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Sample records for enzyme-linked immunoelectrotransfer blot

  1. Antibody Banding Patterns of the Enzyme-Linked Immunoelectrotransfer Blot and Brain Imaging Findings in Patients With Neurocysticercosis.

    Science.gov (United States)

    Arroyo, Gianfranco; Rodriguez, Silvia; Lescano, Andres G; Alroy, Karen A; Bustos, Javier A; Santivañez, Saul; Gonzales, Isidro; Saavedra, Herbert; Pretell, E Javier; Gonzalez, Armando E; Gilman, Robert H; Tsang, Victor C W; Garcia, Hector H

    2018-01-06

    The enzyme-linked immunoelectrotransfer blot (EITB) assay is the reference serological test for neurocysticercosis (NCC). A positive result on EITB does not always correlate with the presence of active infections in the central nervous system (CNS), and patients with a single viable brain cyst may be EITB negative. Nonetheless, EITB antibody banding patterns appears to be related with the expression of 3 protein families of Taenia solium, and in turn with the characteristics of NCC in the CNS (type, stage, and burden of viable cysts). We evaluated EITB antibody banding patterns and brain imaging findings of 548 NCC cases. Similar banding patterns were grouped into homogeneous classes using latent class analysis. The association between classes and brain imaging findings was assessed. Four classes were identified. Class 1 (patients negative or only positive to the GP50 band, related to the protein family of the same name) was associated with nonviable or single viable parenchymal cysticerci; class 2 (patients positive to bands GP42-39 and GP24, related to the T24-42 protein family, with or without anti-GP50 antibodies) was associated with intraparenchymal viable and nonviable infections; classes 3 and 4 (positive to GP50, GP42-39, and GP24 but also responding to low molecular weight bands GP21, GP18, GP14, and GP13, related to the 8 kDa protein family) were associated with extraparenchymal and intraparenchymal multiple viable cysticerci. EITB antibody banding patterns correlate with brain imaging findings and complement imaging information for the diagnosis of NCC and for staging NCC patients. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  2. Detection of early antibodies in human immunodeficiency virus infection by enzyme-linked immunosorbent assay, Western blot, and radioimmunoprecipitation.

    OpenAIRE

    Saah, A J; Farzadegan, H; Fox, R; Nishanian, P; Rinaldo, C R; Phair, J P; Fahey, J L; Lee, T H; Polk, B F

    1987-01-01

    A current concept of the serological response to human immunodeficiency virus (HIV) infection in humans is that antibodies to core antigens (p55, p24, and p15) are detectable earlier during initial stages of antibody production than antibodies against envelope antigens (gp160, gp120, and gp41). Comparative studies of Western blot (immunoblot), radioimmunoprecipitation assay (RIPA), and enzyme-linked immunosorbent assay (ELISA) during initial antibody production are limited to case reports and...

  3. Comparative assessment of enzyme-linked immunosorbent assay and Western blot for the diagnosis of toxocariasis in patients with skin disorders.

    Science.gov (United States)

    Bellanger, A-P; Humbert, P; Gavignet, B; Deschaseaux, A D; Barisien, C; Roussel, S; Millon, L; Aubin, F; Piarroux, R

    2010-01-01

    Background The link between various chronic skin disorders and toxocariasis was previously demonstrated by case reports and several case-control studies. However, these previous studies were based only on the Toxocara canis excretory-secretory-enzyme-linked immunosorbent assay (TES-ELISA) serological technique, which is not specific due to cross-reactivity with parasites of the genera Anisakis or Ascaris. Immunoblot analysis is highly specific and can detect very low levels of Toxocara antibodies. Therefore, this technique may be useful in the identification of Toxocara infection in patients with chronic skin disorders. Objectives Because urticaria and pruritus/prurigo are skin conditions previously associated with toxocariasis, we carried out a prospective study using both TES-ELISA and Toxocara Western blot on 113 patients with either chronic urticaria (n = 84) or chronic pruritus (n = 29). Methods Patients were matched with controls according to gender, age and residence location (rural or urban area). Data were analysed using a Mantel-Haenszel chi(2) test. Results The proportion of positive TES-ELISA results was not significantly different for patients with chronic skin disorders (urticaria or pruritus/prurigo) from that of control subjects. However, the proportion of positive immunoblot results was significantly higher for patients with chronic urticaria than for control subjects (P = 0.009). Conclusions Our study demonstrates the need to perform Western blotting immunodiagnosis, whatever the TES-ELISA result, to improve diagnosis of human toxocariasis in patients with chronic urticaria caused by Toxocara infection.

  4. The Dot Blot ELISA.

    Science.gov (United States)

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  5. Evaluation of an immunodot blot technique for the detection of antibodies against Taenia solium larval antigens.

    Science.gov (United States)

    Salazar-Anton, Fernando; Tellez, Aleyda; Lindh, Johan

    2012-06-01

    Immunodiagnostic tests represent an important tool for diagnosis of cysticercosis, the disease caused by cysticerci of Taenia solium. Accurate diagnosis of neurocysticercosis (NCC) requires costly neuroimaging techniques (magnetic resonance imaging and computed tomography), which are seldom affordable for people in endemic countries. Hence, new low-cost diagnostic methods offering good sensitivity and specificity are needed. Here, we studied four immunodiagnostic tests immunodot blot Tsol-p27, a commercial ELISA, and Western blot Tsol-p27/TsolHSP36, and compared them with a commercial enzyme-linked immunoelectrotransfer blot (EITB) that we regarded as the gold standard method. The analyzed serum samples were obtained from 160 patients: 94 epileptics suspected of NCC, six individuals confirmed NCC-positive, and 60 with positive (30) or negative (30) serology for Chagas diseases. Of the 100 serum samples from epileptic patients, 13 were positive and 87 negative by EITB. Compared to Western blot Tsol-p27, immunodot blot Tsol-p27 offered similar specificity (97.8% vs. 95.6%) but better sensitivity (86.7% vs. 76.4%). The ELISA was similar to the immunodot blot Tsol-p27 regarding both sensitivity and specificity. Western blot TsolHSP36 provided the lowest sensitivity (61.9%) and specificity (86.1%). None of the antibodies in the serum samples from the Chagas control groups were recognized by immunodot blot Tsol-p27. Our results indicate that the immunodot blot Tsol-p27 provides good sensitivity and specificity. Furthermore, considering the simplicity and low cost of this test, it might be preferable as a diagnostic method in poorly equipped laboratories in endemic countries.

  6. [Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens].

    Science.gov (United States)

    Escalante, Hermes; Davelois, Kelly; Ortiz, Pedro; Rodríguez, Hans; Díaz, Enrique; Jara, César

    2011-01-01

    To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot) using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag) for the diagnosis of human fasciolosis. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the finding of parasite eggs in the stool microscopy). Antigens of 10, 12, 17, 23, 27, 30, 36, 43, 66 and 136 kDa were detected and used to develop the Western blot technique. The sensitivity was evaluated using sera from 67 fasciolosis patients, and the specificity using sera from 57 patients with other parasitic diseases, and 10 from healthy individuals. Out of the 67 sera, 64 reacted with the 23 kDa band and 61 with the one of 17 kDa. These two bands were not detected in sera from patients with other parasitic diseases or in those from healthy volunteers and thus could be considered specific and diagnostic. The sensitivity of the test, using the bands of 17 and 23 kDa, was 95.5% for positive reactions to at least one of these two bands, being its specificity 100% with a positive predictive value of 100% and negative predictive value of 95.71%.

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

    African Journals Online (AJOL)

    Determination of histamine in Iranian cheese using enzyme-linked immunosorbent assay (ELISA) method. Mojtaba Rashedi, Mohsen Panahi Dorcheh, Mohammad Salajegheh, Amin Mohammadi, Mohammad Reza Hajimirzaei, Ebrahim Rahimi ...

  8. Comparative application of antigen detection enzyme-linked ...

    African Journals Online (AJOL)

    Antigen-detection enzyme-linked immunosorbent assay (Ag-ELISA) and buffy coat parasitological technique (BCT) were employed for the diagnosis of bovine trypanosomiosis in trade cattle slaughtered at the Bodija Municipal abattoir in Ibadan, Oyo State, between March and November, 2002 and in some sedentary herds ...

  9. Designing of enzyme linked immunosorbent assay (ELISA) kit for ...

    African Journals Online (AJOL)

    The sensitivity of microscopic examination of fecal samples to recognize Giardia parasites is low. In the methods based on antigen scanning of parasites such as enzyme linked immunosorbent assay (ELISA), copro-antigens of parasite will be traced and diagnosed even if the live parasite is absent in the fecal samples.

  10. Comparisons of competitive enzyme-linked immunosorbent assay ...

    African Journals Online (AJOL)

    The aim of this study was to comprise competitive enzyme-linked immunosorbent assay (C-ELISA) with one step RT-PCR test for the detection of BTV in sheep. A total of 770 blood samples were obtained from sheep (265 serum positive samples and 505 serum negative samples in C-ELISA). According to our data, out of ...

  11. Development of an indirect enzyme-linked immunosorbent assay ...

    African Journals Online (AJOL)

    By removing the N-terminal hydrophobic sequence, truncated VP2 (tVP2) genes were cloned into the pET-32a (+) plasmid and subsequently expressed as His fusion proteins. The purified recombinant tVP2 proteins were specific to canine parvovirus (CPV), and one of them was used in an indirect enzyme-linked ...

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

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    Determination of histamine in Iranian cheese using enzyme-linked immunosorbent assay (ELISA) method. Mojtaba Rashedi1*, Mohsen Panahi Dorcheh2, Mohammad Salajegheh2, Amin Mohammadi2,. Mohammad Reza Hajimirzaei2 and Ebrahim Rahimi1,3. 1Young Researchers Club, Faculty of Veterinary Medicine, ...

  14. A competitive enzyme linked immunosorbent assay for the ...

    African Journals Online (AJOL)

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

  15. Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Kizek, René; Havran, Luděk; Billová, Sabina; Fojta, Miroslav

    2002-01-01

    Roč. 469, č. 1 (2002), s. 73-83 ISSN 0003-2670 R&D Projects: GA ČR GV204/97/K084; GA ČR GA204/00/D049 Institutional research plan: CEZ:AV0Z5004920 Keywords : electrochemical sensor for DNA hybridization * enzyme-linked immunoassay of target DNA * magnetic beads with attached DNA probe Subject RIV: BO - Biophysics Impact factor: 2.114, year: 2002

  16. Smartphone instrument for portable enzyme-linked immunosorbent assays

    OpenAIRE

    Long, Kenneth D.; Yu, Hojeong; Cunningham, Brian T.

    2014-01-01

    We demonstrate the utilization of a smartphone camera as a spectrometer that is capable of measuring Enzyme Linked Immunosorbent Assays (ELISA) at biologically-relevant concentrations with the aid of a custom cradle that aligns a diffraction grating and a collimating lens between a light source and the imaging sensor. Two example biomarkers are assayed using conventional ELISA protocols: IL-6, a protein used diagnostically for several types of cancer, and Ara h 1, one of the principle peanut ...

  17. Comparison of T24H-his, GST-T24H and GST-Ts8B2 recombinant antigens in western blot, ELISA and multiplex bead-based assay for diagnosis of neurocysticercosis.

    Science.gov (United States)

    Hernández-González, Ana; Noh, John; Perteguer, María Jesús; Gárate, Teresa; Handali, Sukwan

    2017-05-15

    Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium. Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA

  18. Enzyme-linked immunosorbent assays in murine cryptococcosis.

    Science.gov (United States)

    Scott, E N; Muchmore, H G; Felton, F G

    1981-12-01

    In this investigation, enzyme-linked immunosorbent assay (ElISA) procedures were used to study the time of appearance and the duration of demonstrable antigen and antibody in body fluids of mice with disseminated cryptococcosis. The ELISA antigen procedure detected cryptococcal capsular polysaccharide (CCP) in the serum and urine of infected mice 3 days after infection--4 days before it could be demonstrated by the latex agglutination procedure. ELISA-reactive antibody was present throughout the course of infection (mean death time, 32 days), whereas antibody was not detected by whole cell agglutination after day 20. High serum concentrations of CCP (titers to 64,000) persisted throughout the course of infection, while antibody declined to low levels with progression of disease. ELISA provides a sensitive system for quantitation and monitoring of antigen (CCP) processing and clearance (or storage), and for cryptococcal antibody formation in progressive cryptococcosis.

