Union as well as the Brazilian Agriculture Ministry. All positive samples for enrofloxacin residues detected by LC-MS/MS were also positive by ELISA. These data confirm the efficiency of the ELISA test, and suggest its use as a screening method for enrofloxacin residues in poultry tissues due to its quick results, low price and ease of applicability.

SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

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Ricardo Luiz Dantas MACHADO

1998-09-01

Full Text Available We report an adaptation of a technique for the blood sample collection (GFM as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.Relatamos a adaptação de uma técnica para coleta de amostras (MFV e outra para extração, amplificação de DNA de parasitas da malária para diagnóstico por PCR/ELISA. O método de coleta de amostras requer menos habilidade e economisa tempo e dinheiro, assim reduzindo a mais da metade o custo. O material é também adequado para análise genética em especimens frescos ou estocados, preparados por este método.

Prevalence of entamoeba histolytica and entamoeba dispar by means of microscopy and ELISA in a suburb of Lima

International Nuclear Information System (INIS)

Cornejo, W.; Alva, P.; Suarez, R.; Espinoza, Y.; Huiza, A.; Sevilla, C.; Naquira, C.

1999-01-01

To assess the prevalence of E. histolytica and E. dispar in a marginal population of Callao, Lima, using ELISAs. Two ELISAs were used, one of them for both Entamoeba species (Entamoeba-ELISA), and an E. histolytica-specific ELISA. 128 stool samples from randomized Callao inhabitants were microscopically examined. Thirteen (10%) were microscopically diagnosed as having E. histolytica or E. dispar infection; and 7 (6%) were positive to Entamoeba-ELISA (sensitivity= 54%). Five of the 115 samples without cysts of E. histolytica or E. dispar were Entamoeba ELISA positive (specificity= 96%). All samples were negative to the E. histolytica-specific ELISA. The correlation between the microscopical identification and Entamoeba-ELISA was not good, it may be due to an overvaluation of the of the morphological diagnosis. (authors)

Comparison of microscopy, ELISA, and real-time PCR for detection of Giardia intestinalis in human stool specimens

Science.gov (United States)

Beyhan, Yunus Emre; Taş Cengiz, Zeynep

2017-08-23

Background/aim: This study included patients who had digestive system complaints between August 2015 and October 2015. The research was designed to compare conventional microscopy with an antigen detection ELISA kit and the TaqMan-based real-time PCR (RT-PCR) technique for detection of Giardia intestinalis in human stool specimens. Materials and methods: Samples were concentrated by formalin-ether sedimentation technique and microscopic examinations were carried out on wet mount slides. A commercially available ELISA kit (Giardia CELISA, Cellabs, Brookvale, Australia) was used for immunoassay. DNA was extracted from fecal samples of about 200 mg using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and the LightCycler Nano system (Roche Diagnostics, Mannheim, Germany) was used for the TaqMan-based RT-PCR assay. Results: A total of 94 stool samples, 38 of them diagnosed positive (40.4%) and 56 of them diagnosed negative by microscopy, were selected for evaluation by antigen detection and molecular assays. The prevalence of G. intestinalis infection was found as 46.8% (n: 44) and 79.8% (n: 75) by ELISA and RT-PCR, respectively. RT-PCR revealed by far the highest positivity rate compared to the other two methods. The difference between these methods was found to be statistically significant (P PCR, the sensitivity and specificity of microscopy and ELISA were 50.7% and 100% and 53.3% and 79%, respectively. Conclusion: RT-PCR seems to be much more sensitive and beneficial for rapid and accurate diagnosis of G. intestinalis in human stools.

Frequency of antiphospholipid antibodies in patients with infectious diseases using three different ELISA methods Freqüência de anticorpos antifosfolípides em pacientes com doenças infecciosas usando três diferentes testes de ELISA

Directory of Open Access Journals (Sweden)

Mittermayer Barreto Santiago

2006-02-01

Full Text Available OBJECTIVE: The standard enzyme-linked immunosorbent assay (ELISA for anticardiolipin (aCL antibodies is the most important test for the diagnosis of antiphospholipid syndrome (APS. However, the test is also positive in some infectious diseases and other non-related syndromes. It has been suggested that the detection of antibodies to a mixture of phospholipids or to beta2-glycoprotein I (beta2-GP I has higher specificity for APS than the standard aCL ELISA. The aim of the present work is to compare the diagnostic specificity of three different antiphospholipid (aPL assays in patients with infectious diseases. METHODS: Antiphospholipid antibodies were searched by three ELISA techniques, namely standard aCL, APhL® ELISA kit and anti-beta2-GP I, in sera of patients with infectious diseases, including syphilis (69, leptospirosis (33 and visceral leishmaniasis (30. RESULTS: The frequency of positivity of IgG aPL in patients with syphilis, leptospirosis and Kala-azar was 13/69 (19%, 9/33 (27% and 2/30 (6%, respectively, using standard ELISA, versus only 1/69 (1.4%, 0/33 (0% and 0/30 (0% positivity by the APhL® ELISA kit. The positivity of the isotype IgM aPL was 10/69 (14%, 4/33 (12% and 1/30 (3%, respectively, by the standard ELISA, and 1/69 (1.4%, 0/33 (0% and 0/30 (0% by the APhL® ELISA kit. The presence of significant levels of IgG anti-beta2GPI was observed in 14/69 cases of syphilis (20%, 6/33 cases of leptospirosis (18% and 16/30 cases of Kala-azar (53%. The APhL® ELISA kit had superior performance showing the highest specificity: 97% (95% CI: 92%-99% for IgG compared to 81% (95% CI: 74%-87% for standard ELISA and 72% (95% CI: 64%-79% for anti-beta2 GPI assay. CONCLUSIONS: The APhL® ELISA kit proved to be significantly more specific than the aCL standard ELISA and the anti-beta2GPI ELISA, and it should be used to help in the diagnosis and confirmation of APS.OBJETIVO: O ensaio de enzyme-linked immunosorbent assay (ELISA para a pesquisa de

Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

Science.gov (United States)

Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

2018-06-26

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

Développement et exploitation d'un simulateur électro-optique pour le futur détecteur spatial à ondes gravitationnelles eLISA

OpenAIRE

Gruning , Pierre

2015-01-01

Gravitational waves will offer a new window to the universe. Their detection by ground based detectors should arrive soon and the future space based interferometer eLISA will allow to do gravitational wave astronomy in order to get unique informations on their sources wether black holes, neutron stars, the Big Bang etc. The effect of a gravitational wave is an extremely weak variation of distance, it will require a very performant detector. That’s why eLISA should reach picometer precision wi...

Indirect Inference for Stochastic Differential Equations Based on Moment Expansions

KAUST Repository

Ballesio, Marco; Tempone, Raul; Vilanova, Pedro

2016-01-01

We provide an indirect inference method to estimate the parameters of timehomogeneous scalar diffusion and jump diffusion processes. We obtain a system of ODEs that approximate the time evolution of the first two moments of the process

RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

DEFF Research Database (Denmark)

Dybkaer, Karen; Munch, Mette; Handberg, Kurt Jensen

2003-01-01

A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected wit...... of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard....

Performance of the ELISA test for the diagnosis of cysticercosis using experimentally and naturally cattle infected with metacestode of Taenia saginata

Directory of Open Access Journals (Sweden)

Rafaella Paola Meneguete dos Guimarães-Peixoto

2015-04-01

Full Text Available The bovine taeniasis-cysticercosis complex can be defined as a set of pathological changes caused by the adult form of Taenia saginata in humans and their larval form in animals (Cysticercus bovis. Data on the occurrence of bovine cysticercosis comes from the records of veterinary inspection of meat in slaughterhouses under fiscalization, where some positive cases may go unnoticed, especially in moderate infections. So, it is relevant the use of serological tests that have greater sensitivity than the post-mortem routine exams. Studies have shown the possible application of the ELISA test as a tool for epidemiological studies of the parasitosis and in the identification of animals with cysts. The aim of this study is to determine the detection threshold of the indirect ELISA test, using experimentally and naturally infected animals in detecting cases of cysticercosis. The sensitivity of the test to naturally infected animals using a cut-off 1 and 2 added with 2 standart-deviation (SD was of 12% and 24.4%, respectively. However, when using the cut-off of 1 and 2 added with 3 SD, the test sensitivity dropped, represented by 14.4% and 1.99% of the test sensitivity. As for the samples from experimentally infected animals, using the cut-off 1 and 2 added with 2 SD the sensitivity was 55.9% and 92.5%; and adding 3 SD the values found were 31.2% and 86%, respectively. The specificity of the test in all situations tested was 100%. It is important to take into account the right choice of control sera used in the ELISA test, whereas, according to its application, it is necessary to increase the sensitivity or specificity.

Microchip ELISA coupled with cell phone to detect ovarian cancer HE4 biomarker in urine.

Science.gov (United States)

Wang, ShuQi; Akbas, Ragip; Demirci, Utkan

2015-01-01

Ovarian cancer is a leading cause of death from gynecologic cancers in the USA, and early diagnosis can potentially increase 5-year survival rate. Detection of biomarkers derived from hyperplasia of epithelial tissue by enzyme-linked immunosorbent assay (ELISA) proves to be a practical way of early diagnosis of ovarian cancer. However, ELISA is commonly performed in a laboratory setting, and it cannot be used in a clinical setting for on-site consultation. We have shown a microchip ELISA that detects HE4, an ovarian cancer biomarker, from urine using a cell phone integrated with a mobile application for imaging and data analysis. In microchip ELISA, HE4 from urine was first absorbed on the surface; the primary and secondary antibodies were subsequently anchored on the surface via immuno-reaction; and addition of substrate led to color development because of enzymatic labeling. The microchip after color development was imaged using a cell phone, and the color intensity was analyzed by an integrated mobile application. By comparing with an ELISA standard curve, the concentration of HE4 was reported on the cell phone screen. The presented microchip ELISA coupled with a cell phone is portable as opposed to traditional ELISA, and this method can facilitate the detection of ovarian cancer at the point-of-care (POC).

Ontology-based indirect interaction of mobile robots for joint task solving: a scenario for obstacle overcoming

Directory of Open Access Journals (Sweden)

Petrov Mikhail

2017-01-01

Full Text Available This paper describes an ontology-based approach to interaction of users and mobile robots for joint task solving. The use of ontologies allows supporting semantic interoperability between robots. The ontologies store knowledge about the tasks to be performed, knowledge about the functionality of robots and the current situation factors like a robot location or busyness. Ontologies are published in a smart space which allows indirect interaction between participants. On the basis of the knowledge, a robot can define a task that is to be performed and get the current status of other robots. The paper presents a reference model of the approach to indirect interaction between mobile robots for joint task solving, an ontology model for the knowledge organization, and application of the presented approach for the scenario for obstacle overcoming.

A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

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Larry H. Stanker

2013-11-01

Full Text Available Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT, produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D., ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule and readily detects toxin in those food samples tested.

A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in food.

Science.gov (United States)

Stanker, Larry H; Scotcher, Miles C; Cheng, Luisa; Ching, Kathryn; McGarvey, Jeffery; Hodge, David; Hnasko, Robert

2013-11-18

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A-H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD's) for individual antibodies ranging from 10 to 48 × 10-11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested.

Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

Science.gov (United States)

Xie, Zhen-ming; Yu, Lin; Fang, Li-sha

2015-01-01

The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 μg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

Detection of ciguatoxin in fish tissue using sandwich ELISA and neuroblastoma cell bioassay.

Science.gov (United States)

Empey Campora, Cara; Dierking, Jan; Tamaru, Clyde S; Hokama, Yoshitsugi; Vincent, Douglas

2008-01-01

The applicability of a new enzyme-linked immunoassay (ELISA) for detecting ciguatoxin (CTX) in fish tissue was evaluated by testing three fish species commonly implicated in ciguatera fish poisoning in Hawaii. A total of 164 individual almaco jack (Seriola rivoliana) and greater amberjack (S. dumerili) and a total of 175 individuals of the blue-spotted grouper (Cephalopholis argus) were caught at various locations in the Hawaiian Islands. Muscle tissue from each individual was assessed for the presence of CTX using two methods: a semi-quantitative ELISA that was recently developed for detecting picogram levels of CTX in fish extract and a neuroblastoma (NB) cell assay commonly used to screen for marine toxins in fish. Results of the tests were highly correlated, with the ELISA indicating the presence of CTX in 9.4% of all fish samples, and the NB assay indicating toxicity in 6.8% of the fish samples. We conclude that the ELISA produces reliable and accurate results that are consistent with those provided by the accepted NB assay and that the ELISA has potential for future applications in screening fish populations for CTX.

