40,474 research outputs found
Evaluation of ELISA and Brucellacapt tests for diagnosis of human Brucellosis
No full textBackground and Objectives: Brucellosis is one of the most common zoonotic diseases in Iran and human brucellosis is endemic in all parts of the country. Because of the difficulty in the diagnosis of brucellosis, particularly in endemic areas, the use of new and feasible diagnostic tests seem to be of great importance for resolving the diagnostic obstacles. We evaluated the usefulness of a new serological test based on an immunocapture-agglutination technique in comparison with ELISA test for serological diagnosis of brucellosis.
Materials and Methods: A total of 11 patients with brucellosis, who had positive blood cultures for Brucella species, and 47 suspected patients were included in this study. Serum samples collected from these patients were tested by brucellacapt and ELISA and the results were, consequently, compared.
Results: In patients with positive blood culture, all the samples gave positive results with brucellacapt test while IgM ELISA, IgG ELISA and (IgG + IgM) ELISA tests were positive in 8, 9 and 11 patients, respectively. Out of the 46 suspected patients, (IgG + IgM) ELISA, Brucellacapt, IgG ELISA and IgM ELISA were positive in 37, 15, 34 and 37 patients, respectively.The best cut-off point of ELISA-IgG was 10.78 IU/ml which produced the maximal sensitivity and specificity for the diagnosis of human brucellosis.
Conclusion: Both the (IgG + IgM) ELISA and Brucellacapt tests demonstrate a high specificity in this study. According to the results of the current study, it is found that both tests are valuable tools for diagnosis of brucellosis in Iran as an endemic area of brucellosis. It is strongly suggested that a combination of both tests to be used for the diagnosis of brucellosis
A membrane-based ELISA assay for the herbicide Isoproturon in soil samples
No full textA 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 an affinity membrane sorbent based ELISA where the isoproturon/ovalbumin conjugate was immobilized on commercial membranes. Different porosities and immobilization conditions were utilized to optimize the MELISA, including sensitivity, selectivity, and stability studies. This enabled detection of isoproturon in the range 0.5ngml-1-20µgml-1, with an LLD90 of 0.5ngml-1. The use of acetonitrile extracts from soil samples was found to not overly impair the performance of the MELISA. Good correlation between ELISA and HPLC was obtained for extracts from spiked soil samples
Differentiation of Foot-and-Mouth Disease-Infected pigs from Vaccinated Pigs Using Antibody-Detecting Sandwich ELISA
No full textThe presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for naïve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificit
Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA - development of a novel assay for vancomycin
No full textA simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop ELISA type assays is presented here for the first time. NanoMIPs were synthesized by a solid phase approach with immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering and electron microscopy. Immobilization, blocking and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a HRP-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was three orders of magnitude better than a previously described ELISA based on antibodies. In these experiments nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELIS
Utilizzo delle TIC nella ricerca linguistica sull’arabo scritto contemporaneo: il sistema ipotetico
No full textCon il presente articolo, ci proponiamo di contribuire allo studio dei sistemi condizionali della lingua araba cosiddetta fuṣḥā (“eloquentissima”, “purissima”) scritta contemporanea. Allo scopo di presentarne una descrizione maggiormente aderente alla situazione attuale, il nostro studio prende in esame l’utilizzo scritto della fuṣḥā contemporanea nel web. Attraverso l’applicazione delle TIC all’analisi di corpora digitali è stato possibile confrontare un elevato numero di dati linguistici estrapolati da una selezione di blog e forum arabi di estrema attualità. In ragione della complessità dei sistemi condizionali della lingua araba, la nostra ricerca si concentra essenzialmente sul sistema condizionale potenziale introdotto dall’operatore condizionale ʼin “se”, le cui occorrenze sono state estrapolate dal corpus attraverso l’estrattore terminologico da noi utilizzato, il programma AntConc. Con lo scopo di chiarire il significato socio-pragmatico delle scelte linguistiche evidenziate, la lista delle concordanze è stata analizzata anche in termini sociolinguistici. Completa l’analisi una presentazione diacronica degli studi dei principali grammatici e linguisti arabi, nonché di linguisti occidentali, sull’argomento. Il risultato è il reperimento di nuovi dati estremamente significativi, che in parte contraddicono e in parte sviluppano ulteriormente quanto riportato dalla letteratura scientifica di settore.By the present article we aim at contributing to the study of conditional systems in the so-called fuṣḥā (“most pure”, “most eloquent”) contemporary written Arabic. In