  19. Smartphone instrument for portable enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Long, Kenneth D; Yu, Hojeong; Cunningham, Brian T

    2014-11-01

    We demonstrate the utilization of a smartphone camera as a spectrometer that is capable of measuring Enzyme Linked Immunosorbent Assays (ELISA) at biologically-relevant concentrations with the aid of a custom cradle that aligns a diffraction grating and a collimating lens between a light source and the imaging sensor. Two example biomarkers are assayed using conventional ELISA protocols: IL-6, a protein used diagnostically for several types of cancer, and Ara h 1, one of the principle peanut allergens. In addition to the demonstration of limits of detection at medically-relevant concentrations, a screening of various cookies was completed to measure levels of peanut cross-contamination in local bakeries. The results demonstrate the utility of the instrument for quantitatively performing broad classes of homogeneous colorimetric assays, in which the endpoint readout is the color change of a liquid sample.

  20. Protein blotting with direct blotting electrophoresis.

    Science.gov (United States)

    Beck, S

    1988-05-01

    Direct blotting electrophoresis, a method designed to be of general application for the separation and electroblotting of macromolecules, has been adapted to produce protein blots suitable for subsequent processing by standard techniques such as dye staining or immunological detection. After their separation in a very short gel the protein bands are electrophoresed out of the gel onto an immobilizing matrix. The matrix which is moved across the bottom of the gel by a conveyor belt binds these proteins with high affinity. Once the protein samples have been loaded onto the gel and electrophoresis has been started, no further intervention is needed until the blot is completed. The total expenditure of time for such a direct blot is less than 4 h for a mixture of proteins in the molecular weight range of 14-70 kDa. The staining sensitivity of directly blotted proteins is about 200 ng protein per band as revealed by India ink staining.

  1. Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

    Science.gov (United States)

    Liu, M T; Ram, B P; Hart, L P; Pestka, J J

    1985-01-01

    A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples. PMID:2932054

  2. Development of an enzyme-linked immunosorbent assay for toosendanin.

    Science.gov (United States)

    Zhang, Jing; Feng, Gang; Luo, Li; Yu, Xiang Yang; Ma, Zhi Qing; Feng, Jun Tao; Liu, Xian Jin; Zhang, Xing

    2008-08-01

    Enzyme-linked immunosorbent assays (ELISAs) were developed by using polyclonal antibody for toosendanin (TSN), a biopesticide from Melai toosendan Sieb. et Zucc. Their application in the determination of this analyte in spiked cabbage, tomato and apple samples was studied. The haptens, 28-hemisuccinyl-TSN (TSN-S) and 28-hemiglutaryl-TSN (TSN-G) were synthesized by using esterification. Immunogen and coating antigen were synthesized by using the mixed anhydride reaction and active ester protocol, respectively. Rabbits were immunized with TSN-G-BSA and TSN-S-BSA. Using the selected antibody and coating antigen, an indirect competitive ELISA for TSN was developed, which showed an IC(50) value of 1.023 microg mL(-1), with a detection limit of 0.009 microg mL(-1). A direct competitive ELISA using an enzyme tracer was also developed. The assay showed an IC(50) value of 0.840 microg mL(-1) with a detection limit of 0.014 microg mL(-1). Both assays displayed high cross-reactivity to a closely structurally related compound. Recoveries of TSN from both immunoassays of fortified samples ranged from 76.4% to 113.2% and 75.1% to 132.3%, respectively. Linear regression analysis showed good correlation between the TSN concentrations derived from ELISA and HPLC analyses, which suggested that the ELISA is a convenient supplementary analytical tool for monitoring TSN.

  3. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membrane...... the gap to the more laborious nuclease protection experiments....

  4. The western blot

    Science.gov (United States)

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

  5. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membran...

  6. Microfluidic Western blotting.

    Science.gov (United States)

    Hughes, Alex J; Herr, Amy E

    2012-12-26

    Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10-60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.

  7. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

    Directory of Open Access Journals (Sweden)

    Hermes Escalante

    2011-09-01

    Full Text Available Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 % cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 % con un valor predictivo positivo de 100 % y un valor predictivo negativo de 95,71 %.Objectives. To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag for the diagnosis of human fasciolosis. Materials and methods. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the

  8. Western blotting: an introduction.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.

  9. Detection by enzyme-linked immunosorbent assay of Aujeszky's disease virus in tissues of infected pigs.

    OpenAIRE

    Qvist, P; Meyling, A; Hoff-Jørgensen, R

    1990-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of Aujeszky's disease virus antigen in tissue extracts and in nasal swabs. The enzyme-linked immunosorbent assay is based on two different monoclonal antibodies with specificity for the gII glycoprotein of Aujeszky's disease virus. Viral antigen was detected in 81 of 93 tissue extracts prepared from virus-infected organs. Fifteen outbreaks of Aujeszky's disease were analyzed in this study, and they were all identified by the...

  10. The Western Blot.

    Science.gov (United States)

    Hnasko, Thomas S; Hnasko, Robert M

    2015-01-01

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semiquantitative identification of specific proteins and their molecular weight from a complex mixture. This chapter will outline the requisite steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen.

  11. Western blotting is useful in the salivary diagnosis of Helicobacter pylori infection

    OpenAIRE

    Ballam, L; Mendall, M; Asante, M; Morris, J; Strachan, D; Whincup, P; Cook, D

    2000-01-01

    Background—The salivary diagnosis of Helicobacter pylori infection offers attractive possibilities for the epidemiological study of infection in children. Salivary enzyme linked immunosorbent assay (ELISA) is less reliable then serum ELISA, owing to variable transudation of immunoglobulin. In addition, children are more difficult to study because of lower specific serum antibody concentrations to H pylori. The performance of salivary western blotting in comparison with serum western blotting ...

  12. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing...

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

    DEFF Research Database (Denmark)

    Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten

    2014-01-01

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

  14. Western Blot Techniques.

    Science.gov (United States)

    Kim, Brianna

    2017-01-01

    The Western blot is an important laboratory technique that allows for specific identification and characterization of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins are electophoretically transferred to a polyvinylidene fluoride (PVDF) membrane which is then incubated with specific antibodies, then developed to show the protein of interest. Here, we describe the transfer and detection of Outer surface protein A (OspA), a protein only found on the surface of Borrelia burgdorferi, the bacteria responsible for Lyme disease.

  15. Influence of Anti-FloR Antibody on Florfenicol Accumulation in Florfenicol-Resistant Escherichia coli and Enzyme-Linked Immunosorbent Assay for Detection of Florfenicol-Resistant E. coli Isolates

    OpenAIRE

    Wu, Beibei; Xia, Chun; Du, Xiangdang; Cao, Xingyuan; Shen, Jianzhong

    2006-01-01

    To detect florfenicol-resistant Escherichia coli isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione S-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromato...

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    An enzyme-linked immunosorbent assay for quantification of haptoglobin in porcine serum was evaluated. Tbe detection limit when expressed as the estimated concentration of a blank sample was 0.0003 mg/ml. The precision of the assay was acceptable with intra-assay coefficients of variation below 4...

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

    Science.gov (United States)

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

  18. The Use of Competitive Enzyme Linked Immuno-Sorbent Assay in ...

    African Journals Online (AJOL)

    A total of 500 lung tissues and sera samples of cattle from CBPP endemic areas was used in this study to determine the effectiveness of combining abattoir survey with competitive Enzyme Linked Immunosorbent Assay (cELISA) as an alternative to Complement Fixation Test (CFT) in providing a better and more reliable ...

  19. Comparison of five commercial anti-tetanus toxoid immunoglobulin G enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Perry, A L; Hayes, A J; Cox, H A; Alcock, F; Parker, A R

    2009-12-01

    Five commercially available enzyme-linked immunosorbent assays for the measurement of anti-tetanus toxoid immunoglobulin G (IgG) antibodies were evaluated for performance. The data suggest that there are manufacturer-dependent differences in sensitivity and accuracy for the determination of tetanus toxoid IgG antibodies that could result in different diagnostic interpretations.

  20. Diagnostic Potential of an Enzyme-Linked Immunospot Assay in Tuberculous Pericarditis

    NARCIS (Netherlands)

    Bathoorn, E.; Limburg, A.; Bouwman, J. J.; Bossink, A. W.; Thijsen, S. F.

    Tuberculous pericarditis is a rare disease in developed countries. The diagnosis is difficult to set since there are no robust rapid tests, and culture of pericardial fluid for Mycobacterium tuberculosis is often negative. T-SPOT. TB, an enzyme-linked immunospot (ELISPOT) test, measures the gamma

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

    Science.gov (United States)

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

    2014-12-01

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

  2. Lectin-Array Blotting.

    Science.gov (United States)

    Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

    2017-09-01

    Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  3. Variations in Western blot banding patterns of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.

    OpenAIRE

    Burke, D S; Redfield, R R; Putman, P; Alexander, S S

    1987-01-01

    Serum samples from 27 patients infected with human T-cell lymphotropic virus type III (14 with acquired immune deficiency syndrome [AIDS] and 13 with AIDS-related complex) were examined for antibodies to viral proteins by the Western blot method and with four different commercial solid-phase enzyme-linked immunosorbent assays (ELISAs). Virus-specific bands on blots at molecular masses of 64, 55, 53, 41, 31, 24, and 17 kilodaltons were observed. Rank correlation matrices were calculated to rel...

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

    Science.gov (United States)

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

    2015-08-10

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

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

    DEFF Research Database (Denmark)

    Bergmann, S M; Wang, Q; Zeng, W

    2017-01-01

    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....

  6. TLC blot (far-eastern blot) and its applications.

    Science.gov (United States)

    Taki, Takao; Gonzalez, Tania Valdes; Goto-Inoue, Naoko; Hayasaka, Takahiro; Setou, Mitsutoshi

    2009-01-01

    A simple method for transfer of lipids including phospholipids, glycolipids, and neutral lipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, called TLC blot (far-eastern blot), is presented. Lipids separated on a HPTLC plate are blotted quantitatively. This procedure made it possible to purify individual lipids from a blotted membrane in a short time. Binding study, immunodetection, and mass spectrometric analysis are available for PVDF membrane. Furthermore, the world of molecular species imaging is opened by a scanning analysis with a combination of TLC blot and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (TLC-Blot/MALDI-TOF MS).

  7. Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-04-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

  8. Western blotting using capillary electrophoresis.

    Science.gov (United States)

    Anderson, Gwendolyn J; M Cipolla, Cynthia; Kennedy, Robert T

    2011-02-15

    A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

  9. Valid application of western blotting.

    Science.gov (United States)

    Wu, Liuji; Hu, Xiuli; Tang, Haitao; Han, Zanping; Chen, Yanhui

    2014-05-01

    Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. However, the systematic errors in the application of western blotting analysis are frequently to be found, which may compromise the interpretation of results. To make a valid application of western blotting, it is essential to begin with three independent biological replicates. Subsequently, a more reliable normalization method is in urgent need for western blotting analysis and using reference proteins is the currently preferred method of normalization. Additionally, identification of valid reference proteins is crucial for western blotting analysis and it should be examined carefully in relation to the cell or tissue types when using housekeeping proteins as internal standards.

  10. Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein▿

    OpenAIRE

    Nimmagadda, Sridevi V.; Aavula, Shukra M.; Biradhar, Neelakantam; Rao, Varaprasada Sankarasetty; Shanmugham, Rajalakshmi; Chandran, Dev; Thirumeni, Nagarajan; Singanallur, Nagendrakumar Balasubramanian; Villuppanoor, Srinivasan Alwar

    2010-01-01

    The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (VH) and light chain (VL) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human × mouse heterohybridoma (human MAb R16E5) was...

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  12. An overview of Western blotting for determining antibody specificities for immunohistochemistry.

    Science.gov (United States)

    Kurien, Biji T; Dorri, Yaser; Dillon, Skyler; Dsouza, Anil; Scofield, R Hal

    2011-01-01

    Despite its overall simplicity, protein blotting or Western blotting has been proven to be a powerful procedure for the immunodetection of proteins, especially those that are of low abundance, following electrophoresis. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly since its inception and researchers have a variety of ways and means to carry out this transfer. This procedure is used in combination with other important antibody-based detection methods such as enzyme-linked immunosorbant assay and immunohistochemistry to provide confirmation of results both in research and diagnostic testing. Specificity of antibodies used for immunohistochemistry is of critical importance and therefore Western blot is a "must" to address antibodies' specificity.

  13. Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

    Science.gov (United States)

    Hamm, Melissa; Ha, Sha; Rustandi, Richard R

    2015-06-01

    Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2017-03-15

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

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

    Directory of Open Access Journals (Sweden)

    A. M. Marassá

    2008-01-01

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

  16. Antibody Validation by Western Blotting.

    Science.gov (United States)

    Signore, Michele; Manganelli, Valeria; Hodge, Alex

    2017-01-01

    Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

  17. Single-cell western blotting.

    Science.gov (United States)

    Quadri, Syed M S

    2015-01-01

    Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.