Seroprevalence of Mycoplasma gallisepticum antibody by ELISA and serum plate agglutination test of laying chicken

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Md. Zulfekar Ali

2015-01-01

Full Text Available Aim: Mycoplasma gallisepticum (MG is important avian pathogen responsible for chronic respiratory disease of chicken and turkeys, which result in large economic loss for the poultry industry. The objectives of this study were determination of seroprevalence of MG antibody of commercial layer chicken at laying period in selected areas of Bangladesh. Materials and Methods: A total of 563 blood samples were collected randomly from selected commercial layer chickens at laying period during the period from July to December, 2013. Indirect enzyme linked immunosorbent assay (iELISA and serum plate agglutination (SPA test were performed to detect the presence of antibodies against MG. Results: Of 563 samples, 64.47% and 56.13% showed an overall prevalence of MG antibodies in iELISA and SPA test respectively. Prevalence of MG was recorded the highest (69.63% at 50-55 weeks of age compared with lowest (53.26% at 56-61 weeks of age (p<0.05. Significant (p<0.05 effect of breed were observed in the seroprevalence of MG infection in layer birds in the present study. The overall, 68.77%, 63.74% and 59.37% prevalence were found respectively in sonali, ISA Brown and White leg horn. The prevalence of MG antibodies was the highest (70.13% in December followed by November (68%, October (65.67%, August (63.46%, September (58.54% and July (51.78% month. The seroprevalence of MG antibodies was higher (69.63% in most of the large flocks and lower (56.82% in small flocks. Conclusion: Therefore, might be suggested that the commercial layer farms should be routinely checked to monitor MG infection and the reactor birds should be culled since MG organism has the potential to transmit vertically. The correlation between MG antibody in month and flock size was not significant (p=0.359 and p=0.868, respectively.

Evaluation of a Salmonella Enteritidis vaccine and related ELISA for respective induction and assessment of acquired immunity to the vaccine and/or Echinacea purpurea in Awassi Ewes.

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Barbour, Elie K; Assi, Chibli A Abou; Shaib, Houssam; Hamadeh, Shadi; Murtada, Muhammad; Mahmoud, Ghassan; Yaghmoor, Soonham; Iyer, Archana; Harakeh, Steve; Kumosani, Taha

2015-05-05

The aim of this study was to evaluate an experimental Salmonella Enteritidis (SE) bacterin and an indirect ELISA system to assess quantitatively the acquired immunity in Awassi ewes to the vaccine and/or Echinacea purpurea (EP) dried roots. Four treatments of the ewes were included in the experimental design, with 6 ewes/treatment. The first treatment (T1) had the controls that were non-vaccinated and non-treated with EP. The T2 ewes were only treated with EP. The T3 and T4 ewes were vaccinated at D1 (initiation of trial) and D10, while the T4 ewes were additionally administered the EP dried roots. Blood was collected from the jugular vein of all ewes at D1, D10, D21 and D45. The construction of the vaccine and the ELISA are detailed within the manuscript. The ELISA was able to detect quantitatively the significant acquired primary and secondary immunity to the vaccine in T3 and T4 ewes, compared to their low level of background immunities at initiation of the experiment (p0.05) in T1 control ewes, and in the T2 ewes that were given only the (EP) (p>0.05). Moreover, the ELISA was able to uncover the significant seroconversion of secondary immune response in T4 ewes at D21 compared to that at D10 (pewes. This is the first work in literature that reports the need to supplement the vaccination by the experimental SE bacterin with daily oral intake of 250mg of EP-dried roots, effective the first vaccination day and up to 21 days, for obtaining a statistically significant seroconversion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison between mixed lysate antigen and α-actinin antigen in ELISA for serodiagnosis of trichomoniasis.

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Kim, Seung-Ryong; Kim, Jung-Hyun; Park, Soon-Jung; Lee, Hye-Yeon; Kim, Yong-Suk; Kim, Yu-Mi; Hong, Yeon-Chul; Ryu, Jae-Sook

2015-10-01

The aim of this study was to identify an antigen suitable for ELISA for serodiagnosis of Trichomonas vaginalis (T. vaginalis) infection. Mixed lysate antigen (Ag) from eight strains of T. vaginalis and recombinant α-actinin protein was compared. The sera of three groups were examined by ELISA: 73 women infected with trichomoniasis served as a positive control, 31 male volunteers as a negative control, and 424 women attending an outpatient health screening at Hanyang University Guri Hospital. Based on the cutoff optical density for each Ag obtained with a negative control, the serosensitivity of the mixed lysate Ag (79.5%) was significantly higher than that of the α-actinin (52.1%) in the 73 patients with trichomoniasis. The specificity using lysate Ag and α-actinin was 100% and 96.8%, respectively. On the other hand, the positivity rate in 424 outpatients was 39.2% and 11.8% with mixed lysate and α-actinin Ag, respectively. Taken together, mixed lysate Ag showed higher sensitivity and specificity than α-actinin. Therefore, mixed lysate may be a better Ag than α-actinin for ELISA for the diagnosis of trichomoniasis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

ELISA technique standardization for strongyloidiasis diagnosis

International Nuclear Information System (INIS)

Huapaya, P.; Espinoza, I.; Huiza, A.; Universidad Nacional Mayor de San Marcos, Lima; Sevilla, C.

2002-01-01

To standardize ELISA technique for human Strongyloides stercoralis infection diagnosis a crude antigen was prepared using filariform larvae obtained from positive stool samples cultured with charcoal. Harvested larvae were crushed by sonication and washed by centrifugation in order to obtain protein extracts to be used as antigen. Final protein concentration was 600 μg/mL. Several kinds of ELISA plates were tested and antigen concentration, sera dilution, conjugate dilution and cut off were determined to identify infection. Sera from patients with both hyper-infection syndrome and intestinal infection demonstrated by parasitological examination were positive controls and sera from people living in non-endemic areas with no infection demonstrated by parasitological examination were negative controls. Best values were 5 μg/mL for antigen, 1/64 for sera, 1/1000 for conjugate; optical density values for positive samples were 1,2746 (1,1065 - 1,4206, DS = 0,3284) and for negative samples 0,4457 (0,3324 - 0,5538, DS = 0,2230). Twenty sera samples from positive subjects and one hundred from negative subjects were examined, obtaining 90% sensitivity and 88% specificity. The results show this technique could be useful as strongyloidiasis screening test in population studies

Food allergen analysis for processed food using a novel extraction method to eliminate harmful reagents for both ELISA and lateral-flow tests.

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Ito, Kaori; Yamamoto, Takayuki; Oyama, Yuriko; Tsuruma, Rieko; Saito, Eriko; Saito, Yoshikazu; Ozu, Takeshi; Honjoh, Tsutomu; Adachi, Reiko; Sakai, Shinobu; Akiyama, Hiroshi; Shoji, Masahiro

2016-09-01

Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine food allergens in food products. However, a significant number of ELISAs give an erroneous result, especially when applied to highly processed food. Accordingly, an improved ELISA, which utilizes an extraction solution comprising the surfactant sodium lauryl sulfate (SDS) and reductant 2-mercaptoethanol (2-ME), has been specially developed to analyze food allergens in highly processed food by enhancing analyte protein extraction. Recently, however, the use of 2-ME has become undesirable. In the present study, a new extraction solution containing a human- and eco-friendly reductant, which is convenient to use at the food manufacturing site, has been established. Among three chemicals with different reducing properties, sodium sulfite, tris(3-hydroxypropyl)phosphine, and mercaptoethylamine sodium sulfite was selected as a 2-ME substitute. The protein extraction ability of SDS/0.1 M sodium sulfite solution was comparable to that of SDS/2-ME solution. Next, the ELISA performance for egg, milk, wheat, peanut, and buckwheat was evaluated by using model-processed foods and commercially available food products. The data showed that the SDS/0.1 M sulfite ELISA significantly correlated with the SDS/2-ME ELISA for all food allergens examined (p analysis of food allergens in processed food, showing consistency with the SDS/0.1 M sulfite ELISA results. Accordingly, a harmonized analysis system for processed food comprising a screening LF test and a quantitative ELISA with identical extraction solution has been established. The ELISA based on the SDS/0.1 M sulfite extraction solution has now been authorized as the revised official method for food allergen analysis in Japan.

A 2.9 kDa Fasciola hepatica-recombinant protein based ELISA test for the detection of current-ovine fasciolosis trickle infected.

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Arias, M; Hillyer, G V; Sánchez-Andrade, R; Suárez, J L; Pedreira, J; Lomba, C; Díaz, P; Morrondo, P; Díez-Baños, P; Paz-Silva, A

2006-04-15

The suitability of an enzyme linked immunosorbent assay (ELISA) test with a 2.9 kDa Fasciola hepatica-recombinant protein (FhrAPS) for diagnosing early and current-ovine fasciolosis was analyzed, and compared to that obtained by using a direct ELISA for detecting F. hepatica-circulating FhES antigens and to the coprological sedimentation for fluke egg quantitation. Fourteen Gallega autochthonous breed sheep were experimentally infected with metacercariae by a trickle system (small repetitive infections) and divided into two groups: G-I represented a primary infection for 34 weeks; G-R, animals with primary infection and reinfected 18 w.a.p.i. Seven sheep were left uninfected as the control group (G-C). Serum IgG antibody values against the FhrAPS rose rapidly by 1st w.a.p.i. in all infected sheep. Antibody levels in those with primary infection (G-I, G-C) peaked at 10 weeks, diminishing slightly and levelling from 16 to 34 weeks. Those with primary infection reinfected at 18 weeks had a rebound effect with the highest values observed. Circulating F. hepatica-ES antigens were detected by the 1st w.a.p.i. in all infected groups peaking at 6 weeks, decreasing rapidly to uninfected control values by 10 weeks of infection. Faecal egg-output started 11 weeks after primary infection. An increase in the IgG antibody as well as antigen responses to the FhrAPS and to anti-FhES from the 18 w.a.p.i. was recorded in G-T and G-R after the challenge infection. Antibody levels remained high whereas antigenemia values diminished after 6 weeks. A positive significant correlation between the IgG response against the FhrAPS and the F. hepatica circulating antigens (r2 = 0.428, p = 0.001) was obtained. In conclusion, our standardized diagnostic ELISA for fasciolosis based on the detection of IgG responses to the FhrAPS would be a valuable tool to diagnosis early and current F. hepatica-infections in sheep.

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.

Validation of the ELISA technique for diagnosis of trypanosomiasis in cattle in Uganda

International Nuclear Information System (INIS)

Okuna, N.M.

1992-01-01

ELISA, developed in ILRAD for diagnosis of T. congolense, T. brucei and T. vivax in cattle, has not been validated in Uganda. This study was undertaken to validate the technique. Negative reference sera were collected from 44 cattle in Kapchorwa, a tsetse-free area. The cattle were free of the three trypanosome species T. congolense, T. brucei and T. vivax by the haematocrit buffy coat technique (BCT). But by ELISA, three were positive for T. vivax, one for both T. congolense and T. vivax and one for T. congolense. Sera were collected from the same 44 cattle 10 weeks later. The cattle were again free of T. congolense, T. brucei and T. vivax, both by BCT and by mouse inoculations. Two cattle out of 450 screened at a centre 5 km away had T. vivax by BCT. The ELISA results for the second set of sera were quite similar to the results obtained from the first set of sera. The calculated optical density (D) cut off point was 50 for both T. brucei and T. vivax, but it was 60 for T. congolense. Sera from 5 cattle which had T. theileri and two which had microfilaria were all negative for antigenaemia by ELISA. Positive reference sera were collected form 40 cattle in a high tsetse challenge area. Using the haematocrit buffy coat technique, 5 had T. vivax, two had T. brucei and one had T. congolense. Checked by ELISA for antigenaemia, only 4 cattle were free of all the three trypanosome species, T. congolense, T. vivax and T. brucei. All the 40 cattle were treated with Diminazene aceturate at the rate of 7 mg/kg body weight. Two weeks later, the ELISA test showed that 10 cattle were free of any antigenaemia. Those still positive for antigenaemia had lower OD readings. The ELISA technique is valid. It is much more sensitive compared to parasitological tests. It is specific since none of the 7 cattle with either T. theileri or microfilaria gave positive results by ELISA. The technique would be very useful for epizootiological studies. (author)

On the comprehensibility and perceived privacy protection of indirect questioning techniques.

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Hoffmann, Adrian; Waubert de Puiseau, Berenike; Schmidt, Alexander F; Musch, Jochen

2017-08-01

On surveys that assess sensitive personal attributes, indirect questioning aims at increasing respondents' willingness to answer truthfully by protecting confidentiality. However, the assumption that subjects understand questioning procedures fully and trust them to protect their privacy is rarely tested. In a scenario-based design, we compared four indirect questioning procedures in terms of their comprehensibility and perceived privacy protection. All indirect questioning techniques were found to be less comprehensible by respondents than a conventional direct question used for comparison. Less-educated respondents experienced more difficulties when confronted with any indirect questioning technique. Regardless of education, the crosswise model was found to be the most comprehensible among the four indirect methods. Indirect questioning in general was perceived to increase privacy protection in comparison to a direct question. Unexpectedly, comprehension and perceived privacy protection did not correlate. We recommend assessing these factors separately in future evaluations of indirect questioning.