the attempt to provide a more faithful portrayal of nowadays situation of written fuṣḥā, our study focuses on the contemporary use of written fuṣḥā on the web. Through the application of ICT to corpus-based analysis we have been able to compare a large amount of linguistic data extracted from a selection of Arab blogs and forums dealing with some very topical issues. Given the complexity of conditional systems in the Arabic language, our research only focuses on the conditional hypothetical system introduced by the conditional operator ’in “if”, whose concordances have been extrapolated by means of a term extractor, the freeware AntConc software in our case. In order to fully appreciate the socio-pragmatic implications of the linguistic choices found in our corpus, these have been analyzed also in sociolinguistic terms. Our analysis comes complete with a diachronic presentation of the most important studies on the topic by the main Arab grammarians and linguists, as well as by western scholars in Arabic linguistics. The results of our research present us with extremely interesting new data that partially contradict and partially further develop what has so far been outlined in the scientific literature in the field
Applying a kinetic method to an indirect ELISA measuring Ostertagia ostertagi antibodies in milk
No full textIndirect enzyme-linked immunosorbent assays (ELISAs) are frequently run as endpoint ELISAs (e-ELISAs). However, kinetic ELISAs (k-ELISAs) have certain advantages over e-ELISAs. The objective of this study was to understand the relationship between e-ELISA and k-ELISA results. Specifically, to determine whether it was possible to run both k-ELISA and e-ELISA on the same plate and establish an appropriate time interval for k-ELISA measurements. A normalization method for k-ELISA slopes (slope ratio) is proposed. Using an indirect e-ELISA test measuring antibodies against Ostertagia ostertagi in milk from dairy cattle, we found that running a k-ELISA had no effect on optical density ratio results of an e-ELISA on the same plate, and that agreement was very strong at 10, 15, and 28 min, allowing for a reduction in the total processing time for ELISA tests
Translation of C-ELISA-HRP to the HTS platform.
No full text(A) Europium conjugated secondary antibodies can be used for the cell-based assay. C-ELISA was performed with Europium conjugated rabbit IgG at different dilutions. The error bars represent the ± SEM of three wells in one experiment (*Z’ factor > 0.5). (B, C) The washing steps were done either manually (B) or with an automated wash system (C). The error bars represent the ± SEM of three wells in one experiment (* Z’ factor > 0.5). (D) C-ELISA-Eu detects the increase in α-tubulin nitrotyrosination with time. The error bars represent the ± SEM of three wells in one experiment (*Z’ factor > 0.5). (E, F) Washing steps with PBS can be replaced with the DelphiaTM buffers used for the C_ELISA-Eu. (E) Washing steps before the addition of rabbit IgG were done with PBS, and DELPHIATM buffer was used thereafter. (F) All the washing steps were performed with DELPHIATM buffer. The error bars represent the ± SEM of three wells in one experiment. The y-axis represents the time-resolved fluorescence values at emission/excitation wave lengths of 340/615 nm. The symbol * indicates Z’ factors > 0.5. (TIF)</p
A Leishmania infantum FML-ELISA for the Detection of Symptomatic and Asymptomatic Canine Visceral Leishmaniasis in an Endemic Area of Iran
No full textBackground: Visceral leishmaniasis (VL) is caused by Leishmania infantum in
Mediterranean basin and is an endemic disease in some parts of Iran. Canines are the main
reservoirs of VL in most of the endemic areas. Different serological methods have been
introduced for diagnosis of canine visceral leishmaniasis (CVL). Objective: In this survey a
Fucose-Mannose Ligand (FML) ELISA, using native L. infantum antigen, was developed and
its validity for detection of infected dogs in comparison with direct agglutination test (DAT)
and PCR was evaluated. Methods: Blood samples of sixty ownership dogs (≤ 3 years old)
were collected from Meshkin-shahr district in Ardabil province, North-west of Iran. Sera
were separated for serological assays (DAT and FML-ELISA) and the buffy coats were
collected for molecular evaluation. Results: Two out of the 60 (3.33%) samples were found
to be positive (antibody titer of ≥ 1/320) in DAT while seven of the 60 (11.66%) samples
were positive by FML-ELISA. Nine out of 60 (15%) buffy coat samples showed a band about
680 bp indicative of L. infantum in PCR. Three out of 60 dogs had Kala-azar symptoms and
were positive by PCR and FML-ELISA, while two of these three dogs had antibody titers
>1/320 in their serum samples. The sensitivity and specificity of FML-ELISA for the
detection of CVL in both symptomatic and asymptomatic dogs were found to be 77.8% and
100%, respectively. Conclusion: Considering the acceptable sensitivity and high specificity
of FML-ELISA, use of this serological method can be recommended for epidemiological
surveys of CVL
The Development of ELISA and SPR-Based Immunoassays for the Detection of Heat Shock Proteins.