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

    Science.gov (United States)

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

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  19. An enzyme-linked immunosorbent assay for the convenient serodiagnosis of contagious equine metritis in mares.

    Science.gov (United States)

    Katz, J; Geer, P

    2001-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of contagious equine metritis (CEM), a sexually transmitted disease caused by Taylorella equigenitalis. Antigen preparation was simple, and antigens derived from both classical and atypical forms of T. equigenitalis enabled detection of antibody responses elicted in horses experimentally exposed to either form of the bacterium. Sera serially obtained from these horses from 0 to 63 days postexposure were tested by the traditional complement fixation test (CFT) for CEM and with the ELISA, using both antigens separately. There was close agreement between CFT and ELISA methodologies during the postexposure time period used to detect CEM serodiagnostically in regulatory animal health testing programs. Unlike the CFT, which requires an overnight incubation step, the ELISAs are more convenient and can be completed in 3 hours.

  20. Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Borre, M B; Jepsen, S

    1991-01-01

    A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P....... falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant...... New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control...

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-03-01

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

  3. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Battelli, M G; Abbondanza, A; Musiani, S; Buonamici, L; Strocchi, P; Tazzari, P L; Gramantieri, L; Stirpe, F

    1999-03-01

    Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

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

    International Nuclear Information System (INIS)

    Chembe, E.

    1985-01-01

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

  5. Western blotting using chemiluminescent substrates.

    Science.gov (United States)

    Alegria-Schaffer, Alice

    2014-01-01

    Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Comparative study of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test for the diagnosis of pemphigus.

    Science.gov (United States)

    Zhou, Tingting; Fang, Siyue; Li, Chunlei; Hua, Hong

    2016-11-01

    Pemphigus is one of the potentially fatal autoimmune blistering diseases. An early and accurate diagnosis is important for prognosis and therapy. It may be difficult to diagnosis based on clinical grounds alone. Direct and indirect immunofluorescence, enzyme-linked immunosorbent assay, the Tzanck smear test, or histopathology are all available for the diagnosis of pemphigus. However, there are no generally accepted diagnostic criteria for the diagnosis of this condition at present. To evaluate the diagnostic value of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test for the diagnosis of pemphigus in dental clinics. A single-center retrospective study was conducted, and the clinical data of 33 patients with pemphigus and 61 controls were collected and analyzed from the Department of Oral Medicine, Peking University School of Stomatology, during 2010-2014. The sensitivities and specificities of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test were calculated and compared in two groups. Sensitivities for the Tzanck smear test, indirect immunofluorescence, and enzyme-linked immunosorbent assay were 96.7%, 84.8%, and 84.8%, respectively, whereas the specificities of these tests were 60%, 91.8%, and 96.7%, respectively. The serial tests for the Tzanck smear test and enzyme-linked immunosorbent assay showed 82% sensitivity and 98.7% specificity. The serial test for the Tzanck smear test and enzyme-linked immunosorbent assay may represent a simple, rapid, and reliable way to definitive diagnosis of pemphigus. It is recommended as a common test for the diagnosis of pemphigus in dental clinics. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Detection of Plasmodium Aldolase Using a Smartphone and Microfluidic Enzyme Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Nikhil S. Gopal

    2017-01-01

    Full Text Available Background. Malaria control efforts are limited in rural areas. A low-cost system to monitor response without the use of electricity is needed. Plasmodium aldolase is a malaria biomarker measured using enzyme linked immunosorbent assay (ELISA techniques. A three-part system using ELISA was developed consisting of a microfluidic chip, hand crank centrifuge, and a smartphone. Methods. A circular microfluidic chip was fabricated using clear acrylic and a CO2 laser. A series of passive valves released reagents at precise times based upon centrifugal force. Color change was measured via smartphone camera using an application programmed in Java. The microchip was compared to a standard 96-well sandwich ELISA. Results. Results from standard ELISA were compared to microchip at varying concentrations (1–10 ng/mL. Over 15 different microfluidic patterns were tested, and a final prototype of the chip was created. The prototype microchip was compared to standard sandwich ELISA (n=20 using samples of recombinant aldolase. Color readings of standard ELISA and microfluidic microchip showed similar results. Conclusion. A low-cost microfluidic system could detect and follow therapeutic outcomes in rural areas and identify resistant strains.

  8. An enzyme-linked immunosorbent assay for measuring GPIHBP1 levels in human plasma or serum

    DEFF Research Database (Denmark)

    Miyashita, Kazuya; Fukamachi, Isamu; Nagao, Manabu

    2018-01-01

    antibodies against GPIHBP1 and used the antibodies to establish a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GPIHBP1 levels in human blood. Results: The GPIHBP1 ELISA was linear in the 8 to 500 pg/mL range and allowed the quantification of GPIHBP1 in serum and in pre- and post...... of triglyceride-rich lipoproteins. Objective: Because some GPI-anchored proteins have been detected in plasma, we tested whether GPIHBP1 is present in human blood and whether GPIHBP1 deficiency or a history of cardiovascular disease affected GPIHBP1 circulating levels. Methods: We developed 2 monoclonal......-heparin plasma (including lipemic samples). GPIHBP1 was undetectable in the plasma of subjects with null mutations in GPIHBP1. Serum GPIHBP1 median levels were 849 pg/mL (range: 740-1014) in healthy volunteers (n = 28) and 1087 pg/mL (range: 877-1371) in patients with a history of cardiovascular or metabolic...

  9. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods.

    Science.gov (United States)

    Koppelman, Stef J; Söylemez, Gülsen; Niemann, Lynn; Gaskin, Ferdelie E; Baumert, Joseph L; Taylor, Steve L

    2015-01-01

    Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested). The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame.

  10. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods

    Directory of Open Access Journals (Sweden)

    Stef J. Koppelman

    2015-01-01

    Full Text Available Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested. The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame.

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

    Science.gov (United States)

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

    2013-01-01

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

  12. Enzyme-linked immunosorbent assay and radioimmunoassay of serum pepsinogen A

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-02-01

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

  13. Enzyme-linked immunosorbent assay and radioimmunoassay of serum pepsinogen A

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  15. Design considerations of a hollow microneedle-optofluidic biosensing platform incorporating enzyme-linked assays

    Science.gov (United States)

    Ranamukhaarachchi, Sahan A.; Padeste, Celestino; Häfeli, Urs O.; Stoeber, Boris; Cadarso, Victor J.

    2018-02-01

    A hollow metallic microneedle is integrated with microfluidics and photonic components to form a microneedle-optofluidic biosensor suitable for therapeutic drug monitoring (TDM) in biological fluids, like interstitial fluid, that can be collected in a painless and minimally-invasive manner. The microneedle inner lumen surface is bio-functionalized to trap and bind target analytes on-site in a sample volume as small as 0.6 nl, and houses an enzyme-linked assay on its 0.06 mm2 wall. The optofluidic components are designed to rapidly quantify target analytes present in the sample and collected in the microneedle using a simple and sensitive absorbance scheme. This contribution describes how the biosensor components were optimized to detect in vitro streptavidin-horseradish peroxidase (Sav-HRP) as a model analyte over a large detection range (0-7.21 µM) and a very low limit of detection (60.2 nM). This biosensor utilizes the lowest analyte volume reported for TDM with microneedle technology, and presents significant avenues to improve current TDM methods for patients, by potentially eliminating blood draws for several drug candidates.

  16. Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

    Science.gov (United States)

    Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

    2016-01-01

    Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

  17. Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Akiyama, Mitoshi; Takaishi, Masatoshi; Murakami, Yoshie; Ueda, Ryuzo; Yamakido, Michio; Tsubokura, Tokuo.

    1989-09-01

    Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

  18. Measurement of Inflammatory Biomarkers in Synovial Tissue Extracts by Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Rosengren, Sanna; Firestein, Gary S.; Boyle, David L.

    2003-01-01

    We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1β (IL-1β), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of ≤25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1β, IL-6, IL-8, and TNF-α levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy. PMID:14607859

  19. Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Zhao Hongwei; Xue Changguo; Nan Tiegui; Tan Guiyu; Li Zhaohu; Li, Qing X.; Zhang Qingchuan; Wang Baomin

    2010-01-01

    A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL -1 of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL -1 , respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

  20. Detection of antibodies to the extractable nuclear antigens by enzyme linked immunosorbent assay

    International Nuclear Information System (INIS)

    Aziz, Khalil A.; Fzizal, Abul A.

    2005-01-01

    Anti-extractable nuclear antigen (ENA) antibodies are a group of autoantibodies that are directed against various components of the cell nucleus. Antibodies to these antigens are closely associated with connective tissue disease. Early diagnosis of these diseases can prove very difficult and therefore clinicians rely on the use of anti-ENA antibody testing for the exclusion. Old methods of testing are time consuming and require great skills. For these reasons clinical immunology laboratories are switching to testing for anti-ENA antibodies by enzyme linked immunosorbent assay (ELISA). The latter assays are more sensitive and require little skills. In the present study we have investigated a number of different ELISA preparations. The study was conducted at Birmingham Heartlands Hospital during the period 2003. We tested a number of ENA-positive and negative samples using 3 different commercial ELISA preparations and compared the results with traditional CCIE-assay. The present study revealed that some ELISA preparations can be more sensitive than CCIE method. Laboratories still using later method should switch to ELISA. However it is important that laboratories evaluate a long range of different ELISA preparations before selecting the most optimal one. In addition it is recommended that laboratories then audit results in order to determine true significance of such results. Finally until the true significance of ELISA generated results is known, positive ENA-results should be interpreted in conjunction with the clinical picture and this would require close liaison in between the clinical immunology laboratory and clinicians

  1. A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

    Science.gov (United States)

    Shibahara, Yusuke; Uesaka, Yoshihiko; Wang, Jun; Yamada, Shoichi; Shiomi, Kazuo

    2013-01-15

    Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 μg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices.

    Science.gov (United States)

    Ishii, Masanori; Preechakasedkit, Pattarachaya; Yamada, Kentaro; Chailapakul, Orawon; Suzuki, Koji; Citterio, Daniel

    2018-01-01

    Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL -1 from just a single application of the sample solution.

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

    Science.gov (United States)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  5. An enzyme-linked immunosorbent assay for the detection of mouse polyomavirus-specific antibodies in laboratory mice.

    NARCIS (Netherlands)

    H.W.J. Broeders; J. Groen (Jan); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert)

    1994-01-01

    textabstractAn enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of IgM and IgG serum antibodies to mouse polyomavirus (MPV). To evaluate the potential of this ELISA for the screening of laboratory rodents, serum samples from specific pathogen free (SPF)

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

    NARCIS (Netherlands)

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

    2005-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  10. Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA)

    NARCIS (Netherlands)

    Cliquet, P.; Cox, E.; Haasnoot, W.; Schacht, B.; Goddeeris, B.M.

    2003-01-01

    Sulfonamide-specific polyclonal rabbit antibodies were obtained after immunization with a sulfathiazole derivative (N1-[4-(carboxymethyl)-2-thiazolyl]sulfanilamide = TS) coupled to keyhole lympet hemocyanin. Using these antibodies, two sulfonamide-specific enzyme-linked immunosorbent assays (ELISAs)

  11. Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

    Directory of Open Access Journals (Sweden)

    William Cevallos

    Full Text Available BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans, and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC curve analysis with an area under curve (AUC value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI: 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%. Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

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

    Directory of Open Access Journals (Sweden)

    Sudarisman

    2006-03-01

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

  13. Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes.

    Science.gov (United States)

    Gard, A L; Warrington, A E; Pfeiffer, S E

    1988-05-01

    Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.

  14. Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of cashew nut in foods.

    Science.gov (United States)

    Gaskin, Ferdelie E; Taylor, Steve L

    2011-01-01

    The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 μg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements. © 2011 Institute of Food Technologists®

  15. Helicobacter pylori-related gastroduodenal disease in children. Diagnostic utility of enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Glassman, M S; Dallal, S; Berezin, S H; Bostwick, H E; Newman, L J; Perez-Perez, G I; Blaser, M J

    1990-08-01

    To evaluate the accuracy of IgG and IgA serological tests in establishing a diagnosis of Helicobacter (Campylobacter) pylori gastric infection, 60 children presenting with chronic abdominal pain were prospectively studied. Endoscopic antral biopsies were obtained and analyzed for the presence of H. pylori using three standard methods: culture and identification of bacterial isolates, microscopic examination for morphologically characteristic bacteria, and urease production by the biopsy specimen. Concomitantly obtained serum samples were analyzed for the presence of IgG and IgA antibodies against H. pylori surface antigens using enzyme-linked immunosorbent assay (ELISA). Thirty-four of 60 (56.6%) had histological evidence of chronic active gastritis, eight of whom (13.3%) also had evidence of H. pylori infection by at least one criteria. Six of the eight infected patients had H. pylori demonstrated by all three methods. Of the eight infected patients, seven had IgG antibodies against H. pylori (sensitivity of 87%) and six had IgA antibodies (sensitivity of 75%). Among the six patients who had H. pylori infection confirmed by all three methods, all had IgG antibodies (sensitivity of 100%). In the patients without evidence of H. pylori infection, the IgG ELISA had a specificity of 96% (50/52), and the IgA ELISA had a specificity of 100% (52/52). Our data suggest that serological testing for the presence of antibodies against H. pylori may be a useful diagnostic tool in screening children with chronic abdominal pain for the presence of gastric infection with H. pylori.