Cruel intentions on television and in real life: can viewing indirect aggression increase viewers' subsequent indirect aggression?

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Coyne, Sarah M; Archer, John; Eslea, Mike

2004-07-01

Numerous studies have shown that viewing violence in the media can influence an individual's subsequent aggression, but none have examined the effect of viewing indirect aggression. This study examines the immediate effect of viewing indirect and direct aggression on subsequent indirect aggression among 199 children ages 11 to 14 years. They were shown an indirect, direct, or no-aggression video and their subsequent indirect aggression was measured by negative evaluation of a confederate and responses to a vignette. Participants viewing indirect or direct aggression gave a more negative evaluation of and less money to a confederate than participants viewing no-aggression. Participants viewing indirect aggression gave less money to the confederate than those viewing direct aggression. Participants viewing indirect aggression gave more indirectly aggressive responses to an ambiguous situation and participants viewing direct aggression gave more directly aggressive responses. This study provides the first evidence that viewing indirect aggression in the media can have an immediate impact on subsequent aggression.

Analytical validation of a reference laboratory ELISA for the detection of feline leukemia virus p27 antigen.

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Buch, Jesse S; Clark, Genevieve H; Cahill, Roberta; Thatcher, Brendon; Smith, Peter; Chandrashekar, Ramaswamy; Leutenegger, Christian M; O'Connor, Thomas P; Beall, Melissa J

2017-09-01

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. Immunoassays for the p27 core protein of FeLV aid in the detection of FeLV infections. Commercial microtiter-plate ELISAs have rapid protocols and visual result interpretation, limiting their usefulness in high-throughput situations. The purpose of our study was to validate the PetChek FeLV 15 ELISA, which is designed for the reference laboratory, and incorporates sequential, orthogonal screening and confirmatory protocols. A cutoff for the screening assay was established with 100% accuracy using 309 feline samples (244 negative, 65 positive) defined by the combined results of FeLV PCR and an independent reference p27 antigen ELISA. Precision of the screening assay was measured using a panel of 3 samples (negative, low-positive, and high-positive). The intra-assay coefficient of variation (CV) was 3.9-7.9%; the inter-assay CV was 6.0-8.6%. For the confirmatory assay, the intra-assay CV was 3.0-4.7%, and the inter-assay CV was 7.4-9.7%. The analytical sensitivity for p27 antigen was 3.7 ng/mL for inactivated whole FeLV and 1.2 ng/mL for purified recombinant FeLV p27. Analytical specificity was demonstrated based on the absence of cross-reactivity to related retroviruses. No interference was observed for samples containing added bilirubin, hemoglobin, or lipids. Based on these results, the new high-throughput design of the PetChek FeLV 15 ELISA makes it suitable for use in reference laboratory settings and maintains overall analytical performance.

Confidence Limits for the Indirect Effect: Distribution of the Product and Resampling Methods

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MacKinnon, David P.; Lockwood, Chondra M.; Williams, Jason

2010-01-01

The most commonly used method to test an indirect effect is to divide the estimate of the indirect effect by its standard error and compare the resulting z statistic with a critical value from the standard normal distribution. Confidence limits for the indirect effect are also typically based on critical values from the standard normal distribution. This article uses a simulation study to demonstrate that confidence limits are imbalanced because the distribution of the indirect effect is normal only in special cases. Two alternatives for improving the performance of confidence limits for the indirect effect are evaluated: (a) a method based on the distribution of the product of two normal random variables, and (b) resampling methods. In Study 1, confidence limits based on the distribution of the product are more accurate than methods based on an assumed normal distribution but confidence limits are still imbalanced. Study 2 demonstrates that more accurate confidence limits are obtained using resampling methods, with the bias-corrected bootstrap the best method overall. PMID:20157642

Comparison between microscopic examination, ELISA and quantitative buffy coat analysis in the diagnosis of falciparum malaria in an endemic population.

Science.gov (United States)

Tanpradist, S; Tharavanij, S; Yamokgul, P; Bualombai, P; Wongchotigul, V; Singhasivanon, P; Patarapotikul, J; Thammapalerd, N; Prasittisuk, C; Tantanasrikul, S

1995-03-01

Monoclonal antibody-based ELISA and QBC (quantitative buffy coat analysis) were tested in two endemic areas with low and high incidence of malaria in Kanchanaburi Province, West Thailand with annual parasite incidence in 1992 of 119 and 5 per 1,000 population, respectively. The numbers of individuals positive by thick blood film examination (TBF) for P. falciparum with or without P. vivax, and P. vivax only were 82 and 69, respectively. The detection limit of ELISA was 10 parasites/10(6) red blood cells (RBC) (0.001% parasitemia). Of 1,095 individuals involved in the study at the beginning of the study, ELISA showed sensitivity, specificity, positive predictive value and negative predictive value of 78.1%, 94.9%, 72% and 98.1%, respectively. Nine of 18 (50%) TBF-positive but ELISA-positive individuals had parasitemia of less than 10 parasites/10(6) RBC. High and low incidence areas did not affect the validity of our result. Regression analysis showed good correlation between log parasitemia and ELISA percent OD increase (Y = 0 + 64.9*logX, r = 0.65), and agreement between TBF and ELISA results was 95.9%. In a fortnightly follow-up, in 82 TBF-positive individuals, both ELISA and TBF positive rates correlatively declined with agreement of 96.3%. With samples taken on the first day of the study, the TBF and QBC results were also correlated with agreement of 95.8% for P. falciparum, 95.6% for P. vivax. During 8 week follow-up involving altogether 191 samples, agreement between TBF and QBC results were 87.4% for P. falciparum. QBC detected more cases with P. falciparum infections but detected smaller number of cases with P. vivax infections.

Data-Foraging-Oriented Reconnaissance Based on Bio-Inspired Indirect Communication for Aerial Vehicles

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Josué Castañeda Cisneros

2017-07-01

Full Text Available In recent years, aerial vehicles have allowed exploring scenarios with harsh conditions. These can conduct reconnaissance tasks in areas that change periodically and have a high spatial and temporal resolution. The objective of a reconnaissance task is to survey an area and retrieve strategic information. The aerial vehicles, however, have inherent constraints in terms of energy and transmission range due to their mobility. Despite these constraints, the Data Foraging problem requires the aerial vehicles to exchange information about profitable data sources. In Data Foraging, establishing a single path is not viable because of dynamic conditions of the environment. Thus, reconnaissance must be focused on periodically searching profitable environmental data sources, as some animals perform foraging. In this work, a data-foraging-oriented reconnaissance algorithm based on bio-inspired indirect communication for aerial vehicles is presented. The approach establishes several paths that overlap to identify valuable data sources. Inspired by the stigmergy principle, the aerial vehicles indirectly communicate through artificial pheromones. The aerial vehicles traverse the environment using a heuristic algorithm that uses the artificial pheromones as feedback. The solution is formally defined and mathematically evaluated. In addition, we show the viability of the algorithm by simulations which have been tested through various statistical hypothesis.

Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA

Science.gov (United States)

Zhang, Hao; Zhou, Hui

2017-01-01

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals. PMID:28350826

DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro

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Chaban Christina

2010-11-01

Full Text Available Abstract Background About 10% of all genes in eukaryote genomes are predicted to encode transcription factors. The specific binding of transcription factors to short DNA-motifs influences the expression of neighbouring genes. However, little is known about the DNA-protein interaction itself. To date there are only a few suitable methods to characterise DNA-protein-interactions, among which the EMSA is the method most frequently used in laboratories. Besides EMSA, several protocols describe the effective use of an ELISA-based transcription factor binding assay e.g. for the analysis of human NFκB binding to specific DNA sequences. Results We provide a unified protocol for this type of ELISA analysis, termed DNA-Protein-Interaction (DPI-ELISA. Qualitative analyses with His-epitope tagged plant transcription factors expressed in E. coli revealed that EMSA and DPI-ELISA result in comparable and reproducible data. The binding of AtbZIP63 to the C-box and AtWRKY11 to the W2-box could be reproduced and validated by both methods. We next examined the physical binding of the C-terminal DNA-binding domains of AtWRKY33, AtWRKY50 and AtWRKY75 to the W2-box. Although the DNA-binding domain is highly conserved among the WRKY proteins tested, the use of the DPI-ELISA discloses differences in W2-box binding properties between these proteins. In addition to these well-studied transcription factor families, we applied our protocol to AtBPC2, a member of the so far uncharacterised plant specific Basic Pentacysteine transcription factor family. We could demonstrate binding to GA/TC-dinucleotide repeat motifs by our DPI-ELISA protocol. Different buffers and reaction conditions were examined. Conclusions We successfully applied our DPI-ELISA protocol to investigate the DNA-binding specificities of three different classes of transcription factors from Arabidopsis thaliana. However, the analysis of the binding affinity of any DNA-binding protein to any given DNA

Interpolation of the magnetic field at the test masses in eLISA

International Nuclear Information System (INIS)

Mateos, I; Díaz-Aguiló, M; Ramos-Castro, J; García-Berro, E; Lobo, A

2015-01-01

A feasible design for a magnetic diagnostics subsystem for eLISA will be based on that of its precursor mission, LISA Pathfinder. Previous experience indicates that magnetic field estimation at the positions of the test masses has certain complications. This is due to two reasons. The first is that magnetometers usually back-act due to their measurement principles (i.e., they also create their own magnetic fields), while the second is that the sensors selected for LISA Pathfinder have a large size, which conflicts with space resolution and with the possibility of having a sufficient number of them to properly map the magnetic field around the test masses. However, high-sensitivity and small-sized sensors that significantly mitigate the two aforementioned limitations exist, and have been proposed to overcome these problems. Thus, these sensors will be likely selected for the magnetic diagnostics subsystem of eLISA. Here we perform a quantitative analysis of the new magnetic subsystem, as it is currently conceived, and assess the feasibility of selecting these sensors in the final configuration of the magnetic diagnostic subsystem. (paper)

A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

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Cardoso T.C.

1999-01-01

Full Text Available A liquid phase blocking ELISA (LPB-ELISA was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV. The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926 between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%, specificity (100% and agreement (95.31%.

Utilisation of the buffy coat technique and an antibody-detection ELISA as tools for assessing the impact of trypanosomosis on health and productivity of N'Dama cattle

International Nuclear Information System (INIS)

Faye, J.A.; Mattioli, R.C.

2000-01-01

The buffy coat technique (BCT), a parasitological test, and an indirect antibody ELISA (Ab-ELISA) were used to detect trypanosome infections in blood and serum samples, respectively, collected on N'Dama cattle exposed to natural high tsetse challenge. These two diagnostic tools were also utilized to assess trypanosomal status in sequentially collected blood and serum samples from two groups composed of 5 N'Dama cattle each experimentally challenged with Trypanosoma congolense and T. vivax, In both studies, packed red cell volume (PCV) and live weight were measured. The specificity of the Ab-ELISA was computed by testing approximately 70 serum samples obtained from a cattle population kept under zero tsetse challenge. The specificity was found to be 95.8% for T. vivax and 97. 1 % for T. congolense. In the field study, 3.9% (12/310) of blood samples was parasitologically positive. In corresponding serum samples the prevalence of positive trypanosome sero-reactors was 54.8% (170/310). However, antibodies against trypanosomes persisted in serum when blood samples were no longer parasitologically positive. In both blood and serum samples, T. vivax was found to be the main infecting species. The sensitivity of the Ab-ELISA for T. vivax was 81.8%. Due to the extremely low numbers of T. congolense infection (only one), as detected by BCT, the sensitivity for that trypanosome species was not computed. In the experimentally challenged cattle, 80% (24/30) and 33.3% (10/30) of blood samples were BCT positive for T. congolense and T. vivax, respectively. Antibodies in corresponding sera were present in 69% (20/29) and 96.3% (26/27) of animals challenged with T. congolense and T. vivax, respectively. The serological assay for T. congolense antibody detection exhibited high cross-reactivity with T. vivax antigens, as assessed in sera collected from T. vivax infected animals. In the field study, cattle showing the presence of antibodies against T. congolense and/or T. vivax had

Constraining early and interacting dark energy with gravitational wave standard sirens: the potential of the eLISA mission

International Nuclear Information System (INIS)

Caprini, Chiara; Tamanini, Nicola

2016-01-01

We perform a forecast analysis of the capability of the eLISA space-based interferometer to constrain models of early and interacting dark energy using gravitational wave standard sirens. We employ simulated catalogues of standard sirens given by merging massive black hole binaries visible by eLISA, with an electromagnetic counterpart detectable by future telescopes. We consider three-arms mission designs with arm length of 1, 2 and 5 million km, 5 years of mission duration and the best-level low frequency noise as recently tested by the LISA Pathfinder. Standard sirens with eLISA give access to an intermediate range of redshift 1 ∼< z ∼< 8, and can therefore provide competitive constraints on models where the onset of the deviation from ΛCDM (i.e. the epoch when early dark energy starts to be non-negligible, or when the interaction with dark matter begins) occurs relatively late, at z ∼< 6. If instead early or interacting dark energy is relevant already in the pre-recombination era, current cosmological probes (especially the cosmic microwave background) are more efficient than eLISA in constraining these models, except possibly in the interacting dark energy model if the energy exchange is proportional to the energy density of dark energy.