No full textHeat shock proteins (Hsps) are highly conserved molecules in all eukaryotes and prokaryotes. They are named on the basis of their molecular weight and their synthesis is up-regulated by stress conditions such as inflammation, oxidative stress and exposure to high temperature. They function by assisting the folding of newly synthesized proteins and thus prevent aggregation or damage to other cellular components. Beside the role in intracellular protein folding, heat shock proteins can particularly act as intercellular signals with a wide variety of biological effects, such as to stimulate cells to produce proinflammatory cytokines, like TNFα, and other proteins involved in immunity and inflammation, or they are also thought to be involved in the pathogenesis of a wide range of diseases such as diabetes mellitus and cardiovascular disease. Thus, screening for aberrant expression of this protein could be an easy and useful tool to detect at risk individuals for developing and those more susceptible towards developing these diseases.
Currently, the Enzyme-Linked ImmunoSorbent Assay (ELISA) is the main immunoassay method used for the measurement of heat shock proteins levels in both clinical and research laboratories. It is relatively more rapid and sensitive than RadioImmunoassay (RIA), and most importantly, it is safer because enzymes are used as labels instead of harmful radioactive substances. However, a newly developed biosensor method, the Surface Plasmon Resonance (SPR), offers a number of advantageous over ELISA. For example, it is much more simple and rapid because a lot of steps can be set up automatically and no labels are required for SPR immunoassays.
In this study, a comparison of different types of ELISA assays was made. The results showed that the Sandwich format of ELISA is much more sensitive than Indirect ELISA for detection of the concentrations of Heat Shock Protein 70 (Hsp70), and this sensitivity can be further improved by applying an Avidin-Biotin system together with Sandwich ELISA under certain conditions.
To develop the SPR protocol for the detection of heat shock protein concentrations, two Hsp70 binding curves using different assay formats, the Sandwich SPR immunoassay and the Competitive SPR immunoassay, were set up by using CM 5 sensor chip. Another sensor chip, the mixed Self-Assembled Monolayer (mSAM), was also examined using Sandwich SPR format. No subsequent experimental steps were carried on for SPR studies since the regeneration conditions of these tests for both sensor chips need to be well studied. Thus, this study suggests that in order to set up a SPR immunoassay protocol that is a better choice for the detection of heat shock protein levels in complex matrixes than ELISA, experimental conditions, such as the choice of regeneration buffer and the duration of regeneration cycle, need to be well optimized
A collaborative study on larval excretory/secretory antigens of Toxocara canis for the immunodiagnosis of human toxocariasis with ELISA.
No full textTwo batches of excretory/secretory (E/S) antigens from second stage larvae of Toxocara canis maintained in vitro were prepared independently in two different laboratories (Zürich and Basel) and analysed in order to obtain information for future efforts to standardize the enzyme-linked immunosorbent assay (ELISA) used for the serodiagnosis of human toxocariasis. SDS-PAGE and "Western-blotting" revealed at least 10 different antigenic components common to the two antigen preparations. However, distinct qualitative and quantitative differences among the two E/S-antigens were observed, since one antigen had a more complex composition than the other. Despite these differences, an accordance of serodiagnosis was obtained in 80% of 25 sera from patients with suspected Toxocara infection tested independently in two different ELISA systems (Basel and Zürich) with the corresponding E/S-antigens. The specificity was 93% as determined (BS-antigen, BS-ELISA) by testing 46 out of 3396 sera from patients with parasitologically proven extra-intestinal helminthic infections. Cross-reactions occurred mainly with sera from patients infected with filariae (5 from 13 cases) exhibiting very high extinction values in their homologous ELISA-system. The reproducibility (intra- and inter-test variations) of two ELISA systems using the corresponding E/S-antigens varied from 5-15%. The results demonstrate that T. canis E/S-antigens may well be applicable for standardization of the ELISA used for the serodiagnosis of human toxocariasis
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