  16. Two new automated, compared with two enzyme-linked immunosorbent, antimüllerian hormone assays.

    Science.gov (United States)

    Nelson, Scott M; Pastuszek, Ewa; Kloss, Grzegorz; Malinowska, Iwona; Liss, Joanna; Lukaszuk, Aron; Plociennik, Lukasz; Lukaszuk, Krzysztof

    2015-10-01

    To compare new automated antimüllerian hormone (AMH) assay performance characteristics from the new automated Elecsys AMH (Roche; Elecsys) and Access AMH (Beckman Coulter; Access) assays with the existing AMH Gen II ELISA (enzyme-linked immunosorbent assay; Gen II; Beckman Coulter) and AMH ELISA (Ansh Labs) assays. Prospective assay evaluation. University-affiliated clinical chemistry laboratory. Patients referred for serum AMH measurement (n = 83) before start of in vitro fertilization cycle between September 2014 and October 2014. None. Serum AMH concentration. Intra-assay coefficients of variation were low; Ansh ≤ 9.0%; Gen II ≤ 5.8%; Access ≤ 10.7%; and Elecsys ≤ 2.8%. The Passing-Bablok regression equations (pmol/L) were y (Access) = 0.128 + (0.781 × Gen II); and y (Access) = 0.302 + (0.742 x Ansh). For y (Elecys) = 0.087 + (0.729 x Gen II) and y (Elecys) = 0.253 + (0.688 x Ansh Labs). For y (Elecys) = 0.943 - (0.037 × Access). For all the assays, AMH exhibited a moderate positive correlation with AFC (r = 0.62-0.64); number of cumulus oocyte complexes (r = 0.60-0.64); and metaphase II oocytes (r = 0.48-0.50). Accuracy of pregnancy prediction, as determined by area under the receiver operating characteristic curve, was uniformly low for all assays (0.62-0.63). The novel automated assays exhibit strong concordance in calibration, but derived values are substantially lower than those obtained from pre-existing assays, with assay-specific interpretation required for routine clinical use. These results highlight the need for an international standard of measurement of AMH. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. Serum biotin in Japanese children: Enzyme-linked immunosorbent assay measurement.

    Science.gov (United States)

    Wakabayashi, Kenji; Kodama, Hiroko; Ogawa, Eishin; Sato, Yasuhiro; Motoyama, Kahoko; Suzuki, Mitsuyoshi

    2016-09-01

    Biotin deficiency has been reported in Japanese infants fed special formulas for medical reasons, including those with milk allergy and congenital metabolic diseases, because these formulas contain little biotin. Serum biotin measurement is useful for diagnosing biotin deficiency. We applied a simple and rapid method to analyze serum biotin, and established normal ranges for children and adults. Serum biotin in 188 healthy Japanese children aged 0-4 years and in 25 healthy adults was analyzed using a Biotin ELISA Kit (immundiagnostik). The effects of various conditions on the measurement of serum biotin were also examined. Median biotin in children aged 0-4 years was 10.4 ng/dL (IQR, 7.9-13.4 ng/dL), and that in adults was 12.9 ng/dL (IQR, 10.8-15.8 ng/dL). Normal range was 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults (calculated using two-sided 95%CI). Measurements obtained with this method were not affected by frozen storage, freeze-thaw, or hemolysis, indicating that serum biotin can be analyzed accurately under these conditions, with a possible application to plasma samples. Serum biotin was significantly lower in children than in adults, with the normal range being 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults. This simple and accurate enzyme-linked immunosorbent assay method is useful for diagnosing biotin deficiency. © 2016 The Authors. Pediatrics International published by John Wiley & Sons Australia, Ltd on behalf of Japan Pediatric Society.

  18. Development of a direct competitive enzyme-linked immunosorbent assay for parathion residue in food samples.

    Science.gov (United States)

    Gui, Wen-Jun; Liu, Yi-Hua; Wang, Chun-Mei; Liang, Xiao; Zhu, Guo-Nian

    2009-10-01

    A heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) for parathion residue determination is described based on a monoclonal antibody and a new competitor. The effects of several physicochemical factors, such as methanol concentration, ionic strength, pH value, and sample matrix, on the performance of the ELISA were optimized for the sake of obtaining a satisfactory assay sensitivity. Results showed that when the assay medium was in the optimized condition (phosphate buffer solution [PBS] containing 10% [v/v] methanol and 0.2 mol/L NaCl at a pH value of 5.0), the sensitivity (estimated as the IC(50) value) and the limit of detection (LOD, estimated as the IC(10) value) were 1.19 and 0.08 ng/ml, respectively. The precision investigation indicated that the intraassay precision values all were below 10% and that the interassay precision values ranged from 4.89 to 19.12%. In addition, the developed ELISA showed a good linear correlation (r(2)=0.9962) to gas chromatography within the analyte's concentration range of 0.1 to 16 ng/ml. When applied to the fortified samples (parathion adding level: 5-15 microg/kg), the developed ELISA presented mean recoveries of 127.46, 122.52, 91.92, 124.01, 129.72, 99.37, and 87.17% for tomato, cucumber, banana, apple, orange, pear, and sugarcane, respectively. Results indicated that the established ELISA is a potential tool for parathion residue determination.

  19. Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

    Science.gov (United States)

    Wu, John T.Y.; Dreger, Sally; Chow, Eva Y.W.; Bowlby, Evelyn E.

    2002-01-01

    Abstract This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV−) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV− values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures. PMID:12418782

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

    Directory of Open Access Journals (Sweden)

    Susann Neiser

    2016-07-01

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

  1. Diagnostic value of enzyme linked immuno-sorbent assay for cytomegalovirus disease.

    Directory of Open Access Journals (Sweden)

    Priya K

    2002-07-01

    Full Text Available BACKGROUND: Since interpretation of results of enzyme linked immuno-sorbent assay (ELISA for diagnosis of Cytomegalovirus (CMV infection in India is difficult, its diagnostic value required evaluation. AIMS: To evaluate the diagnostic value of ELISA against polymerase chain reaction (PCR in CMV disease. SETTINGS AND DESIGN: Results of ELISA test for CMV antibodies in CMV-DNA PCR positive and negative patients and normal healthy blood donors were analysed. METHODS AND MATERIAL: Anti-CMV antibodies were assayed by ELISA on the sera of 26 CMV PCR positive and 21 PCR negative patients and 35 normal healthy blood donors. STATISTICAL ANALYSIS: Chi square and Fischer exact test were used for statistical analysis. RESULTS: Anti-CMV antibodies (IgG or IgG and IgM were present in 20 (76.9% of 26 PCR positive and 13 (61.9% of 21 PCR negative patients. ELISA was negative in six (23.1% of 26 PCR positive patients. Of the 28 paediatric patients, ELISA was positive in 14 (73.7% of 19 PCR positive and three (33.3% of nine PCR negative patients showing a statistically significant difference (Chi square test, P value 0.038. Among the 19 patients having complications after organ transplant, ELISA showed anti-CMV antibodies in six (85.7% of seven PCR positive and 11 (91.7% of 12 PCR negative patients showing no significant difference. CMV-DNA was not detected in the buffy coat of 35 sero-positive blood donors. CONCLUSION: ELISA has no diagnostic value in the detection of CMV activation although it may help in the differential diagnosis of CMV infection in the paediatric age group.

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

    Science.gov (United States)

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

    2016-12-01

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

  3. New Approaches to Quantitative Western Blotting

    OpenAIRE

    Hagner-McWhirter, A.; Soderquist, K.; Grimsby, S.; Winkvist, M.

    2011-01-01

    Fluorescent detection in Western blotting offers high sensitivity, broad dynamic range and stability of signals. This makes it highly suitable for quantitative Western blotting. Here we show how fluorescent Western blotting can be used for simultaneously detection of up to three different proteins on the same blot at the same time and for detection of proteins of the same molecular weight without stripping and reprobing. We also demonstrate how fluorescent Western blotting with 3 layer probin...

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

    DEFF Research Database (Denmark)

    Barington, T; Sparholt, S; Juul, L

    1992-01-01

    A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary...

  5. Recombinant diabody-based immunocapture enzyme-linked immunosorbent assay for quantification of rabies virus glycoprotein.

    Science.gov (United States)

    Nimmagadda, Sridevi V; Aavula, Shukra M; Biradhar, Neelakantam; Rao, Varaprasada Sankarasetty; Shanmugham, Rajalakshmi; Chandran, Dev; Thirumeni, Nagarajan; Singanallur, Nagendrakumar Balasubramanian; Villuppanoor, Srinivasan Alwar

    2010-08-01

    The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V(H)) and light chain (V(L)) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human x mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r(2) value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.

  6. Single-Cell Western Blotting.

    Science.gov (United States)

    Sinkala, Elly; Herr, Amy E

    2015-01-01

    Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.

  7. Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format.

    Science.gov (United States)

    Kornegay, J R; Shepard, A P; Hankins, C; Franco, E; Lapointe, N; Richardson, H; Coutleé, F

    2001-10-01

    We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses

  8. Evaluation of an enzyme-linked immunosorbent assay based on crude Leishmania histone proteins for serodiagnosis of human infantile visceral leishmaniasis.

    Science.gov (United States)

    Lakhal, Sami; Mekki, Salima; Ben-Abda, Imène; Mousli, Mohamed; Amri, Fethi; Aoun, Karim; Bouratbine, Aïda

    2012-09-01

    Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crude Leishmania histone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were biochemically purified from promastigote nuclear extracts. Their reactivities were analyzed by Western blotting (WB) using rabbit polyclonal antibodies against Leishmania recombinant histones and sera from VL patients, respectively. Then, the diagnostic potential of CLH proteins was validated by the CLH-based ELISA using 42 infantile VL patients' sera and 70 control subjects. The CLH-based ELISA performance was compared to that of the soluble Leishmania antigen (SLA)- and the recombinant K39 (rK39)-based ELISAs. Analysis of the WB profile with the use of polyclonal antibodies confirmed the histone origin of low molecular mass proteins (12 to 16 kDa). All VL samples tested presented antibodies reacting against different antigen fractions; however, recognition patterns were different depending on the reactivity of each serum. CLH-based ELISA showed an excellent ability to discriminate between VL cases and healthy controls (97.6% sensitivity and 100% specificity). It had a diagnostic performance similar to that of rK39-based ELISA (97.6% sensitivity and 97.1% specificity, P = 0.5) and a better serodiagnosis accuracy than the SLA-based ELISA (85.7% sensitivity and 90% specificity, P histone extract could be a valuable antigen for clinical use.

  9. Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

    DEFF Research Database (Denmark)

    Rimhanen-Finne, R.; Enemark, Heidi L.; Kolehmainen, J.

    2007-01-01

    The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5) oocysts/g, while the detection limit for Giardia ELISA was 10(4) cysts/g. The Cryptosporidium ELISA showed 94% specificity...... zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs...

  10. Detection of immunoglobulin M and G antibodies against cytomegalovirus early and late antigens by enzyme-linked immunosorbent assay.

    OpenAIRE

    Middeldorp, J M; Jongsma, J; ter Haar, A; Schirm, J; The, T H

    1984-01-01

    A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) is described for the detection of immunoglobulin M and antibodies with specifity for human cytomegalovirus (CMV) early (CMV-EA) and late (CMV-LA) antigens. The emphasis is on the production of high-quality CMV antigens, CMV-EA and CMV-LA separately, and conditions for their application in the ELISA. The induction of CMV-EA and -LA in infected cell extracts was studied in detail by using human sera with defined antibody spe...

  11. Application of an enzyme-linked immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and potatoes

    International Nuclear Information System (INIS)

    Van Emon, J.; Seiber, J.; Hammock, B.

    1987-01-01

    The USDA Food Safety and Inspection Service (FSIS) has included paraquat on its list of compounds to be considered for monitoring in foods. However, present methods do not easily accommodate the processing of large numbers of samples, thus limiting routine monitoring of the compound. The conventional method, based on spectrophotometry of reduced paraquat solutions, requires time-consuming sample preparation. Although the advantages of immunoassays for pesticide residue analysis have been pointed out, the reported immunoassays for paraquat have only been applied to cases of clinical poisoning or human exposure assessment. In this study, spiked milk, potato, and beef were analyzed directly, without prior cleanup, by an enzyme-linked immunosorbent assay (ELISA)

  12. A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

    International Nuclear Information System (INIS)

    Layton, G.T.