Multi-Layer Organic Squaraine-Based Photodiode for Indirect X-Ray Detection

Science.gov (United States)

Iacchetti, Antonio; Binda, Maddalena; Natali, Dario; Giussani, Mattia; Beverina, Luca; Fiorini, Carlo; Peloso, Roberta; Sampietro, Marco

2012-10-01

The paper presents an organic-based photodiode coupled to a CsI(Tl) scintillator to realize an X-ray detector. A suitable blend of an indolic squaraine derivative and of fullerene derivative has been used for the photodiode, thus allowing external quantum efficiency in excess of 10% at a wavelength of 570 nm, well matching the scintillator output spectrum. Thanks to the additional deposition of a 15 nm thin layer of a suitable low electron affinity polymer, carriers injection from the metal into the organic semiconductor has been suppressed, and dark current density as low as has been obtained, which is comparable to standard Si-based photodiodes. By using a collimated X-ray beam impinging onto the scintillator mounted over the photodiode we have been able to measure current variations in the order of 150 pA on a dark current floor of less than 50 pA when operating the X-ray tube in switching mode, thus proving the feasibility of indirect X-ray detection by means of organic semiconductors.

Development and validation of a meat juice ELISA for the diagnosis of Fasciola hepatica in cattle in Cuba

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Amilcar Arenal

2016-08-01

Full Text Available Objective: To establish and validate a home-made ELISA for determination of antibodies against excretory-secretory proteins of Fasciola hepatica in bovine meat juice samples. Methods: The validity criteria of the assay were defined based on standards of ISO. The following parameters were evaluated: excretion/secretion antigen concentrations for coating, anti-bovine immunoglobulin G dilution, linearity, accuracy and precision. Results: The assay was validated on 126 meat juice samples with known infection status. Using the receiver operating characteristic (n = 126 the optimal cut-off for the ELISA assay was 0.78, above this value the probability for an animal to have fasciolosis was 11 times. And the specificity and sensitivity were 100% and 90.91% respectively. The repeatability of the intra- and inter-assay tests had coefficients of variation lower than 10% and 20% respectively. Conclusions: The ELISA is a suitable test for further use in studies towards the epidemiology of Fasciola hepatica in Cuba.

Development of a sandwich ELISA for quantification of immunoglobulin G in mink blood

DEFF Research Database (Denmark)

Mathiesen, Ronja; Chriél, Mariann; Struve, T.

2016-01-01

early immunity and thus their resistance against pathogenic agents found in the environment. This study describes a sandwich ELISA for quantification of the concentration of total immunoglobulin G in mink blood. The ELISA was validated with serum samples from females (n=8) and their kits (litters of 4...

Measles serodiagnosis: standardization and evaluation of a dot-ELISA Diagnóstico sorológico do sarampo: padronização e avaliação do teste Dot-ELISA

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Lourdes R. A. Vaz de Lima

1994-04-01

Full Text Available A Dot-ELISA using a measles virus (MV antigen obtained by sodium deoxycholate treatment was standardized and evaluated for IgM and IgG antibody detection in measles patients and measles-vaccinated subjects. A total of 192 serum samples were studied, comprising 47 from patients with acute and convalescent measles, 55 from 9-month old children prior to measles vaccination and 41 from children of the same age after vaccination, and 49 from patients with unrelated diseases. The diagnostic performances of the IgG Dot-ELISA and IgG immuno fluorescence test (IFT were found to be close, varying from 0.97 to 1.00 in sensitivity and the specificities were maximum (1.00. Nevertheless, the sensitivity of the IgM Dot-ELISA (0.85 was higher than that (0.63 of the IgM IFT, although both assays had comparably high (1.00 specificities. The IgM Dot-ELISA in particular proved to be more sensitive in relation to other assays studied by revealing antibodies in 80.0% (12/15 of vaccinated children on the 15th day after immunization. In contrast the IgM IFT, failed to detect antibodies in the same group of vaccinated children. The stability of the MV antigen was longer than that of the IFT antigen, and the reproducibility of the Dot-Elisa was satisfactory.A técnica de Dot-ELISA (DE para detecção de anticorpos IgM e IgG anti vírus do sarampo foi padronizada e avaliada utilizando-se antígeno viral obtido por tratamento com desoxicolato de sódio (DOC. Foram estudadas 192 amostras de soros, compreendendo 47 amostras de 22 pacientes com sarampo nas fases aguda e convalescente, 55 amostras de soros de crianças antes da vacinação, tendo 9 meses de idade, 41 amostras de soros de crianças da mesma idade colhidas após vacinação e 49 amostras de soros de pacientes com outras patologias. O desempenho diagnóstico da técnica de Dot-ELISA-IgG foi semelhante ao de Imunofluorescência indireta (IFI IgG cujos índices de sensibilidade variaram de 0,97 a 1,00 e os de

Rabbit haemorrhagic disease: advantages of cELISA in assessing immunity in wild rabbits (Oryctolagus cuniculus).

Science.gov (United States)

Zheng, Tao; Parkes, John P

2011-12-15

Rabbit haemorrhagic disease (RHD) is an acute fatal disease of domestic and wild European rabbits (Oryctolagus cuniculus) caused by RHD virus (RHDV). Accurate assessment of immunity is of great importance for the conservation and control of wild rabbits. We evaluated a competitive ELISA (cELISA) against isotype ELISAs for assessing the protective immunity against the disease by challenging 50 wild-caught rabbits with a lethal dose of RHDV. Death or survival to the challenge was used as a criterion to determine the performance characteristics of the assay for the assessment of immunity in rabbits. At 1:10 dilution, a serum exhibiting ≥ 25% inhibition (1:10(25)) was regarded as the presence of RHDV-specific antibodies. Eleven of 16 (68.8%) rabbits with antibodies at 1:10(25) (<1:40) died of RHD. When the cut-off was moved from 25% to 50% inhibition (1:10(50)) at 1:10 serum dilution, the assay sensitivity, specificity and accuracy for the protective immunity were improved from 84%, 54.2% and 69.4% to 84%, 100% and 91.8%, respectively. We also demonstrated at the epitope amino acid sequence level why the presence of the RHDV-cross reactive benign rabbit calicivirus, which interfered with isotype ELISAs, had little impact on the specificity of the cELISA for the diagnosis of RHDV infection. The presence of RHDV-specific antibody at 1:10(50) by the cELISA is a reliable indicator for the protective immunity. In contrast to isotype ELISAs, the cELISA is a valuable specific tool for monitoring the herd immunity to RHD for the conservation and management of wild rabbits in the field. Copyright © 2011 Elsevier B.V. All rights reserved.

DETECTION OF SERIAL SERUM P-185 LEVEL IN PATIENTS WITH MALIGNANCY USING ELISA

Institute of Scientific and Technical Information of China (English)

ZHANG Zhi-hui; WU Ping; LI Li; MIAO Yi-meng; LI Jun

2006-01-01

Objective: To investigate P-185 contents in serum of the normal person and cancer patients and it's the significance on prognosis of diseases. Methods: We used ELISA method to evaluate P-185 levels in 193 normal persons, 133 malignancies and 34 hepatocirrhosises were evaluated using ELISA method. Results: Normal person had lower expression of P-185. However, malignancy and hepatocirrhosis patients had a significantly higher expression level of P-185 than normal (P＜0.05). Conclusion: ELISA method is an easy and reliable way to measure the level of P-185 in serum. Being a cancer marker, P-185 overexpression can be used for early diagnosis and prognosis of cancer patients.

Early antihepatitis C virus response with second-generation C200/C22 ELISA

NARCIS (Netherlands)

van der Poel, C. L.; Bresters, D.; Reesink, H. W.; Plaisier, A. A.; Schaasberg, W.; Leentvaar-Kuypers, A.; Choo, Q. L.; Quan, S.; Polito, A.; Houghton, M.

1992-01-01

Detection of early antibody to hepatitis C virus (HCV) by a new second-generation C200/C22 anti-HCV enzyme-linked immunosorbent assay (ELISA) and a four-antigen recombinant immunoblot assay (4-RIBA) was compared with the first-generation anti-HCV C100 ELISA using sequential serum samples of 9

Consistency of direct microscopic examination and ELISA in detection of Giardia in stool specimen among children

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Zohreh Torabi

2014-09-01

Full Text Available Objective: To investigate the consistency of direct microscopic examination and ELISA for determination of Giadia in stool specimen. Method: Study population consisted of children with any clinical symptoms of Giardia infestation since last two weeks. Fresh stool specimen was collected from each child. The stools specimens were assessed by two methods of direct microscopic examination and ELISA.The degree of agreement between direct stool exam and ELISA was calculated by Cohen's kappa coefficient. Results: In this study, 124 children with age range 2-12 years were investigated. A total of 64 (61.7% and 79 (65.7% of children had Giardia by direct stool exam and ELISA test respectively. There was association between frequency of constipation and Giardia infection (P=0.036. The Cohen's kappa coefficient calculated for degree of agreement between direct stool exam and ELISA showed κ=0.756 (P<0.001. Conclusions: The frequency of Giardia infection in symptomatic children was high and there was high agreement rate between ELISA and direct stool smear.

A new ELISA for the quantification of equine procalcitonin in plasma as potential inflammation biomarker in horses.

Science.gov (United States)

Rieger, Martin; Kochleus, Christian; Teschner, Dana; Rascher, Daniela; Barton, Ann Kristin; Geerlof, Arie; Kremmer, Elisabeth; Schmid, Michael; Hartmann, Anton; Gehlen, Heidrun

2014-09-01

In human medicine, procalcitonin (PCT) is a very common and well-established biomarker for sepsis. Even though sepsis is also a leading cause of death in foals and adult horses, up to now, no data about the role of equine PCT in septic horses has been available. Based on monoclonal antibodies targeted against human PCT, we report here the development of a sandwich ELISA for the quantification of equine PCT in equine plasma samples. The ELISA was characterized for intra- and interassay variance and a working range from 25 to 1,000 ng mL(-1) was defined as within this range; both intra- and interassay variances were below 15 %. The target recovery ranged between 73 and 106 %. The ELISA was used to determine the equine PCT concentration in 24 healthy and 5 septic horses to show the potential for clinical evaluation of equine PCT. Significantly different (P = 0.0006) mean equine PCT concentrations were found for the healthy control group and the sepsis group (47 and 8,450 ng mL(-1)).

eLISA eccentricity measurements as tracers of binary black hole formation

OpenAIRE

Nishizawa, Atsushi; Berti, Emanuele; Klein, Antoine; Sesana, Alberto

2016-01-01

Up to hundreds of black hole binaries individually resolvable by eLISA will coalesce in the Advanced LIGO/Virgo band within ten years, allowing for multi-band gravitational wave observations. Binaries formed via dynamical interactions in dense star clusters are expected to have eccentricities $e_0\\sim 10^{-3}$-$10^{-1}$ at the frequencies $f_0=10^{-2}$ Hz where eLISA is most sensitive, while binaries formed in the field should have negligible eccentricity in both frequency bands. We estimate ...

Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Science.gov (United States)

Cao, Biyun; He, Guangzhao; Yang, Hong; Chang, Huafang; Li, Shuqun; Deng, Anping

2013-10-15

Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive. © 2013 Elsevier B.V. All rights reserved.

Indirect taxation in the European Union

OpenAIRE

Ene, Sebastian; Micuda, Dan

2007-01-01

Indirect taxes are levied on the production and consumption of goods and services. They influence the retail price, and hence affect patterns of trade and consumption. Indirect taxes are ultimately paid by the final consumer. Sales and turnover taxes, excise duties and tariffs are the basic indirect taxes. In contrast with direct taxes, indirect taxes are seldom progressive. The principles for the levying of these taxes will be considered before the analysis of indirect taxes.