    1980-01-01

    Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10 -3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  14. Markers of human immunodeficiency virus infection in high-risk individuals seronegative by first generation enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Pedersen, C; Lindhardt, B O; Lauritzen, E

    1989-01-01

    A total of 228 stored serum samples from 140 high risk individuals was examined for serological markers of human immunodeficiency virus (HIV) infection by second generation enzyme-linked immunosorbent assay, immunoblot, and HIV antigen assay. All the samples were negative in first generation enzyme...... are common in high risk individuals seronegative by first generation ELISA. However, HIV infection do occur in subjects negative by first generation ELISA, which emphasises the need for more sensitive screening assays and/or the use of antigen detection as part of screening in high risk individuals...

  15. β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

    Science.gov (United States)

    Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

    2012-08-15

    Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Multianalyte on-chip native Western blotting.

    Science.gov (United States)

    Tia, Samuel Q; He, Mei; Kim, Dohyun; Herr, Amy E

    2011-05-01

    We introduce and characterize multiplexed native Western blotting in an automated and unified microfluidic format. While slab gel Western blotting is slow and laborious, conventional multiplexed blotting ("reblotting": probing one sample with multiple antibodies) requires even more resources. Here we detail three key advances that enable an automated and rapid microfluidic alternative to slab gel reblotting. First, we introduce both assay and microdevice designs that integrate protein blotting against multiple antibody blotting regions with native polyacrylamide gel electrophoresis. This microfluidic integration strategy overcomes nonspecific material losses inherent to harsh antibody stripping steps typically needed for conventional reblotting; said conditions can severely limit analyte quantitation. Second, to inform rational design of the multiplexed microfluidic device we develop an analytical model for analyte capture on the blotting regions. Comparison to empirical observations is reported, with capture efficiencies of >85%. Third, we introduce label free detection that makes simultaneous and quantitative multiplexed measurements possible without the need for prelabeling of sample. Assay linear dynamic range spans 8-800 nM with assay completion in 5 min. Owing to the speed, automation, enhanced quantitation capability, and the difficulty of conventional slab gel Western reblotting, microfluidic multiplexed native Western blotting should find use in systems biology, in particular in analyses of protein isoforms and multimeric protein complexes.

  17. Influence of Anti-FloR Antibody on Florfenicol Accumulation in Florfenicol-Resistant Escherichia coli and Enzyme-Linked Immunosorbent Assay for Detection of Florfenicol-Resistant E. coli Isolates

    Science.gov (United States)

    Wu, Beibei; Xia, Chun; Du, Xiangdang; Cao, Xingyuan; Shen, Jianzhong

    2006-01-01

    To detect florfenicol-resistant Escherichia coli isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione S-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromatography, respectively. Western blotting using the anti-FloR1 antibody showed specific binding of the antibody to the florfenicol-resistant FloR protein. Preincubation of florfenicol-resistant strains with the antibody significantly increased the intracellular accumulation of florfenicol and enhanced the bacterial susceptibility to florfenicol, suggesting that antibody binding to the FloR protein inhibited the activity of the efflux protein conferred by the floR gene. Analyses of florfenicol-resistant and -sensitive isolates by ELISA using the anti-FloR1 antibody showed good correlation between FloR protein expression and the floR genotype. The anti-FloR1 antibody-based ELISA is a useful tool for the detection of florfenicol-resistant bacteria harboring the floR gene. PMID:16455887

  18. Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Alcaide, M; Reina, D; Frontera, E; Navarrete, I

    2005-06-01

    A serodiagnostic test for the diagnosis of infestation by the sheep nasal bot fly, Oestrus ovis (Linné) was examined. The enzyme-linked immunosorbent assay (ELISA) technique was used to analyze and compare the production of immunoglobulin G (IgG) antibodies against excretory-secretory products (ESP) and crude extract (CE) antigens from all the different larval stages of O. ovis in the sera of 276 adult sheep sampled in summer (n = 135) and winter (n = 141). ESP from first stage larvae was the most sensitive, coating antigen in winter and ESP from second stage larvae during summer. The most specific values were obtained by ESP against L1 in winter and by CE against L3 in summer. These results show that the stage of larval development has a significant impact on the humoral immune response over the course of a season. A significant correlation (P oestrosis immunodiagnosis.

  19. Immunodiagnosis of fascioliasis using sandwich enzyme-linked immunosorbent assay for detection of Fasciola gigantica paramyosin antigen

    Science.gov (United States)

    Abou-Elhakam, Hany Mohamed Adel; Bauomy, Ibraheem Rabia; El Deeb, Somaya Osman; El Amir, Azza Mohamed

    2013-01-01

    Background: Many immunological techniques have been developed over years using the different Fasciola antigens for diagnosis of parasitic infection and to replace the parasitological techniques, which are time consuming and usually lack sensitivity and reproducibility. Materials and Methods: In this study, Fasciola gigantica paramyosin (Pmy) antigen was early detected in cattle sera using sandwich enzyme-linked immunosorbent assay (ELISA), to evaluate the Pmy antigen performance in diagnosis. This work was conducted on 135 cattle blood samples, which were classified according to parasitological investigation into, healthy control (30), fascioliasis (75), and other parasites (30) groups. Results: The sensitivity of Sandwich ELISA was 97.33%, and the specificity was 95%, in comparison with parasitological examination, which recorded 66.66% sensitivity and 100% specificity, respectively. Conclusions: It was clear that the native F. gigantica Pmy is considered as a powerful antigen in early immunodiagnosis of fascioliasis, using a highly sensitive and specific sandwich ELISA technique. PMID:23961441

  20. Evaluation of a chemiluminescent enzyme-linked immunosorbent assay for the diagnosis of Trypanosoma cruzi infection in a nonendemic setting

    Directory of Open Access Journals (Sweden)

    Luis Izquierdo

    2013-11-01

    Full Text Available The disappearance of lytic, protective antibodies (Abs from the serum of patients with Chagas disease is accepted as a reliable indicator of parasitological cure. The efficiency of a chemiluminescent enzyme-linked immunosorbent assay based on a purified, trypomastigote-derived glycosylphosphatidylinositol-anchored mucin antigen for the serologic detection of lytic Abs against Trypanosoma cruzi was evaluated in a nonendemic setting using a panel of 92 positive and 58 negative human sera. The technique proved to be highly sensitive {100%; 95% confidence interval (CI = 96-100} and specific (98.3%; 95% CI = 90.7-99.7, with a kappa score of 0.99. Therefore, this assay can be used to detect active T. cruzi infection and to monitor trypanosomicidal treatment.

  1. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens...... in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test, The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...

  2. The development of an Enzyme Linked Immunosorbent Assay for detecting Injectious laryngotrachitis viral antibodies in chicken serum

    Directory of Open Access Journals (Sweden)

    Risa Indriani

    2002-06-01

    Full Text Available The aim of this study was to develop an Enzyme-linked immunosorbent assay (ELISA for detection of antibody against gallid herpes virus, the causal agent of infectious laryngotracheitis (ILT in chicken. Its application in experimental chicken under laboratory condition was also evaluated. Results showed that ELISA for ILT was developed successfully with sensitivity and specificity was 98% and 97,14% respectively. The ELISA was able to determine the dynamic of antibodies respond in experimental chickens following vaccination and artificial infection with ILT virus. It was concluded that this ELISA offers a simple, sensitive and specific antibody assay for detection of antibodies against ILT virus in chickens arising from vaccination or infection.

  3. An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Ajjouri, Gitte; Handberg, Kurt

    2013-01-01

    of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV...... during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98. CONCLUSIONS: The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only......BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    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......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...... 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...

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  6. Sandwich enzyme-linked immunosorbent assay for the quantification of human serum albumin fragment 408-423 in bodily fluids.

    Science.gov (United States)

    Mohr, Katharina B; Zirafi, Onofrio; Hennies, Mark; Wiese, Sebastian; Kirchhoff, Frank; Münch, Jan

    2015-05-01

    Urinary levels of human serum albumin (hSA) fragment 408-423 have been proposed to represent an early marker for graft-versus-host disease (GvHD) and chronic kidney diseases. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of hSA(408-423). The sandwich ELISA has a detection limit of 0.5ng/ml and is highly specific for hSA(408-423) because it does not cross-react with other albumin fragments or the full-length precursor. This ELISA allows rapid and convenient quantification of hSA(408-423) in bodily fluids, further clarifying the prognostic and diagnostic value of this peptide in GvHD, kidney disease, and other disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

    Directory of Open Access Journals (Sweden)

    Roldán William

    2006-01-01

    Full Text Available A dot enzyme-linked immunosorbent assay (dot-ELISA was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.

  8. Rapid Enzyme-Linked Immunosorbent Assay for the Detection of Hantavirus-Specific Antibodies in Divergent Small Mammals

    Directory of Open Access Journals (Sweden)

    Karla Cautivo

    2014-05-01

    Full Text Available We assessed the utility of an enzyme-linked immunosorbent assay (ELISA for the detection of hantavirus-specific antibodies from sera of Oligoryzomys longicaudatus, the principal reservoir of Andes virus (ANDV, using an antigen previously developed for detection of antibodies to Sin Nombre virus (SNV in sera from Peromyscus maniculatus. The assay uses a protein A/G horseradish peroxidase conjugate and can be performed in as little as 1.5 hours. Serum samples from Oligoryzomys longicaudatus collected in central-south Chile were used and the assay identified several that were antibody positive. This assay can be used for the rapid detection of antibodies to divergent hantaviruses from geographically and phylogenetically distant rodent species.

  9. Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

    DEFF Research Database (Denmark)

    Chow, E.Y.W.; Wu, J.T.Y.; Jauho, E.S.

    2004-01-01

    in 5 laboratories from Europe and North America. Comparison with culture results showed that 88.5% of 26 culture-positive animals were ELISA positive, as were 55% of 60 animals from 2 culture-positive pig herds. Of 90 animals from 2 high health farms with no clinical symptoms of salmonellosis, 98......In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested.......9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between...

  10. Western blot: technique, theory, and trouble shooting.

    Science.gov (United States)

    Mahmood, Tahrin; Yang, Ping-Chang

    2012-09-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

  11. Use of Polymerase Chain Reaction Enzyme Linked Oligonucleotide Sorbent Assay (Pcr-Elosa for Detection of Disease Agents

    Directory of Open Access Journals (Sweden)

    Simson Tarigan

    2016-03-01

    Full Text Available Diagnostic tool comprises one of the vital components in the control of infectious diseases. One of the most common techniques in the diagnosis of infectious disease currently available is the polymerase chain reaction (PCR because this technique is very sensitive, specific, and rapid. This technique requires an adjunct technique to indicate the formation of the right reaction product. Agarose gel electrophoresis has been the most common technique to visualise the PCR product or amplicon. Enzyme linked oligonucleotide sorbent assay (ELOSA is an alternative technique which is more sensitive and gives more important identity of the amplicon. This technique can be more than 100 times as sensitive as a gel agarose electrophoresis, and very specific since confirmation of the amplicon is carried out by DNA hibridisation. The capacity of the ELOSA can also be extended to the detection of disease-causal agent at subtype level, or detection of mutation at particular location in a gene. Since the equipment used for ELOSA is similar to that for ELISA (enzyme linked immunosorbent assay, a large number of samples can be accomplished rapidly. As in ELISA, a number of variation can be made in ELOSA depend on the requirement. Nucleotide can be immobilised on the microwell plate either by passive adsorbtion, by first conjugation of nucleotide with biotin then immobilisation on streptavidin-coated microwell plate, or immobilisaion by covalent bonding. The PCR and ELOSA can be performed at separate or in a single tube by first immobilising the PCR primers on the surface of microwell plates.

  12. Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey.