Padronização de três ELISAs polivalentes com lipopolissacarídeos de cadeia longa dos sorotipos 1 e 5, 2, 3 e 7 ou 10 e 12 de Actinobacillus pleuropneumoniae Standardization of three polyvalent ELISA based on long chain lipopolysaccharides of serotypes 1 and 5, 2, 3 and 7, or 10 and 12 of Actinobacillus pleuropneumoniae

Directory of Open Access Journals (Sweden)

S.S. Kuchiishi

2008-04-01

Full Text Available Três ELISAs polivalentes baseados em lipopolissacarídeos de cadeia longa (LPS-CL foram estabelecidos para detectar anticorpos para todos os sorotipos prevalentes de Actinobacillus pleuropneumoniae. Foram testadas amostras provenientes do banco de soros de suínos experimentalmente inoculados com todos os sorotipos de A. pleuropneumoniae. Os ELISAs foram sensíveis à detecção de anticorpos contra todos os LPS-CL. Foram observadas reações cruzadas no ELISA polivalente produzido com os sorotipos 1 e 5, com anti-soros específicos para os sorotipos 9 e 11, pois os sorotipos 1, 9 e 11 apresentaram antígenos somáticos comuns. No polivalente com os sorotipos 2, 3 e 7, observaram-se reações com anti-soros dos sorotipos 4, 6 e 8, devido à presença de antígenos somáticos entre os sorotipos 3, 6 e 8 e entre os sorotipos 4 e 7. Amostras de soros de animais infectados com Mycoplasma hyopneumoniae, Mycoplasma flocculare e Haemophilus parasuis, agentes que acometem o sistema respiratório dos suínos, não apresentaram reações cruzadas com os antígenos baseados em LPS-CL.Three polyvalent ELISA based on long chain lipopolysaccharides (LC-LPS were established to detect all prevalent serotypes of Actinobacillus pleuropneumoniae. Samples from a serum bank of experimentally inoculated animals with all serotypes of A. pleuropneumoniae were tested. Antibodies specific to LC-LPS of each serotype were detected. Cross-reactions were observed in the polyvalent ELISA produced with serotypes 1 and 5, with specific antisera to serotypes 9 and 11 due to common somatic antigens presence in serotypes 1, 9, and 11. In the polyvalent with serotypes 2, 3 and 7 reactions were observed with antisera of serotypes 4, 6, and 8, due to the presence of somatic antigens in serotypes 3, 6, and 8 and serotypes 4 and 7. Experimentally infected animals with respiratory agents of swine Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Haemophilus parasuis did not present

Ingroup favoritism and intergroup cooperation under indirect reciprocity based on group reputation.

Science.gov (United States)

Masuda, Naoki

2012-10-21

Indirect reciprocity in which players cooperate with unacquainted other players having good reputations is a mechanism for cooperation in relatively large populations subjected to social dilemma situations. When the population has group structure, as is often found in social networks, players in experiments are considered to show behavior that deviates from existing theoretical models of indirect reciprocity. First, players often show ingroup favoritism (i.e., cooperation only within the group) rather than full cooperation (i.e., cooperation within and across groups), even though the latter is Pareto efficient. Second, in general, humans approximate outgroup members' personal characteristics, presumably including the reputation used for indirect reciprocity, by a single value attached to the group. Humans use such a stereotypic approximation, a phenomenon known as outgroup homogeneity in social psychology. I propose a model of indirect reciprocity in populations with group structure to examine the possibility of ingroup favoritism and full cooperation. In accordance with outgroup homogeneity, I assume that players approximate outgroup members' personal reputations by a single reputation value attached to the group. I show that ingroup favoritism and full cooperation are stable under different social norms (i.e., rules for assigning reputations) such that they do not coexist in a single model. If players are forced to consistently use the same social norm for assessing different types of interactions (i.e., ingroup versus outgroup interactions), only full cooperation survives. The discovered mechanism is distinct from any form of group selection. The results also suggest potential methods for reducing ingroup bias to shift the equilibrium from ingroup favoritism to full cooperation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antibody profiling using a recombinant protein-based multiplex ELISA array accelerates recombinant vaccine development: Case study on red sea bream iridovirus as a reverse vaccinology model.

Science.gov (United States)

Matsuyama, Tomomasa; Sano, Natsumi; Takano, Tomokazu; Sakai, Takamitsu; Yasuike, Motoshige; Fujiwara, Atushi; Kawato, Yasuhiko; Kurita, Jun; Yoshida, Kazunori; Shimada, Yukinori; Nakayasu, Chihaya

2018-05-03

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development and validation of an epitope-blocking ELISA using an anti-haemagglutinin monoclonal antibody for specific detection of antibodies in sheep and goat sera directed against peste des petits ruminants virus.

Science.gov (United States)

Bodjo, Sanne Charles; Baziki, Jean-de-Dieu; Nwankpa, Nick; Chitsungo, Ethel; Koffi, Yao Mathurin; Couacy-Hymann, Emmanuel; Diop, Mariame; Gizaw, Daniel; Tajelser, Idris Badri Adam; Lelenta, Mamadou; Diallo, Adama; Tounkara, Karim

2018-07-01

Peste des petits ruminants (PPR) is a contagious and economically important disease affecting production of small ruminants (i.e., sheep and goats). Taking into consideration the lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is now targeted by the international veterinary community as the next animal disease to be eradicated. To support the African continental programme for the control of PPR, the Pan African Veterinary Vaccine Centre of the African Union (AU-PANVAC) is developing diagnostics tools. Here, we describe the development of a blocking enzyme-linked immunosorbent assay (bELISA) that allows testing of a large number of samples for specific detection of antibodies directed against PPR virus in sheep and goat sera. The PPR bELISA uses an anti-haemagglutinin (H) monoclonal antibody (MAb) as a competitor antibody, and tests results are interpreted using the percentage of inhibition (PI) of MAb binding generated by the serum sample. PI values below or equal to 18% (PI ≤ 18%) are negative, PI values greater than or equal to 25% (PI ≥ 25%) are positive, and PI values greater than 18% and below 25% are doubtful. The diagnostic specificity (DSp) and diagnostic sensitivity (DSe) were found to be 100% and 93.74%, respectively. The H-based PPR-bELISA showed good correlation with the virus neutralization test (VNT), the gold standard test, with a kappa value of 0.947. The H-based PPR-bELISA is more specific than the commercial kit ID Screen® PPR Competition (N-based PPR-cELISA) from IDvet (France), but the commercial kit is slightly more sensitive than the H-based PPR-bELISA. The validation process also indicated good repeatability and reproducibility of the H-based PPR-bELISA, making this new test a suitable tool for the surveillance and sero-monitoring of the vaccination campaign.

The use of the antigen ELISA for monitoring tsetse and trypanosomosis control programmes in Zimbabwe

Energy Technology Data Exchange (ETDEWEB)

Ries, R; Nqindi, J [Central Veterinary Lab., Causeway, Harare (Zimbabwe)

1997-02-01

Blood and serum samples from cattle originating from tsetse free and tsetse infested areas were analyzed using the Buffy Coat technique and an ELISA to detect trypanosomes and trypanosomal antigens, respectively. The results of the two tests were compared and apparent sensitivity and trypanosomal prevalence were calculated. The BCT seemed to be the most suitable test to detect acute infections, while the antigen capture ELISA (Ag-ELISA) was able to detect more chronic infections. The specificity of the Ag-ELISA was found to be very good, but the sensitivity of the test should be improved. One way to detect more T. congolense and T. vivax infections was to lower the cut-off point of percent positivity from 10 to 5%. (author). 1 ref., 1 fig., 7 tabs.

The use of the antigen ELISA for monitoring tsetse and trypanosomosis control programmes in Zimbabwe

International Nuclear Information System (INIS)

Ries, R.; Nqindi, J.

1997-01-01

Blood and serum samples from cattle originating from tsetse free and tsetse infested areas were analyzed using the Buffy Coat technique and an ELISA to detect trypanosomes and trypanosomal antigens, respectively. The results of the two tests were compared and apparent sensitivity and trypanosomal prevalence were calculated. The BCT seemed to be the most suitable test to detect acute infections, while the antigen capture ELISA (Ag-ELISA) was able to detect more chronic infections. The specificity of the Ag-ELISA was found to be very good, but the sensitivity of the test should be improved. One way to detect more T. congolense and T. vivax infections was to lower the cut-off point of percent positivity from 10 to 5%. (author). 1 ref., 1 fig., 7 tabs

Influence of affinity on antibody determination in microtiter ELISA systems

International Nuclear Information System (INIS)

Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

1986-01-01

Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of 125 I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of 125 I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations

DETECTION OF HUMAN BLOODSTAINS BY ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA

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GHOLAM-HOSSEIN EDRISSIAN

1982-07-01

Full Text Available The microplate method of e nzyme-l i nke d i rnmuno s o r be n t a s s ay (ELISA , usi ng alkaline phosphatase anti-human I gG conj ugate , was modified and appl ied in t he dete c t i on of the human b lood specimens among the blood samples prep a r ed fr om human and laborat or y anima l s i n t he form of small dr ied b loodst a i ns on f ilt e r paper . The modi f i e d ELISA t e chnique wa s compa red wit h t he agar double d i f f us ion me t hod of prec ipitin test ."nThe results of this primary study showed t ha t the ELISA i n compare to gel diffusi o n t e st is sensitive and specific. It is also enough repr oducible and practical t o be consider as a competent technique for i de nt i f i c ation o f human bloodstains in medicolegal laboratories .

Scene independent real-time indirect illumination

DEFF Research Database (Denmark)

Frisvad, Jeppe Revall; Christensen, Niels Jørgen; Falster, Peter

2005-01-01

A novel method for real-time simulation of indirect illumination is presented in this paper. The method, which we call Direct Radiance Mapping (DRM), is based on basal radiance calculations and does not impose any restrictions on scene geometry or dynamics. This makes the method tractable for rea...

Shear bond strength of a denture base acrylic resin and gingiva-colored indirect composite material to zirconia ceramics.

Science.gov (United States)

Kubochi, Kei; Komine, Futoshi; Fushiki, Ryosuke; Yagawa, Shogo; Mori, Serina; Matsumura, Hideo

2017-04-01

To evaluate the shear bond strengths of two gingiva-colored materials (an indirect composite material and a denture base acrylic resin) to zirconia ceramics and determine the effects of surface treatment with various priming agents. A gingiva-colored indirect composite material (CER) or denture base acrylic resin (PAL) was bonded to zirconia disks with unpriming (UP) or one of seven priming agents (n=11 each), namely, Alloy Primer (ALP), Clearfil Photo Bond (CPB), Clearfil Photo Bond with Clearfil Porcelain Bond Activator (CPB+Act), Metal Link (MEL), Meta Fast Bonding Liner (MFB), MR. bond (MRB), and V-Primer (VPR). Shear bond strength was determined before and after 5000 thermocycles. The data were analyzed with the Kruskal-Wallis test and Steel-Dwass test. The mean pre-/post-thermalcycling bond strengths were 1.0-14.1MPa/0.1-12.1MPa for the CER specimen and 0.9-30.2MPa/0.1-11.1MPa for the PAL specimen. For the CER specimen, the ALP, CPB, and CPB+Act groups had significantly higher bond strengths among the eight groups, at both 0 and 5000 thermocycles. For the PAL specimen, shear bond strength was significantly lower after thermalcycling in all groups tested. After 5000 thermocycles, bond strengths were significantly higher in the CPB and CPB+Act groups than in the other groups. For the PAL specimens, bond strengths were significantly lower after thermalcycling in all groups tested. The MDP functional monomer improved bonding of a gingiva-colored indirect composite material and denture base acrylic resin to zirconia ceramics. Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases

Science.gov (United States)

Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

2015-01-01

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines. PMID:26184194

Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA for Infectious Diseases

Directory of Open Access Journals (Sweden)

Harpal Singh

2015-07-01

Full Text Available Enzyme-linked Immunosorbent Assay (ELISA-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

Science.gov (United States)

Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

2015-07-08

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

Use of ELISA in the diagnosis of infection and evaluation of treatment with Cymelarsan in camels infected with Trypanosoma evansi in Kenya

International Nuclear Information System (INIS)

Olaho-Mukani, W.; Omuse, J.K.; Nyang'ao, J.M.N.; Mutugi, M.W.; Ngaira, J.M.; Luckins, A.G.; Jeffires, P.

1993-01-01

A polyclonal based antigen detection enzyme immunoassay (Ag-ELISA) was validated and used for the diagnosis of camel trypanosomiasis in five localities in Kenya. The Ag-ELISA results were correlated with those of parasitological tests, namely, Buffy Coat Technique (BCT) and rodent subinoculation (RI). More animals showed evidence of infection using Ag-ELISA than when using BCT and RI, and showed infection rates ranging from 45.3% to 86.2% in sampled herds, compared with 0.8% to 18% using parasitologial tests. An overall apparent sensitivity of 74% by Ag-ELISA was obtained after screening sera from three camel herds. Higher ELISA values were observed in those herds with the highest rate of patent infection or where drug intervention was not being practised. However, the study revealed that trypanosomiasis was endemic in all the sampled herds and although treatment with quinapyramine sulphate suppressed parasitaemia, the presence of circulating trypanosomal antigens indicated the probable persistence of infection due to resistance to this drug. In experimental camels, the use of Ag-ELISA in evaluating the efficacy of Cymelarsan revealed that in more than 80% of the treated camels there appeared to be cure, characterized by the absence of both parasitaemia and circulating trypanosomal antigens. However, in a few animals relapse in parasitaemia occurred, and in a number of camels, antigenaemia persisted throughout the study period. Treatment on the basis of a positive Ag-ELISA reduced the proportion of Ag-positive animals from 75% to 26% and parasite positive animals were no longer present at the end of the experiment. (author). 13 refs, 4 figs, 3 tabs

Comparison of R5 and G12 Antibody-Based ELISA Used for the Determination of the Gluten Content in Official Food Samples

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Rupert Hochegger

2015-11-01

Full Text Available Celiac Disease (CD is one of the most common food intolerances. It comes along with serious damage of the mucosa in the small intestine and is caused by the storage proteins—termed “gluten”—of wheat, rye, barley and possibly oats. Sensitive individuals need to stick to a strict gluten-free diet. The gluten level in food products labeled as “gluten-free”, must not exceed 20 mg/kg. It is obvious that effective test methods are needed to accurately determine the gluten concentration in foods. The determination of the presence of gluten in foodstuffs is mainly done by means of an immunochemical method called ELISA (enzyme-linked immunosorbent assay. To check the suitability of a G12 antibody-based gluten detection kit for its use in official control systems a number of routine samples were tested in parallel with two different test kits, as would be done in a routine lab. The determination of the gluten content was performed on samples entering the official laboratory including samples from official control plans, commercially available and private samples to request gluten-free labels. The results obtained with the G12 antibody ELISA assay were comparable to the official R5 method. A validation of the two different methods was not part of this study.

The evaluation of a nucleoprotein ELISA for the detection of equine influenza antibodies and the differentiation of infected from vaccinated horses (DIVA).

Science.gov (United States)

Galvin, Pamela; Gildea, Sarah; Arkins, Sean; Walsh, Cathal; Cullinane, Ann

2013-12-01

Antibodies against equine influenza virus (EIV) are traditionally quantified by haemagglutination inhibition (HI) or single radial haemolysis (SRH). To evaluate an ELISA for the detection of antibodies against influenza nucleoprotein in the diagnosis and surveillance of equine influenza (EI). The ELISA was compared with the SRH and HI tests. Serial serum samples from 203 naturally and 14 experimentally infected horses, from 60 weanlings following primary vaccination with five different vaccines (two whole inactivated vaccines, two ISCOM-based subunit vaccines and a recombinant canarypox virus vaccine) and from 44 adult horses following annual booster vaccination with six different vaccines were analysed. Fewer seroconversions were detected in clinical samples by ELISA than by SRH or HI but ELISA was more sensitive than SRH in naïve foals post-experimental infection. The ELISA did not detect the antibody response to vaccination with the recombinant canarypox virus vaccine confirming the usefulness of the combination of this kit and vaccine to differentiate between naturally infected and vaccinated horses, that is, DIVA. No DIVA capacity was evident with the other vaccines. The results suggest that this ELISA is a useful supplementary test for the diagnosis of EI although less sensitive than HI or SRH. It is an appropriate test for EI surveillance in a naïve population and may be combined with the recombinant canarypox virus vaccine but not with other commercially available subunit vaccines, in a DIVA strategy. © 2013 Blackwell Publishing Ltd.

Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

Science.gov (United States)

Zhou, Lijun; Lv, Zhiqiang; Shao, Jing; Xu, Ying; Luo, Xiaohong; Zhang, Yuming; Hu, Yang; Zhang, Wenji; Luo, Shuhong; Fang, Jianmin; Wang, Ying; Duan, Chaohui; Huang, Ruopan

2016-09-01

The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas. © 2015 Wiley Periodicals, Inc.

Influence of clinical and laboratory variables on faecal antigen ELISA results in dogs with canine parvovirus infection.

Science.gov (United States)

Proksch, A L; Unterer, S; Speck, S; Truyen, U; Hartmann, K

2015-06-01

False negative faecal canine parvovirus (CPV) antigen ELISA results in dogs with CPV infection are common, but the factors that lead to these false negative results are still unknown. The aim of this study was to investigate whether dogs with a false negative faecal CPV antigen ELISA result have milder clinical signs and laboratory changes, a lower faecal virus load, higher faecal and serum CPV antibody titres and a faster recovery than dogs with a positive result. Eighty dogs with CPV infection, confirmed by the presence of clinical signs and a positive faecal CPV polymerase chain reaction (PCR), were assigned to two groups according to their faecal antigen ELISA result. Time until presentation, severity of symptoms, laboratory parameters, faecal virus load, faecal and serum antibody titres, and CPV sequencing data were compared between both groups. In 38/80 dogs that were hospitalised until recovery, the time to recovery, mortality, and the course of the disease were compared between dogs with positive and negative faecal antigen ELISA results. Of the 80 dogs included, 41 (51.3%) had a false negative faecal antigen ELISA result. ELISA-negative dogs had a significantly shorter time until presentation, lower frequency of defaecation, lower faecal virus load, and higher serum antibody concentrations than ELISA-positive dogs. Laboratory changes, CPV shedding, and outcomes were not associated with faecal antigen ELISA results. In conclusion, low faecal CPV load and antibodies binding to CPV antigen in faeces are likely to be important reasons for false negative faecal antigen ELISA results. Dogs with clinical signs of CPV infection should be retested by faecal PCR. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

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Fang He

Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

Science.gov (United States)

Novo, Pedro; Prazeres, Duarte Miguel França; Chu, Virginia; Conde, João Pedro

2011-12-07

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.

Detection of Trypanosoma congolense type savannah in field samples of buffy coats of bovins using PCR-ELISA

International Nuclear Information System (INIS)

Sidibe, I.

2007-01-01

PCR-ELISA was set up to detect strain of Trypanosoma congolense type savannah in field samples of buffy coats. Results of PCR-ELISA and PCR were compared and the sensibility and specificity of both techniques were also compared with those of the method of Murray [1] for the detection of TCS in 257 samples. The PCR products were labelling with DIG-dUTP during amplification cycles of the repetitive satellite DNA. A DNA biotinyled capture probe was used to detect the amplicon by ELISA in streptavidine coated microplates. Both of PCR-ELISA and PCR were more sensible and more specific than the method of Murray. Indeed, for the 257 samples analysed by the three techniques, PCR-ELISA and PCR have detected TCS in 98 and 97 samples respectively, whereas the method of Murray has detected TCS in only 39 samples. In addition, PCRELISA and PCR had almost the same sensibility and specificity. So, PCR-ELISA and PCR have respectively detected TCS in 38.62% and 39.22% of all the 334 samples analysed by both techniques during this study. At the end of this study, the cost of analyse by PCR-ELISA of a sample of buffy coat, was evaluated at 1993 FCFA or Euro 3,04. (author) [fr

Indirect Self-Destructiveness and Emotional Intelligence.

Science.gov (United States)

Tsirigotis, Konstantinos

2016-06-01

While emotional intelligence may have a favourable influence on the life and psychological and social functioning of the individual, indirect self-destructiveness exerts a rather negative influence. The aim of this study has been to explore possible relations between indirect self-destructiveness and emotional intelligence. A population of 260 individuals (130 females and 130 males) aged 20-30 (mean age of 24.5) was studied by using the Polish version of the chronic self-destructiveness scale and INTE, i.e., the Polish version of the assessing emotions scale. Indirect self-destructiveness has significant correlations with all variables of INTE (overall score, factor I, factor II), and these correlations are negative. The intensity of indirect self-destructiveness differentiates significantly the height of the emotional intelligence and vice versa: the height of the emotional intelligence differentiates significantly the intensity of indirect self-destructiveness. Indirect self-destructiveness has negative correlations with emotional intelligence as well as its components: the ability to recognize emotions and the ability to utilize emotions. The height of emotional intelligence differentiates the intensity of indirect self-destructiveness, and vice versa: the intensity of indirect self-destructiveness differentiates the height of emotional intelligence. It seems advisable to use emotional intelligence in the prophylactic and therapeutic work with persons with various types of disorders, especially with the syndrome of indirect self-destructiveness.

Evolutionary stability and resistance to cheating in an indirect reciprocity model based on reputation

Science.gov (United States)

Martinez-Vaquero, Luis A.; Cuesta, José A.

2013-05-01

Indirect reciprocity is one of the main mechanisms to explain the emergence and sustainment of altruism in societies. The standard approach to indirect reciprocity is reputation models. These are games in which players base their decisions on their opponent's reputation gained in past interactions with other players (moral assessment). The combination of actions and moral assessment leads to a large diversity of strategies; thus determining the stability of any of them against invasions by all the others is a difficult task. We use a variant of a previously introduced reputation-based model that let us systematically analyze all these invasions and determine which ones are successful. Accordingly, we are able to identify the third-order strategies (those which, apart from the action, judge considering both the reputation of the donor and that of the recipient) that are evolutionarily stable. Our results reveal that if a strategy resists the invasion of any other one sharing its same moral assessment, it can resist the invasion of any other strategy. However, if actions are not always witnessed, cheaters (i.e., individuals with a probability of defecting regardless of the opponent's reputation) have a chance to defeat the stable strategies for some choices of the probabilities of cheating and of being witnessed. Remarkably, by analyzing this issue with adaptive dynamics we find that whether an honest population resists the invasion of cheaters is determined by a Hamilton-like rule, with the probability that the cheat is discovered playing the role of the relatedness parameter.

Characterization of thermophysical properties of phase change materials for non-membrane based indirect solar desalination application

International Nuclear Information System (INIS)

Sarwar, J.; Mansoor, B.

2016-01-01

Highlights: • Thermal cycling of paraffin waxes phase change materials. • Differential Scanning Calorimetry and thermogravimetric study of the materials. • Characterization of the phase change materials via Temperature History Method. • Investigation of suitability of materials for indirect solar desalination system. • Paraffin waxes are suitable for non-membrane indirect solar desalination system. - Abstract: Phase change material as a thermal energy storage medium has been widely incorporated in various technologies like solar air/water heating, buildings, and desalination for efficient use and management of fluctuating solar energy. Temperature and thermal energy requirements dictate the selection of an appropriate phase change material for its application in various engineering systems. In this work, two phase change materials belonging to organic paraffin wax class have been characterized to obtain their thermophysical properties. The melting/solidification temperatures, latent heat of fusion and heat capacities of the phase change materials have been investigated using Differential Scanning Calorimetry, Thermogravimetric analysis and Temperature History Method. Thermal cycles up to 300 are performed to investigate melting and solidification reversibility as well as degradation over time. It is shown that the selected paraffin waxes have reversible phase change with no degradation of thermophysical properties over time. It is also shown that melting/solidification temperature and thermal energy storage capabilities make them suitable for their application as a thermal energy storage medium, in high temperature vapour compression, multi-stage flash and multi-effect distillation processes of non-membrane based indirect desalination systems.

Design of three-well indirect pumping terahertz quantum cascade lasers for high optical gain based on nonequilibrium Green's function analysis

Science.gov (United States)

Liu, Tao; Kubis, Tillmann; Jie Wang, Qi; Klimeck, Gerhard

2012-03-01

The nonequilibrium Green's function approach is applied to the design of three-well indirect pumping terahertz (THz) quantum cascade lasers (QCLs) based on a resonant phonon depopulation scheme. The effects of the anticrossing of the injector states and the dipole matrix element of the laser levels on the optical gain of THz QCLs are studied. The results show that a design that results in a more pronounced anticrossing of the injector states will achieve a higher optical gain in the indirect pumping scheme compared to the traditional resonant-tunneling injection scheme. This offers in general a more efficient coherent resonant-tunneling transport of electrons in the indirect pumping scheme. It is also shown that, for operating temperatures below 200 K and low lasing frequencies, larger dipole matrix elements, i.e., vertical optical transitions, offer a higher optical gain. In contrast, in the case of high lasing frequencies, smaller dipole matrix elements, i.e., diagonal optical transitions are better for achieving a higher optical gain.

ELISA with double antigen sandwich for screening specific serum anti-TP antibody in blood donors

International Nuclear Information System (INIS)

Wang Yiqing; Shi Zhixu

2002-01-01

Objective: To select a sensitive and specific laboratory examination suitable for screening serum anti-TP antibody in blood donors. Methods: The serum anti-TP antibody in 11271 blood donors were detected using ELISA with double antigen sandwich and the outcomes were compared with those using RPR assay. The conflicting specimen were confirmed by repeating the test with TPHA assay. Results: The positive rates of serum anti-TP antibody by ELISA with double antigen sandwich and RPR was 0.36% (41/11271) and 0.26% (29/11271), respectively. The coincidence of the detecting outcomes by ELISA with double antigen sandwich and RPR with TPHA was 97.5% (40/41) and 63.41%(26/41) respectively. Conclusion: Compared with RPR assay, ELISA with double antigen sandwich has higher sensibility and specificity for screening serum anti-TP antibody in blood donors

Evaluation of a novel Dot-ELISA assay utilizing a recombinant protein for the effective diagnosis of Taenia pisiformis larval infections.