    Science.gov (United States)

    Sakru, Nermin; Korkmaz, Metin; Ozbel, Yusuf; Ertabaklar, Hatice; Sengul, Mustafa; Toz, Seray Ozensoy

    2007-01-01

    In Turkey, Leishmania infantum is responsible for human visceral leishmaniasis (VL), which is seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions. This study aimed to determine asymptomatic infections in an endemic area of VL in Turkey using the western blot technique. A total of 82 persons including children and adults were chosen randomly in Denizli province which is one of the endemic sites for VL. Serum samples were collected and screened using indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB). One year later, 35 of the 82 persons were sampled and screened serologically for the second time. Seven out of 82 samples were found to be positive by western blot analysis with the presence of 14 and/or 18 kDa bands. Two of these seven sera were also positive by IFAT, but only one of these two was positive by ELISA. Only one person showing seropositivity with all three tests had clinical symptoms and was diagnosed as VL with the presence of amastigotes in bone marrow aspirate. Because six people, including the one found to be seropositive in all two tests, had no clinical symptoms, they were accepted as asymptomatic carriers. The ratio of asymptomatic infection was calculated as 7.41% (6/81) in the region. In the second sampling, the western blot revealed antibodies against the same antigens in all seven subjects. Our findings showed that the presence of antibodies against 14 and 18 kDa antigens are important for the diagnosis of symptomatic and asymptomatic infections. Western blot was found to be effective in the detection of asymptomatic persons in the epidemiological studies in endemic areas.

  13. [Conservation of Malassezia strains in blotting paper].

    Science.gov (United States)

    Ramos, Laura; Ramadán, Silvana; López, Clara; Bulacio, Lucía; Mellado, Soledad

    2006-06-01

    Reference strains belonging to the genus Malassezia were analyzed to evaluate, by comparison, different preservation systems such us subculture, freezing at -80 degrees C in glycerol, and blotting paper-disc conservation. The viability, phenotypic and genotypic characteristics of the strains used in this study was evaluated. The blotting paper method was found to be advantageous to preserve Malassezia spp strains due to both, its simple implementation in the laboratory and its efficiency.

  14. Western Blot: Technique, Theory, and Trouble Shooting

    OpenAIRE

    Mahmood, Tahrin; Yang, Ping-Chang

    2012-01-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blo...

  15. Western Blotting of the Endocannabinoid System.

    Science.gov (United States)

    Wager-Miller, Jim; Mackie, Ken

    2016-01-01

    Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

  16. enzyme-linked

    African Journals Online (AJOL)

    Material and methods. Isolation of HBsAg . HBsAg was prepared from donated blood heavily: contami- nated with hepatitis B virus. Typically, HBsAg in I litre ofserum was precipitated by addition of PEG 6000 (11% w/v) and the precipitate recovered by centrifugation at I 000g for 10 minutes. The precipitate was redissolved in ...

  17. Studying protein-protein interactions via blot overlay/far western blot.

    Science.gov (United States)

    Hall, Randy A

    2015-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  18. Studying protein-protein interactions via blot overlay or Far Western blot.

    Science.gov (United States)

    Hall, Randy A

    2004-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  19. Improved quantification of a commercial enzyme-linked immunosorbent assay kit for measuring anti-MDA5 antibody.

    Science.gov (United States)

    Gono, Takahisa; Okazaki, Yuka; Murakami, Akihiro; Kuwana, Masataka

    2018-04-09

    To compare the quantitative performance for measuring anti-MDA5 antibody titer of two enzyme-linked immunosorbent assay (ELISA) systems: an in-house ELISA and the commercial MESACUP TM anti-MDA5 test. Anti-MDA5 antibody titer was measured in sera from 70 patients with dermatomyositis using an in-house ELISA and the MESACUP TM anti-MDA5 test side-by-side. For the commercial ELISA kit, serum samples diluted 1:101 were used according to the manufacturer's protocol, but serial dilutions of sera were also examined to identify the optimal serum dilution for quantification. The anti-MDA5 antibody titers measured by the in-house and commercial ELISAs were positively correlated with each other (r = 0.53, p = .0001), but the antibody titer measured by the commercial ELISA was less sensitive to change after medical treatment, and 37 (80%) of 46 anti-MDA5-positive sera had antibody titer exceeding the quantification range specified by the manufacturer (≥150 index). Experiments using diluted serum samples revealed that diluting the sera 1:5050 improved the quantitative performance of the MESACUP TM anti-MDA5 test, including a better correlation with the in-house ELISA results and an increased sensitivity to change. We improved the ability of the commercial ELISA kit to quantify anti-MDA5 antibody titer by altering its protocol.

  20. Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae; Jeon, Bo-Young; Yoon, Byoung-Su

    2014-05-01

    Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

  1. Development of enzyme-linked immunosorbent assays for the hepatotoxic alkaloids riddelliine and riddelliine N-oxide.

    Science.gov (United States)

    Lee, S T; Schoch, T K; Stegelmeier, B L; Gardner, D R; Than, K A; Molyneux, R J

    2001-08-01

    Pyrrolizidine alkaloid-containing plants are widely distributed throughout the world and are particularly common in the genus Senecio. The structural types and concentrations of the alkaloids vary among plant species. In addition, within a species of plant, concentrations vary with environment and location. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and in humans. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine the particular plants and conditions under which livestock are likely to be poisoned. In this study, two competitive inhibition enzyme-linked immunosorbent assays for riddelliine, riddelliine N-oxide, and other closely related pyrrolizidine alkaloids were developed using polyclonal antibodies. One assay is class specific toward the free base forms of the pyrrolizidine alkaloids; the other assay showed cross-reactivity to both the free base and N-oxide forms of the alkaloids. The assay with the lowest limit of detection had an I(50) of 803.9 pg with a limit of detection of 47.5 pg for riddelliine. Spike and recovery studies for riddelliine in bovine blood ranged from 45 to 74%. The assay that showed cross-reactivity between the N-oxide and free base forms of the pyrrolizidine alkaloids allowed estimation of the total pyrrolizidine alkaloid content in Senecio riddellii in admixture with alfalfa. These findings suggest that these techniques will be excellent tools to diagnose poisoned animals and identify highly toxic plants.

  2. An enzyme-linked immunosorbent assay for the epidemiological survey of Dermatophilus congolensis infection in camels (Camelus dromedarius).

    Science.gov (United States)

    Gitao, C G

    1993-06-01

    The breeding of camels (Camelus dromedarius) is especially important in arid and semi-arid areas of Africa, where drought and famine frequently occur. A number of diseases which impair camel production have recently been described, including dermatophilosis (caused by Dermatophilus congolensis). However, it is not possible to determine the prevalence of infection from clinical cases alone. An enzyme-linked immunosorbent assay has therefore been developed to determine the epidemiological prevalence of D. congolensis infection in sera of camels. Whole-cell antigen was used on microplates and the test serum was added. Horseradish peroxidase-conjugated sheep antibodies against heavy and light chains of camel immunoglobulin (Ig)G were then added, followed by substrate. The test was used to trace the antibody profile of twelve experimentally-infected camels. Peak antibody levels in serum occurred within twenty-one days following infection. It is planned to use this test to determine the epidemiological prevalence of D. congolensis infection in camels reared in a pastoral area of Kenya.

  3. Analytical characterization and clinical evaluation of an enzyme-linked immunosorbent assay for measurement of afamin in human plasma.

    Science.gov (United States)

    Dieplinger, Benjamin; Egger, Margot; Gabriel, Christian; Poelz, Werner; Morandell, Elisabeth; Seeber, Beata; Kronenberg, Florian; Haltmayer, Meinhard; Mueller, Thomas; Dieplinger, Hans

    2013-10-21

    Comparative proteomics has recently identified afamin, the newest member of the albumin gene family, as a potential biomarker for ovarian cancer. The aim of this study was the analytical and clinical evaluation of a sandwich enzyme-linked immunosorbent assay for the determination of afamin in human plasma. We evaluated precision, linearity, and detection limit of the assay, analyte stability and biological variability, determined reference values and quantified afamin concentrations in various diseases. Within-run and total coefficients of variation were heart failure. Patients with pneumonia or sepsis exhibited markedly decreased afamin plasma concentrations. However, patients with chronic renal disease or chronic obstructive pulmonary disease showed no difference in afamin plasma concentrations as compared to healthy individuals. Correlation analyses revealed an inverse association between afamin and inflammatory biomarkers. The afamin assay meets quality specifications for laboratory medicine. The results of the clinical assay evaluation revealed novel insights with respect to afamin as a potential negative acute phase protein and should encourage further studies. © 2013.

  4. Screening of Dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay.

    Directory of Open Access Journals (Sweden)

    Andrea Cristine Koishi

    Full Text Available Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization.

  5. Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk

    Directory of Open Access Journals (Sweden)

    Yuanming Sun

    2011-06-01

    Full Text Available An indirect competitive enzyme-linked immunosorbent assay (icELISA with enhanced specificity for melamine in milk was developed. Three haptens of melamine with different spacer-arms were used to prepare different plate coating antigens. It was found that the icELISA show best sensitivity and specificity to melamine when using the coating antigen prepared by coupling 3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthiopropanoic acid (Hapten C with ovalbumin (OVA. The 50% inhibitory concentration (IC50 value was 35.4 ng·mL−1, the limit of detection (LOD was 8.9 ng·mL−1 and the detectable working range (20–80% inhibitory concentration was from 14.9 to 108.5 ng·mL−1, respectively. Compared to the ELISA results previously reported, the developed icELISA in the present study showed a much lower cross-reactivity to cyromazine, a fly-killing insecticide widely used in vegetables and stables. Recoveries obtained from milk samples in this study were in agreement with those obtained using the HPLC-MS method, indicating the detection performance of the icELISA could meet the requirement of the residue limit set by the Codex Alimentarius Commission. Therefore, the developed immunoassay can be applied for the analysis of melamine presented in milk.

  6. ENZYME-LINKED IMMUNOSORBENT ASSAY FOR SCREENING DIOXIN SOIL CONTAMINATION BY UNCONTROLLED COMBUSTION DURING INFORMAL RECYCLING IN SLUMS

    Science.gov (United States)

    Trindade, Mirta; Nording, Malin; Nichkova, Mikaela; Spinnel, Erik; Haglund, Peter; Last, Michael S.; Gee, Shirley; Hammock, Bruce; Last, Jerold A.; González-Sapienza, Gualberto; Brena, Beatriz M.

    2010-01-01

    Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly. PMID:18522475

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

    Directory of Open Access Journals (Sweden)

    Gortázar Christian

    2008-11-01

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

  8. Enzyme-linked immunosorbent assay of relaxin-like gonad-stimulating peptide in the starfish Patiria (Asterina) pectinifera.

    Science.gov (United States)

    Mita, Masatoshi; Katayama, Hidekazu

    2018-03-01

    A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we succeeded in obtaining specific antibodies against P. pectinifera RGP (PpeRGP). In this study, the antibodies were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of PpeRGP. A biotin-conjugated peptide that binds to peroxidase-conjugated streptavidin is specifically detectable using 3,3',5,5'-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate; therefore, biotin-conjugated RGP (biotin-PpeRGP) was synthesized chemically. Similarly to PpeRGP, synthetic biotin-PpeRGP bound to the antibody against PpeRGP. In binding experiments with biotin-PpeRGP using wells coated with the antibody, a displacement curve was obtained using serial concentrations of PpeRGP. The ELISA system showed that PpeRGP could be measured in the range 0.01-10pmol per 50µl assay buffer. On the contrary, the B-chains of PpeRGP, Asterias amurensis RGP, Aphelasterias japonica RGP, and human relaxin showed minimal cross-reactivity in the ELISA, except that the A-chain of PpeRGP affected it slightly. These results strongly suggest that this ELISA system is highly specific and sensitive with respect to PpeRGP. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

    Rasmussen, Mette; Dahl, Mette; Buus, Søren

    2014-01-01

    The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative...... splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects....... We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide...

  10. An enzyme-linked immunosorbent assay and a gold-nanoparticle based immuno chromatographic test for amatoxins using recombinant antibody

    International Nuclear Information System (INIS)

    He, Kuo; Zhao, Ruiping; Wang, Lixia; Feng, Tingting; Wei, Dong; Zhang, Xiuyuan

    2016-01-01

    The authors describe two kinds of rapid assays for the determination of amatoxins in mushrooms. The first is an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase. The second is a rapid immuno chromatographic assay that uses colloidal gold as a red label (CG-ICA). Both are based on the use of a well-characterized recombinant single chain variable fragment antibody (named scFv-A4). The half-maximum inhibition concentrations (IC50) of α-amanitin, β-amanitin and γ-amanitin are 78, 85 and 90 ng⋅mL -1 , and the limits of detection (LODs; for IC15) are 1.9, 2.1 and 2.8 ng⋅mL -1 . The method was applied to the determination of amanitins in mushrooms, and the LODs for α-amanitin, β-amanitin and γ-amanitin in mushroom samples were found to be 4.9, 6.4 and 8.3 ng⋅mL -1 . The visual minimum detection limits of the optimized CGIA are 4 and 6 ng⋅mL -1 for mushroom samples. The test can be performed within 10 min. The results of the analysis of spiked samples showed that the CG-IA can rapidly and semi-quantitatively quantify amatoxins in mushroom samples on site and at low costs. (author)

  11. Screening of Dengue Virus Antiviral Activity of Marine Seaweeds by an In Situ Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Koishi, Andrea Cristine; Zanello, Paula Rodrigues; Bianco, Éverson Miguel; Bordignon, Juliano; Nunes Duarte dos Santos, Claudia

    2012-01-01

    Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV) infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa) were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization. PMID:23227238

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

    Science.gov (United States)

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

    2016-09-01

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

  13. Improved Canine and Human Visceral Leishmaniasis Immunodiagnosis Using Combinations of Synthetic Peptides in Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Costa, Míriam Maria; Penido, Marcos; dos Santos, Mariana Silva; Doro, Daniel; de Freitas, Eloísa; Michalick, Marilene Susan Marques; Grimaldi, Gabriel; Gazzinelli, Ricardo Tostes; Fernandes, Ana Paula

    2012-01-01

    Background Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39. Methodology/Principal Findings Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals. Conclusions/Significance The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL. PMID:22629475

  14. Evaluation of an enzyme linked immunosorbent assay kit for the detection of Babesia bovis antibodies in cattle in Argentina

    International Nuclear Information System (INIS)

    Echaide, S.; Echaide, I.E.; Mangold, A.J.; Lugaresi, C.I.; Guglielmone, A.A.; Gaido, A.B.

    1998-01-01

    An enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to Babesia bovis was evaluated by using sera of 874 cattle carrying B. bovis antibodies, 700 sera of uninfected cattle, and 357 sera from calves from 16 herds subjected to different B. bovis inoculation rates. The seropositive/ seronegative cut-off point set as double the mean percent positivity of negative cattle sera (= 16%). The sensitivity of the ELISA (four trials) ranged from 97.1% to 100% and the specificity (three trials) varied from 92.0% to 97.0%. The agreement between ELISA and immunofluorescent antibody test was ≥ 90.0% in 18 of 23 evaluations and it ranged from 86.0% to 88.0% in the remainder. The correlation coefficient between percentage of sera positive to ELISA and IFA test in 16 herds was 0.9958 (P<0.001). The ELISA has the advantages of a high sensitivity, objectivity and capacity to test large number of samples in short period of time and could replace the IFA test specially for epidemiological studies. (author)

  15. Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate.

    Science.gov (United States)

    Spies, Peter; Chen, Guo Jun; Gygax, Daniel

    2008-11-01

    A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 microl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n=96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.

  16. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

    Science.gov (United States)

    Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

    2014-02-06

    Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

    Science.gov (United States)

    Rodak, L; Babiuk, L A; Acres, S D

    1982-07-01

    The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

  18. Field assessment of the enzyme linked immunosorbent assay for foot-and-mouth disease virus diagnosis and typing

    International Nuclear Information System (INIS)

    Smitsaart, E.; Fondevila, N.; Compaired, D.; Maradei, E.; Fernandez, E.

    1998-01-01

    The objective of the present study was to evaluate the enzyme linked immunosorbent assay (ELISA) in comparison with the complement fixation test (CFT) for the diagnosis and typing of foot-and-mouth disease (FMD) virus (FMDV). Diagnostic material was epithelium from either suspected cases of FMD or from animals experimentally infected with FMDV. Epithelial suspensions and supernatant fluids from cell culture passage were assayed by CFT and ELISA. The superiority of the ELISA over the CFT was demonstrated: 1) the detection rate was 23% higher than that of CFT on original (epithelial) suspensions (OS) submissions of all sample (positive and negative) and 30% higher on supernatant fluids from cell culture passage, 2) the detection rate of ELISA on OS of confirmed positive samples was 28% higher than that of CFT, 3) no significant differences were observed in the detection and typing rates between the PANAFTOSA and FAO/IAEA ELISA kits (P<0.05) and 4) the sensitivity of the ELISA was 16 to 85 times higher than that of CFT when serial dilutions of sample homogenates were examined. (author)

  19. Identification of phlebotomine sandfly bloodmeals from Baringo District, Kenya, by direct enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Ngumbi, P M; Lawyer, P G; Johnson, R N; Kiilu, G; Asiago, C

    1992-10-01

    Direct enzyme-linked immunosorbent assay (ELISA) was used to identify the sources of bloodmeals in phlebotomine sandflies from Baringo District, Rift Valley Province, Kenya. Some bloodmeals had been stored for over 4 years before being analysed. Among 356 sandflies identified, 62.9% were Phlebotomus martini, 14.8% Sergentomyia antennatus, 10% S.schwetzi, 6% S.clydei, 1.9% S.adleri, 1.6% P.duboscqi, 1.4% S.africanus and 0.8% S.bedfordi. Out of 224 P.martini bloodmeals, host source was identified for 69. The order of host preference for P.martini was: goat 28.5%, rabbit 22.7%, human 8.9% and others 8.9%. Evidence of mixed feeding was shown by four species comprising sixteen specimens, twelve of which were P.martini. The most effective methods for trapping bloodfed P. martini were sticky paper traps in termite hills, followed by light-traps. Of the 224 P.martini trapped, 58.9% were collected with traps in termite hills, and 22.7% with light traps. Roles of the three most popular hosts for P.martini should be investigated to ascertain whether they act as reservoirs in the transmission of Leishmania donovani causing visceral leishmaniasis in Kenya.

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

    International Nuclear Information System (INIS)

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

    2000-01-01

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

  1. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

    Science.gov (United States)

    Ribotta, M J; Higgins, R; Gottschalk, M; Lallier, R

    2000-01-01

    Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.

  2. Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and PCR in diagnostics of enzootic bovine leukosis

    Directory of Open Access Journals (Sweden)

    Malovrh Tadej

    2005-01-01

    Full Text Available Bovine leukaemia virus (BLV is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gpSI served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELiSA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV.

  3. Onderzoek naar toepasbaarheid van Enzyme-Linked Immunosorbent Assay (ELISA) voor het bepalen van aflatoxinen in voedings- en voedermiddelen 1e interimrapport: Orientatie en taakstelling

    NARCIS (Netherlands)

    Egmond; H.P. van; Paulsch; W.E.; Sizoo; E.A.

    1987-01-01

    Immunochemische methoden van onderzoek voor het bepalen van mycotoxinen komen langzamerhand in de belangstelling te staan, vooral Enzyme-Linked Immunosorbent Assay (ELISA). Om een beeld te krijgen van de praktische en wetenschappelijke karakteristieken van ELISA's in het

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

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

    OpenAIRE

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

    2003-01-01

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

  6. Comparison of an enzyme linked immunosorbent assay (ELISA) and a radioallergosorbent test (RAST) for detection of IgE antibodies to Brugia malayi

    NARCIS (Netherlands)

    Wahyuni, Sitti; van Ree, Ronald; Mangali, Andarias; Supali, Taniawati; Yazdanbakhsh, Maria; Sartono, Erliyani

    2003-01-01

    The enzyme linked immunosorbent assay (ELISA) for specific IgE antibodies to Brugia malayi was compared with the radioallergosorbent test (RAST) for use in immunoepidemiological studies of lymphatic filariasis. Sera used were from individuals (aged 5-82 years) living in an area endemic for lymphatic

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

    NARCIS (Netherlands)

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

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

  8. Simultaneous determination of 13 fluoroquinolone and 22 sulfonamide residues in milk by a dual-colorimetric enzyme-linked immunosorbent assay

    Science.gov (United States)

    Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor the two classes of antimicrobial residues in different food matrices. In th...

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

    NARCIS (Netherlands)

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

    1991-01-01

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

  10. Use of rapid dipstick and latex agglutination tests and enzyme-linked immunosorbent assay for serodiagnosis of Amebic liver abscess, amebic colitis, and Entamoeba histolytica cyst passage

    NARCIS (Netherlands)

    van Doorn, H. Rogier; Hofwegen, Henk; Koelewijn, Rob; Gilis, Henk; Peek, Ron; Wetsteyn, Jose C. F. M.; van Genderen, Perry J. J.; Vervoort, Tony; van Gool, Tom

    2005-01-01

    A homemade enzyme-linked immunosorbent assay (ELISA) and a dipstick assay (Dipstick) for the detection of anti-Entamoeba histolytica antibodies in serum were developed and evaluated together with a commercially available latex agglutination test (LAT; Laboratoires Fumouze) for their use in

  11. Single cell-resolution western blotting.

    Science.gov (United States)

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2016-08-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

  12. Single cell–resolution western blotting

    Science.gov (United States)

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2017-01-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

  13. Multiplexed Western Blotting Using Microchip Electrophoresis.

    Science.gov (United States)

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-05

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

  14. HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

    Science.gov (United States)

    Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

    2012-01-01

    that HIV-2 Western blot be included in the diagnostic algorithm for HIV-1/2 to followup negative or indeterminate HIV-1 Western blot results. KEYWORDS Diagnosis, laboratory techniques and procedures, antibodies, HIV-2, Western blot, enzyme-linked immunosorbent assay, algorithm, Cuba.

  15. Modified Western blotting for insulin and other diabetes-associated peptide hormones.

    Science.gov (United States)

    Okita, Naoyuki; Higami, Yoshikazu; Fukai, Fumio; Kobayashi, Masaki; Mitarai, Miku; Sekiya, Takao; Sasaki, Takashi

    2017-07-31

    Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.

  16. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

    Science.gov (United States)

    Taki, Takao

    2015-01-01

    A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

  17. Enzyme-linked immunosorbent assay to quantitate serum ferritin in black and white ruffed lemurs (Varecia variegata variegata).

    Science.gov (United States)

    Andrews, Gordon A; Chavey, Patricia Sue; Crawford, Graham

    2005-12-01

    Lemurs in captivity progressively accumulate iron deposits in a variety of organs (hemosiderosis) including duodenum, liver, and spleen throughout their lives. When excessive, the toxic effects of intracellular iron on parenchymal cells, particularly the liver, can result in clinical disease and death. The pathogenesis of excessive iron storage in these species has been attributed to dietary factors related to diets commonly fed in captivity. Tissue iron stores can be directly estimated by tissue biopsy and histologic examination, or quantitated by chemical analysis of biopsy tissue, However, expense and risk associated with anesthesia and surgery prevent routine use of tissue biopsy to assess iron status. A noninvasive means of assessing total body iron stores is needed to monitor iron stores in lemurs to determine whether dietary modification is preventing excessive iron deposition, and to monitor potential therapies such as phlebotomy or chelation. Serum ferritin concentration correlates with tissue iron stores in humans, horses, calves, dogs, cats, and pigs. Serum ferritin is considered the best serum analyte to predict total body iron stores in these species and is more reliable than serum iron or total iron binding capacity, both of which may be affected by disorders unrelated to iron adequacy or excess including hypoproteinemia, chronic infection, hemolytic anemia, hypothyroidism, renal disease, and drug administration. We have developed an enzyme-linked immunosorbent assay to measure serum ferritin in lemurs. The assay uses polyclonal rabbit anti-human ferritin antibodies in a sandwich arrangement. Ferritin isolated from liver and spleen of a black and white ruffed lemur (Varecia variegata variegata) was used as a standard. Ferritin standards were linear from 0 to 50 microg/L. Recovery of purified ferritin from lemur serum varied from 95% to 110%. The within-assay variability was 4.5%, and the assay-to-assay variability for three different samples ranged

  18. Comparison of enzyme-linked immunosorbent assay test with immunoblot assay in the diagnosis of pemphigus in Indian patients

    Directory of Open Access Journals (Sweden)

    Khandpur Sujay

    2010-01-01

    Full Text Available Background: The diagnosis of pemphigus vulgaris (PV and pemphigus foliaceous (PF rests upon clinical, histological and immunofluorescence features. Enzyme-linked immunosorbent assay (ELISA test and immunoblot (IB assay have shown variable sensitivity and specificity. Aims: We compared the utility of ELISA and IB in pemphigus patients. Methods: Sixty-six pemphigus cases (PV-54, PF-12 and 72 controls (other vesicobullous disorders and healthy controls were inducted. ELISA for anti-Dsg 3 and 1 antibodies and IB assay were performed. Results: On ELISA, both mean anti-Dsg 1 and 3 titers were raised in PV and PF. Mean anti-Dsg 1 in mucocutaneous PV was significantly higher than in mucosal PV and mean anti-Dsg 3 was significantly raised in PV than in PF. Anti-Dsg 1 and 3 in the control group were negative. Sensitivity and specificity of ELISA in PV was 98.14% and 90.5% while in PF it was 91.6% and 61.1%, respectively.On IB in PV, 36 cases (66.67% showed the 130 kDa and 160 kDa antigen bands, 12 (22.2% only the 130 kDa and six (11.1% only the 160 kDa band. Eight of the nine pure mucosal cases (88.8% showed only the 130 kDa. In PF, only the 160 kDa antigen was detected. These antigens were not identified in the control group. Sensitivity and specificity of IB in PV was 88.9% and 100% and in PF it was 100% and 95.2%, respectively. Conclusion: Both tests could differentiate pemphigus from other dermatoses, including other blistering disorders. ELISA could not make a distinction between PV and PF or between the various clinical phenotypes of PV. IB differentiated between PV and PF and the different clinical variants of PV.