Science.gov (United States)

Chen, Lin; Yang, Deying; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2014-08-29

Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in domestic breeds of the rabbit Oryctolagus cuniculus that results in economic losses. At present, there is no convenient and effective method for the rapid detection of T. pisiformis larvae. Here, we developed and tested the efficacy of a Dot-ELISA assay for the diagnosis of T. pisiformis larval infections in rabbits, based on the expression of the recombinant fusion protein (rTp1) from the Tp1 gene. Rapid amplification of cDNA ends (RACE) was used to amplify the 3' ends of the Tp1 gene, based on the unigene similar to Ts1 gene (EU009656.1) which comes from transcriptome sequencing of T. pisiformis. The Tp1 gene was successfully amplified, cloned and expressed in BL21 (DE3). Western blot analysis revealed that the recombinant Tp1 protein is specifically recognized by rabbit T. pisiformis cysticercosis antisera. This purified recombinant fusion protein, rTp1, was probed by Dot-ELISA with sera from rabbits infected with T. pisiformis larvae and with other parasitic infections. Results showed that this Dot-ELISA assay had both high sensitivity (92.9-97.6%) and specificity (95.2-98.4%) to detect T. pisiformis larval infections. We also found very low levels of cross-reaction with other parasitic infections. This study has revealed that our novel Dot-ELISA assay utilizing the recombinant fusion protein, rTp1, has a strong potential for the effective diagnosis of T. pisiformis infections in rabbits. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of a novel chemiluminescence enzyme immunoassay (CLEIA) with enzyme-linked immunosorbent assay (ELISA) for the determination of MPO-ANCA in patients with ANCA-associated vasculitis.

Science.gov (United States)

Hirose, Orie; Itabashi, Mitsuyo; Takei, Takashi; Nitta, Kosaku

2015-03-01

Myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) represents the serological hallmark of ANCA-associated vasculitis (AAV). We evaluated the analytical and diagnostic accuracy of chemiluminescence enzyme immunoassay (CLEIA) versus enzyme-linked immunosorbent assay (ELISA) for the detection of MPO-ANCA. A total of 242 sera obtained from 51 patients with AAV and 103 patients without AAV were tested for MPO-ANCA by ELISA (NephroScholor MPOANC II) and CLEIA (the STACIA MEBLux test). Disease activity in the patients with AAV was determined based on the Birmingham Vasculitis Activity Score. We analyzed the correlations between the MPO-ANCA titers determined by the CLEIA and those determined by the ELISA, and also between the MPO-ANCA titers and the disease activity. The MPO-ANCA titers determined by the CLEIA (x) were strongly correlated with those determined by the ELISA (y). The correlation could be expressed by the following equation in this study: y = 1.8x + 7.7 (r = 0.96; p ELISA yielded positive test results in 57 of the 242 sera (23.6%). The CLEIA yielded false-positive test results in 4 of the 120 sera obtained from the non-AAV patients (3.3%), whereas the ELISA yielded a false-positive result in only 1 of the 120 sera obtained from the non-AAV patients (0.8%). The sensitivity and specificity of the CLEIA for the diagnosis of AAV were 100% and 96.7%, respectively, while those of the ELISA were 94.3% and 99.2%, respectively. The sensitivity and specificity of the CLEIA for the prediction of active disease were 100% and 64.4%, respectively, while those of the ELISA were 94.3% and 73.6%, respectively. The false positivity rate of the CLEIA for MPO-ANCA tended to be high as compared with that of the ELISA. Also, according to the correlation coefficient between the results of the CLEIA and the ELISA calculated in this study, it is necessary to pay attention to the differences in the sensitivity and specificity between CLEIA and ELISA.

Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture.

Science.gov (United States)

Panneum, S; Rukkwamsuk, T

2017-03-01

For preventive and control strategies of Caprine Arthritis Encephalitis Virus (CAEV) infection in dairy goats, performance of the available diagnostic tests was described as one of the most important and necessary aspects. The study aimed at evaluating the diagnostic test performance, including PCR, ELISA and viral culture, for CAEV infection in dairy goats in Thailand. Blood samples of 29 dairy goats from five low- to medium-prevalence herds and one very low-prevalence herd were collected for PCR and ELISA methods. The performance of these two diagnostic methods was evaluated by comparing with cytopathic effects (CPE) in the co-cultivation of CAEV and primary synovial cells. Results indicated that sensitivity, specificity were, respectively, 69.6%, 100%, for PCR; and 95.7%, 83.3% for ELISA. The PCR assay tended to have lower sensitivity and higher specificity than ELISA. When multiple tests were applied, parallel testing provided sensitivity and specificity of 98.7% and 83.3%, while series testing showed sensitivity and specificity of 66.6% and 100% respectively. These results indicated that combination of ELISA and PCR provided some advantages and possibly offered optimal methods to detect CAEV-infected goats. Kappa value of the agreement between PCR and ELISA test was 0.34, indicating fair agreement. Regarding the possibility of antigenic variation between CAEV strains used in both PCR and ELISA assays, the actual circulating CAEV strain should be reviewed in order to develop and enhance the diagnostic tests using the CAE viral antigens derived from specific local strains of Thailand.

ELISA Cut-off Point for the Diagnosis of Human Brucellosis; a Comparison with Serum Agglutination Test

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Anahita Sanaei Dashti

2012-03-01

Full Text Available Background: Brucellosis is a world-wide disease, which has a diverse clinical manifestation, and its diagnosis has to be proven by laboratory data. Serum agglutination test (SAT is the most-widely used test for diagnosing brucellosis. The enzyme linked immunosorbent assay (ELISA can also determine specific antibody classes against brucella. It is a sensitive, simple and rapid test, which could be an acceptable alternative to SAT with fewer limitations, however, like any other new test it should be further evaluated and standardized for various populations. This study was planned to determine an optimal cut-off point, for ELISA which would offer maximum sensitivity and specificity for the test when compared to SAT.Methods: Four hundred and seven patients with fever and other compatible symptoms of brucellosis were enrolled in the study. Serum agglutination test, 2-Mercaptoethanol test, and ELISA were performed on their sera. Results: The cut-off point of 53 IU/ml of ELISA-IgG yielded the maximal sensitivity and specificity comparing to the other levels of ELISA-IgG, and was considered the best cut off-point of ELISA-IgG to diagnose acute brucellosis. At this cut-off, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were 84.09%, 85.38%, 62.20, 94.90, 5.75, 0.18, respectively.Conclusion: The best cut-off point of ELISA-IgG is 53 IU/ml, which yields the maximal sensitivity and specificity to diagnose acute brucellosis.

Social Dilemmas and Indirect Reciprocity: A systematic review

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Cristian Acosta

2014-08-01

Full Text Available Indirect reciprocity is an evolutionary theory of altruism and cooperation in social dilemmas. Its explanation of cooperation incorporates information on the reputation people hold of other people regarding whether they are cooperative or not. This study systematically reviewed the current empirical literature on indirect reciprocity in social dilemma situations. The following databases were consulted: ScienceDirect, EBSCO, and OVID, using has key words "indirect reciprocity" AND "dilemma" OR "cooperation" OR "charity". Twenty-three articles were selected which met the inclusion criteria (papers written in English and studies whose methodology was based on experimental games and the theories, objectives, the main types of samples used, and main results were analyzed. The results show that the games used were the indirect reciprocity and public goods games, which may vary according to the objectives. It was found that reputation is a key variable that helps to promote cooperation. In turn, information on previous cooperation helps to build reputation, which is subsequently involved in the decision to cooperate with others. Finally, we highlight the need for more experimental studies that incorporate other explanatory variables and evaluate them in new contexts.

A comparison of sperm agglutination and immobilization assays with a quantitative ELISA for anti-sperm antibody in serum.

Science.gov (United States)

Lynch, D M; Leali, B A; Howe, S E

1986-08-01

An enzyme-linked immunosorbent assay (ELISA) that quantitates antisperm antibody in serum was compared with standard sperm agglutination and immobilization assays with the use of sera from 40 normal and 292 subfertile individuals. Quantitation of the assay was accomplished by standardizing assay parameters, including the incorporation of a standard reference curve, the number of whole target sperm, the optimal dilution of serum, the selection of microtiter plate, and the time and temperatures involved in the adsorption and incubation phases. With this method, the level of antisperm antibody binding to target sperm in 40 normal fertile individuals was found to be 2.3 (+/- 1.1 standard deviation [SD]) fg immunoglobulin (Ig)/sperm. An increased mean level of 7.4 +/- 3.7 fg Ig/sperm was determined in 84 infertile patients with positive agglutination and/or immobilization tests. In 208 individuals with negative agglutination and immobilization tests the mean concentration of antisperm antibody was 2.5 +/- 1.3 fg Ig/sperm. Postvasectomy patients assayed by this method had a mean Ig binding value of 7.1 +/- 2.4 fg Ig/sperm. The infertile group with positive agglutination and/or immobilization tests had a significantly higher mean antisperm antibody level than the normal fertile group, according to the Student's t-test for independent samples (P less than 0.001). This indirect serum-based assay reproducibly quantitates antisperm antibody binding to whole target sperm, suggests the normal and abnormal levels of antisperm antibody, and correlates with standard functional assays.

Immunoglobulin subclass in experimental murine Toxocara cati infection

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Kusnoto

2017-11-01

Full Text Available Aim: The aim of this study was to detect specific immunoglobulin (Ig that could be used to determine monoclonal antibody in conjugate-making an effort for the indirect enzyme-linked immunosorbent assay (ELISA diagnostic kit of toxocariasis in human. Materials and Methods: The study was conducted to assess the Ig profile, based on ELISA-isotyping, in mice infected with second stage larvae eggs of Toxocara cati. The optical density values of anti-T. cati mice serum IgG subclasses were analyzed by applying ANOVA factorial. Results: The specific IgG subclass in mice infected with T. cati mice was found to be IgG2β. Conclusion: Subclass of IgG, especially IgG2β, can provide leads about the use of the monoclonal antibody in conjugate making an effort for the indirect ELISA diagnostic kit.

Methodology for determination of plasma cortisol in fish using Competitive Enzyme-Linked Immunosorbent Assay (ELISA)

DEFF Research Database (Denmark)

Velasco-Santamaría, Yohana M.; Cruz-Casallas, Pablo E.

2007-01-01

Objective. To determine plasma cortisol procedure in fish using competitive enzymelinked immunosorbent assay (ELISA). Materials and methods. Two plasma samples of juveniles rainbow trout Oncorhynchus mykiss were analized by using ELISA human kit for cortisol assay. For standard curve calibration...

Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera

Czech Academy of Sciences Publication Activity Database

Vaisocherová, Hana; Faca, V.M.; Taylor, A. D.; Hanash, S.; Jiang, S.

2009-01-01

Roč. 24, č. 7 (2009), s. 2143-2148 ISSN 0956-5663 Institutional research plan: CEZ:AV0Z20670512 Keywords : SPR * ELISA * Cancer biomarkers Subject RIV: CE - Biochemistry Impact factor: 5.429, year: 2009

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.

Explicit calculation of indirect global warming potentials for halons using atmospheric models

Directory of Open Access Journals (Sweden)

D. J. Wuebbles

2009-11-01

Full Text Available The concept of Global Warming Potentials (GWPs has been extensively used in policy consideration as a relative index for comparing the climate impact of an emitted greenhouse gas (GHG, relative to carbon dioxide with equal mass emissions. Ozone depletion due to emission of chlorinated or brominated halocarbons leads to cooling of the climate system in the opposite direction to the direct warming contribution by halocarbons as GHGs. This cooling is a key indirect effect of the halocarbons on climatic radiative forcing, which is accounted for by indirect GWPs. With respect to climate, it is critical to understand net influences considering direct warming and indirect cooling effects especially for Halons due to the greater ozone-depleting efficiency of bromine over chlorine. Until now, the indirect GWPs have been calculated using a parameterized approach based on the concept of Equivalent Effective Stratospheric Chlorine (EESC and the observed ozone depletion over the last few decades. As a step towards obtaining indirect GWPs through a more robust approach, we use atmospheric models to explicitly calculate the indirect GWPs of Halon-1211 and Halon-1301 for a 100-year time horizon. State-of-the-art global chemistry-transport models (CTMs were used as the computational tools to derive more realistic ozone depletion changes caused by an added pulse emission of the two major Halons at the surface. The radiative forcings on climate from the ozone changes have been calculated for indirect GWPs using an atmospheric radiative transfer model (RTM. The simulated temporal variations of global average total column Halons after a pulse perturbation follow an exponential decay with an e-folding time which is consistent with the expected chemical lifetimes of the Halons. Our calculated indirect GWPs for the two Halons are much smaller than those from past studies but are within a single standard deviation of WMO (2007 values and the direct GWP values derived

Full automation and validation of a flexible ELISA platform for host cell protein and protein A impurity detection in biopharmaceuticals.

Science.gov (United States)

Rey, Guillaume; Wendeler, Markus W

2012-11-01

Monitoring host cell protein (HCP) and protein A impurities is important to ensure successful development of recombinant antibody drugs. Here, we report the full automation and validation of an ELISA platform on a robotic system that allows the detection of Chinese hamster ovary (CHO) HCPs and residual protein A of in-process control samples and final drug substance. The ELISA setup is designed to serve three main goals: high sample throughput, high quality of results, and sample handling flexibility. The processing of analysis requests, determination of optimal sample dilutions, and calculation of impurity content is performed automatically by a spreadsheet. Up to 48 samples in three unspiked and spiked dilutions each are processed within 24 h. The dilution of each sample is individually prepared based on the drug concentration and the expected impurity content. Adaptable dilution protocols allow the analysis of sample dilutions ranging from 1:2 to 1:2×10(7). The validity of results is assessed by automatic testing for dilutional linearity and spike recovery for each sample. This automated impurity ELISA facilitates multi-project process development, is easily adaptable to other impurity ELISA formats, and increases analytical capacity by combining flexible sample handling with high data quality. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus.

Science.gov (United States)

Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun

2011-07-01

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of dengue NS1 antigen rapid tests and ELISA kits using clinical samples.

Directory of Open Access Journals (Sweden)

Subhamoy Pal

Full Text Available Early diagnosis of dengue virus (DENV infection can improve clinical outcomes by ensuring close follow-up, initiating appropriate supportive therapies and raising awareness to the potential of hemorrhage or shock. Non-structural glycoprotein-1 (NS1 has proven to be a useful biomarker for early diagnosis of dengue. A number of rapid diagnostic tests (RDTs and enzyme-linked immunosorbent assays (ELISAs targeting NS1 antigen (Ag are now commercially available. Here we evaluated these tests using a well-characterized panel of clinical samples to determine their effectiveness for early diagnosis.Retrospective samples from South America were used to evaluate the following tests: (i "Dengue NS1 Ag STRIP" and (ii "Platelia Dengue NS1 Ag ELISA" (Bio-Rad, France, (iii "Dengue NS1 Detect Rapid Test (1st Generation" and (iv "DENV Detect NS1 ELISA" (InBios International, United States, (v "Panbio Dengue Early Rapid (1st generation" (vi "Panbio Dengue Early ELISA (2nd generation" and (vii "SD Bioline Dengue NS1 Ag Rapid Test" (Alere, United States. Overall, the sensitivity of the RDTs ranged from 71.9%-79.1% while the sensitivity of the ELISAs varied between 85.6-95.9%, using virus isolation as the reference method. Most tests had lower sensitivity for DENV-4 relative to the other three serotypes, were less sensitive in detecting secondary infections, and appeared to be most sensitive on Day 3-4 post symptom onset. The specificity of all evaluated tests ranged from 95%-100%.ELISAs had greater overall sensitivity than RDTs. In conjunction with other parameters, the performance data can help determine which dengue diagnostics should be used during the first few days of illness, when the patients are most likely to present to a clinic seeking care.

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...

Comment traduire en japonais les styles indirect et indirect libre de Madame Bovary ?

Directory of Open Access Journals (Sweden)

Hisaki Sawasaki

2012-03-01

Full Text Available Parmi les difficultés rencontrées lors de la traduction des textes littéraires occidentaux, en japonais, nous examinons le problème des styles indirect et indirect libre. Pour cela, en effectuant une petite mise au point grammaticale, nous comparons sept traductions de Madame Bovary de Gustave Flaubert, dont les dates de parution s’étendent sur une cinquantaine d’années. Cette période s’apparente, de notre point de vue, à un long itinéraire pour assimiler la notion occidentale des styles direct et indirect, tout en la conciliant avec les particularités du japonais. D’un autre côté, ce travail acharné des traducteurs a influencé quelque peu la langue japonaise. On trouve dans l’annexe tous les textes en japonais examinés.We will examine the difficulties met when translating Western literary texts in Japanese, in particular the problem of indirect and free indirect styles. We will define the grammatical issue and compare seven translations of Madame Bovary, published over a fifty year period. This time can be likened to a long path taken to digest the Western notion of direct and indirect styles, while reconciling it with Japanese language characteristics. On the other hand the translators’ relentless work has somewhat influenced the Japanese language. The annex will present all the Japanese texts examined.

Indirect methods for reference interval determination - review and recommendations.

Science.gov (United States)

Jones, Graham R D; Haeckel, Rainer; Loh, Tze Ping; Sikaris, Ken; Streichert, Thomas; Katayev, Alex; Barth, Julian H; Ozarda, Yesim

2018-04-19

Reference intervals are a vital part of the information supplied by clinical laboratories to support interpretation of numerical pathology results such as are produced in clinical chemistry and hematology laboratories. The traditional method for establishing reference intervals, known as the direct approach, is based on collecting samples from members of a preselected reference population, making the measurements and then determining the intervals. An alternative approach is to perform analysis of results generated as part of routine pathology testing and using appropriate statistical techniques to determine reference intervals. This is known as the indirect approach. This paper from a working group of the International Federation of Clinical Chemistry (IFCC) Committee on Reference Intervals and Decision Limits (C-RIDL) aims to summarize current thinking on indirect approaches to reference intervals. The indirect approach has some major potential advantages compared with direct methods. The processes are faster, cheaper and do not involve patient inconvenience, discomfort or the risks associated with generating new patient health information. Indirect methods also use the same preanalytical and analytical techniques used for patient management and can provide very large numbers for assessment. Limitations to the indirect methods include possible effects of diseased subpopulations on the derived interval. The IFCC C-RIDL aims to encourage the use of indirect methods to establish and verify reference intervals, to promote publication of such intervals with clear explanation of the process used and also to support the development of improved statistical techniques for these studies.

The use of antigen-detection ELISA for the diagnosis of bovine trypanosomosis in Cote d'Ivoire

International Nuclear Information System (INIS)

Kone, P.; Komoin-Oka, C.; N'Depo, A.

1997-01-01

An Antigen ELISA (Ag-ELISA) detecting circulating antigens of trypanosomes was evaluated in the central region of Cote d'Ivoire for the serodiagnosis of cattle trypanosomosis. Of 1423 sera examined, only 43 were positive in the MHCT/BCT, 105 (7%) were detected using stained blood smears, and 74 (5%) were found positive using the Ag-ELISA. The predominant trypanosome species was T. brucei, being present in 84% of the positive samples as detected by the BCT, in 96% using stained bloodsmears, and in 72% by Ag-ELISA. T.vivax was detected less frequently. The serological (ELISA) test did not detect all positive animals as found by the haematological techniques. However, the two techniques should be used in a complementary way to improve the diagnosis of the disease. The results confirm that the prevalence of trypanosomes in cattle is low in the study area. The low prevalence can be due to prophylaxis and therapy of livestock in combination with successful tsetse trapping. (author). 4 refs, 2 figs, 4 tabs

The use of antigen-detection ELISA for the diagnosis of bovine trypanosomosis in Cote d`Ivoire

Energy Technology Data Exchange (ETDEWEB)

Kone, P [Laboratoire Regional de Pathologie Animale de Bouake (LANADA), Bouake (Cote d` Ivoire); Komoin-Oka, C; N` Depo, A [Laboratoire Central de Pathologie Animale de Bingerville (LANADA), Bingerville (Cote d` Ivoire)

1997-02-01

An Antigen ELISA (Ag-ELISA) detecting circulating antigens of trypanosomes was evaluated in the central region of Cote d`Ivoire for the serodiagnosis of cattle trypanosomosis. Of 1423 sera examined, only 43 were positive in the MHCT/BCT, 105 (7%) were detected using stained blood smears, and 74 (5%) were found positive using the Ag-ELISA. The predominant trypanosome species was T. brucei, being present in 84% of the positive samples as detected by the BCT, in 96% using stained bloodsmears, and in 72% by Ag-ELISA. T.vivax was detected less frequently. The serological (ELISA) test did not detect all positive animals as found by the haematological techniques. However, the two techniques should be used in a complementary way to improve the diagnosis of the disease. The results confirm that the prevalence of trypanosomes in cattle is low in the study area. The low prevalence can be due to prophylaxis and therapy of livestock in combination with successful tsetse trapping. (author). 4 refs, 2 figs, 4 tabs.

Language shifts in free indirect discourse

NARCIS (Netherlands)

Maier, Emar

Free indirect discourse is a way of reporting what a protagonist thinks or says that is distinct from both direct and indirect discourse. In particular, while pronouns and tenses are presented from the narrator's perspective, as in indirect discourse, other indexical and expressive elements reflect

Directe en indirecte werknemersparticipatie in Europa

NARCIS (Netherlands)

van Houten, Gijs; Akkerman, Agnes; Sluiter, Roderick; Jansen, Giedo; Vermeylen, Greet

2016-01-01

This study looks at different forms of direct and indirect employee participation in the EU. The research questions are: (1) which forms of direct and indirect employee participation can we distinguish?; (2) to what extent do forms of direct and indirect employee participation coincide within

Indirect reciprocity with trinary reputations.

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Tanabe, Shoma; Suzuki, Hideyuki; Masuda, Naoki

2013-01-21

Indirect reciprocity is a reputation-based mechanism for cooperation in social dilemma situations when individuals do not repeatedly meet. The conditions under which cooperation based on indirect reciprocity occurs have been examined in great details. Most previous theoretical analysis assumed for mathematical tractability that an individual possesses a binary reputation value, i.e., good or bad, which depends on their past actions and other factors. However, in real situations, reputations of individuals may be multiple valued. Another puzzling discrepancy between the theory and experiments is the status of the so-called image scoring, in which cooperation and defection are judged to be good and bad, respectively, independent of other factors. Such an assessment rule is found in behavioral experiments, whereas it is known to be unstable in theory. In the present study, we fill both gaps by analyzing a trinary reputation model. By an exhaustive search, we identify all the cooperative and stable equilibria composed of a homogeneous population or a heterogeneous population containing two types of players. Some results derived for the trinary reputation model are direct extensions of those for the binary model. However, we find that the trinary model allows cooperation under image scoring under some mild conditions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Spatially indirect excitons in coupled quantum wells

Energy Technology Data Exchange (ETDEWEB)

Lai, Chih-Wei Eddy [Univ. of California, Berkeley, CA (United States)

2004-03-01

Microscopic quantum phenomena such as interference or phase coherence between different quantum states are rarely manifest in macroscopic systems due to a lack of significant correlation between different states. An exciton system is one candidate for observation of possible quantum collective effects. In the dilute limit, excitons in semiconductors behave as bosons and are expected to undergo Bose-Einstein condensation (BEC) at a temperature several orders of magnitude higher than for atomic BEC because of their light mass. Furthermore, well-developed modern semiconductor technologies offer flexible manipulations of an exciton system. Realization of BEC in solid-state systems can thus provide new opportunities for macroscopic quantum coherence research. In semiconductor coupled quantum wells (CQW) under across-well static electric field, excitons exist as separately confined electron-hole pairs. These spatially indirect excitons exhibit a radiative recombination time much longer than their thermal relaxation time a unique feature in direct band gap semiconductor based structures. Their mutual repulsive dipole interaction further stabilizes the exciton system at low temperature and screens in-plane disorder more effectively. All these features make indirect excitons in CQW a promising system to search for quantum collective effects. Properties of indirect excitons in CQW have been analyzed and investigated extensively. The experimental results based on time-integrated or time-resolved spatially-resolved photoluminescence (PL) spectroscopy and imaging are reported in two categories. (i) Generic indirect exciton systems: general properties of indirect excitons such as the dependence of exciton energy and lifetime on electric fields and densities were examined. (ii) Quasi-two-dimensional confined exciton systems: highly statistically degenerate exciton systems containing more than tens of thousands of excitons within areas as small as (10 micrometer)² were

Newly established ELISA for N-ERC/mesothelin improves diagnostic accuracy in patients with suspected pleural mesothelioma.

Science.gov (United States)

Sato, Tadashi; Suzuki, Yohei; Mori, Takanori; Maeda, Masahiro; Abe, Masaaki; Hino, Okio; Takahashi, Kazuhisa

2014-10-01

Pleural mesothelioma is an aggressive tumor, commonly caused by exposure to asbestos. The prognosis of mesothelioma remains disappointing despite multimodal treatment. We reported previously that N-ERC/mesothelin could be a useful biomarker for the early diagnosis of pleural mesothelioma and developed an enzyme-linked immunosorbent assay (ELISA) system for its detection. However, the reproducibility of our previous 7-16 ELISA system has been revealed to be unsatisfactory. To measure N-ERC/mesothelin more precisely, we developed a new 7-20 ELISA system. The subjects of this study were patients who were referred to our department with suspected pleural mesothelioma. The current study demonstrated that the newly established 7-20 ELISA system improved the sensitivity and specificity for diagnosing pleural mesothelioma compared with the previous system. Moreover, the 7-20 ELISA system showed better reproducibility and displayed the tendency of both higher sensitivity and higher specificity in plasma than in serum. Particularly for the epithelioid type, the area under the curve (AUC) and the diagnostic accuracy of N-ERC/mesothelin were excellent; the AUC was 0.91, the sensitivity was 0.95, and the specificity was 0.76 in plasma. In conclusion, assessment of N-ERC/mesothelin with our newly established 7-20 ELISA system is clinically useful for the precise diagnosis of pleural mesothelioma. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.