  19. Salivary Desmoglein Enzyme-Linked Immunosorbent Assay for Diagnosis of Pemphigus Vulgaris: A Noninvasive Alternative Test to Serum Assessment

    Directory of Open Access Journals (Sweden)

    Hossein Mortazavi

    2015-01-01

    Full Text Available Background. Serum desmoglein enzyme-linked immunosorbent assay (ELISA is used for the diagnosis and monitoring of pemphigus diseases. Objectives. To compare the diagnostic accuracy of salivary antidesmoglein (Dsg 1 and 3 ELISA in the diagnosis of pemphigus vulgaris (PV patients with that of serum desmogleins ELISA. Methods. Eighty-six untreated PV patients and 180 age- and sex-matched PV-free controls were recruited in this case-control study. PV was diagnosed based on clinical, histopathological, and direct immunofluorescence findings. After processing, serum and salivary anti-Dsg 1 and 3 were measured by the ELISA method using Euroimmun kit (Lübeck, Germany. Results. Using the cut-off point of 20 relative units (RU/mL, the serum anti-Dsg 1 and 3 ELISA were positive in 62 (72.1% and 83 (96.5% patients, respectively, and the salivary anti-Dsg 1 and 3 ELISA were positive in 31 (36.1% and 63 (73.3% patients, respectively. The specificity of salivary anti-Dsg 1 and anti-Dsg 3 were both 98.9%. Optimal cut-off values of 7.7 and 13.4 RU/mL were determined for the salivary anti-Dsg 1 and anti-Dsg 3 ELISA, respectively. Conclusion. Salivary anti-Dsg 1 and 3 ELISA with high specificities (98.9% could be suggested as safe and noninvasive methods for the diagnosis of PV when obtaining a blood sample is difficult.

  20. Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): a comparative study.

    Science.gov (United States)

    Oboh, Ganiyu; Agunloye, Odunayo M; Adefegha, Stephen A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

    2015-03-01

    Chlorogenic acid is a major phenolic compound that forms a substantial part of plant foods and is an ester of caffeic acid and quinic acid. However, the effect of the structures of both chlorogenic and caffeic acids on their antioxidant and antidiabetic potentials have not been fully understood. Thus, this study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid with α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in vitro. The inhibitory effect of the phenolic acids on α-amylase and α-glucosidase activities was evaluated. Thereafter, their antioxidant activities as typified by their 1,1-diphenyl-2 picrylhydrazyl radical scavenging ability and ferric reducing antioxidant properties were determined. The results revealed that both phenolic acids inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (2-8 μg/mL). However, caffeic acid had a significantly (pchlorogenic acid (α-amylase IC50=9.10 μg/mL and α-glucosidase IC50=9.24 μg/mL). Furthermore, both phenolic acids exhibited high antioxidant properties, with caffeic acid showing higher effects. The esterification of caffeic acid with quinic acid, producing chlorogenic acid, reduces their ability to inhibit α-amylase and α-glucosidase activities. Thus, the inhibition of α-amylase and α-glucosidase activities by the phenolic acids could be part of the possible mechanism by which the phenolic acids exert their antidiabetic effects.

  1. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  2. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins.

    Science.gov (United States)

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2015-12-28

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC50 values for aflatoxins G₁ and G₂ were 17.18 ng·mL(-1) and 19.75 ng·mL(-1), respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL(-1). To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.

  3. Detection of immunoglobulin M and G antibodies against cytomegalovirus early and late antigens by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Middeldorp, J M; Jongsma, J; ter Haar, A; Schirm, J; The, T H

    1984-10-01

    A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) is described for the detection of immunoglobulin M and antibodies with specifity for human cytomegalovirus (CMV) early (CMV-EA) and late (CMV-LA) antigens. The emphasis is on the production of high-quality CMV antigens, CMV-EA and CMV-LA separately, and conditions for their application in the ELISA. The induction of CMV-EA and -LA in infected cell extracts was studied in detail by using human sera with defined antibody specificity for CMV-EA and CMV-LA. This resulted in the development of a simple whole cell extraction procedure that provided a high yield of CMV antigens with reproducible antigen quality. The antigens were specific for the detection of anti-CMV antibodies. The influence of autoantibodies on the determination of CMV-specific antibodies was investigated. Parallel analysis of 322 human sera by indirect immunofluorescence and ELISA showed a high correlation between both assays (r = 0.9674 for CMV-EA and 0.9362 for CMV-LA). Antibody titers determined by ELISA were equal to (for CMV-EA) or slightly higher (for CMV-LA) that those determined by immunofluorescence but significantly higher (20- to 5,120-fold) than those determined by complement fixation. From 191 sera positive by ELISA (titer greater than or equal to 40) 4 (2.1%) were negative by immunofluorescence (titer less than 40), and from 61 ELISA-positive sera 12 (19.6%) were negative (titer less than 8) when tested by complement fixation. Consequently, ELISA for CMV may prove to be more reliable for the selection of CMV-seronegative blood donors than these other methods. The use of high-quality antigens allows more economic handling of large-scale serum determinations. Possibilities for further automation are discussed.

  4. Memory T-cell response to rotavirus detected with a gamma interferon enzyme-linked immunospot assay.

    Science.gov (United States)

    Kaufhold, Robin M; Field, Jodie A; Caulfield, Michael J; Wang, Su; Joseph, Heather; Wooters, Melissa A; Green, Tina; Clark, H Fred; Krah, David; Smith, Jeffrey G

    2005-05-01

    Measurements of serum-neutralizing antibody and anti-rotavirus immunoglobulin A (IgA) are the current standard for assessing immune responses following rotavirus vaccination. However, there is ongoing debate as to whether antibody titers correlate with protection against rotavirus gastroenteritis. Children recovering from rotavirus gastroenteritis have increased gamma interferon release from cultured peripheral blood mononuclear cells (PBMCs), suggesting that cell-mediated immunity (CMI) may play a role in viral clearance and protection from subsequent gastroenteritis. We have developed a gamma interferon enzyme-linked immunospot (ELISPOT) assay for evaluation of CMI responses to rotavirus using frozen PBMCs obtained from healthy adults. Responses to three different rotavirus antigen types were analyzed-a peptide pool based on the human VP6 sequence; reassortant human:bovine vaccine strains; and cell culture-adapted (CCA) human G1, G2, G3, G4, and bovine (WC3) G6 strains. The reassortant strains consist of a bovine WC3 genome background expressing the human rotavirus surface proteins VP7 (G1, G2, G3, or G4) or VP4 (P1). Responses to titrations of the peptide pool as well as CCA and reassortant strains were assessed. Gamma interferon ELISPOT responses were similar for CCA and reassortant strains, whether live or UV inactivated, and when tested either individually or pooled. For most subjects, responses to the VP6 peptide pool positively correlated with responses to CCA and reassortant strains. Cell depletion studies indicate the memory responses detected with these frozen adult PBMCs were primarily due to the CD4+ T-cell population. This gamma interferon ELISPOT assay provides a new tool to apply in clinical studies for the characterization of natural or vaccine-induced CMI to rotavirus.

  5. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    International Nuclear Information System (INIS)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A.

    2014-01-01

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings

  6. Comparison of Seven Commercial Antigen and Antibody Enzyme-Linked Immunosorbent Assays for Detection of Acute Dengue Infection

    Science.gov (United States)

    Jarman, Richard G.; Gibbons, Robert V.; Tanganuchitcharnchai, Ampai; Mammen, Mammen P.; Nisalak, Ananda; Kalayanarooj, Siripen; Bailey, Mark S.; Premaratna, Ranjan; de Silva, H. Janaka; Day, Nicholas P. J.; Lalloo, David G.

    2012-01-01

    Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection. PMID:22441389

  7. Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Matsuo, Toshihiro; Miyagawa, Jun-Ichiro; Kusunoki, Yoshiki; Miuchi, Masayuki; Ikawa, Takashi; Akagami, Takafumi; Tokuda, Masaru; Katsuno, Tomoyuki; Kushida, Akira; Inagaki, Takashi; Namba, Mitsuyoshi

    2016-05-01

    The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

  8. Large-Scale Survey of Campylobacter Species in Human Gastroenteritis by PCR and PCR–Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Lawson, A. J.; Logan, J. M. J.; O'neill, G. L.; Desai, M.; Stanley, J.

    1999-01-01

    A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured “Campylobacter spp.” from 464 samples. The PCR methodologies detected 492 Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli (PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections with C. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. lari suggests that it is less important in this context. PMID:10565897

  9. HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis.

    Science.gov (United States)

    Jenkins, M C; Fetterer, R; Schares, G; Björkman, C; Wapenaar, W; McAllister, M; Dubey, J P

    2005-08-10

    The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.

  10. Quantitative computerized western blotting in detail.

    Science.gov (United States)

    Talmi-Frank, Dalit; Jaffe, Charles L; Baneth, Gad

    2015-01-01

    The analysis of antibody reactivity against multiple antigens separated according to their molecular weights is facilitated by western blotting. The distinction between immune dominant and recessive antigens is often difficult and carried out by qualitative or empirical means. Quantitative computerized western blotting (QCWB) analyzes reactivity to specific antigens by providing a statistically measurable value for each band allowing differentiation between immunodominant and immunorecessive determinants. QCWB is useful for both single time point analysis and longitudinal studies where multiple time points are evaluated and the relativities against individual bands compared. This technique can be employed to study humoral responses to complex antigenic mixtures such as allergens and infectious agents, or identify serologic markers for early diagnosis of cancer, autoimmune or infectious diseases, or to monitor patient's clinical status.

  11. Methodological considerations for improving Western blot analysis.

    Science.gov (United States)

    MacPhee, Daniel J

    2010-01-01

    The need for a technique that could allow the determination of antigen specificity of antisera led to the development of a method that allowed the production of a replica of proteins, which had been separated electrophoretically on polyacrylamide gels, on to a nitrocellulose membrane. This method was coined Western blotting and is very useful to study the presence, relative abundance, relative molecular mass, post-translational modification, and interaction of specific proteins. As a result it is utilized routinely in many fields of scientific research such as chemistry, biology and biomedical sciences. This review serves to touch on some of the methodological conditions that should be considered to improve Western blot analysis, particularly as a guide for graduate students but also scientists who wish to continue adapting this now fundamental research tool. Copyright 2009 Elsevier Inc. All rights reserved.

  12. Subcellular western blotting of single cells.

    Science.gov (United States)

    Yamauchi, Kevin A; Herr, Amy E

    2017-01-01

    Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.

  13. Glycosaminoglycan blotting and detection after electrophoresis separation.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca

    2015-01-01

    Separation of glycosaminoglycans (GAGs) by electrophoresis and their characterization to the microgram level are integral parts of biochemical research. Their blotting on membranes after electrophoresis offers the advantage to perform further analysis on single separated species such as identification with antibodies and/or recovery of single band. A method for the blotting and immobilizing of several nonsulfated and sulfated complex GAGs on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose-gel electrophoresis is illustrated. This approach to the study of these complex macromolecules utilizes the capacity of agarose-gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses. Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary blotted after their separation in agarose-gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100 % and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 μg. Nonsulfated polyanions, for example hyaluronic acid (HA), may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 μg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes used for immunological detection or other applications.

  14. Recent Advances in Microscale Western Blotting.

    Science.gov (United States)

    Sanders, Brittany J; Kim, Daniel C; Dunn, Robert C

    2016-10-21

    Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues. While the technique has evolved since its inception, the last decade has witnessed a paradigm shift in Western blotting technology. The introduction of capillary and microfluidic platforms has significantly decreased time and sample requirements while enabling high-throughput capabilities. These advances have enabled Western analysis down to the single cell level in highly parallel formats, opening vast new opportunities for studying cellular heterogeneity. Recent innovations in microscale Western blotting are surveyed, and the potential for enhancing detection using advances in label-free biosensing is briefly discussed.

  15. Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

    Science.gov (United States)

    Gilda, Jennifer E; Ghosh, Rajeshwary; Cheah, Jenice X; West, Toni M; Bodine, Sue C; Gomes, Aldrin V

    2015-01-01

    Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

  16. Evaluation of three enzyme-linked immunosorbent assays for sarcoptic mange diagnosis and assessment in the Iberian ibex, Capra pyrenaica

    Directory of Open Access Journals (Sweden)

    Arián Ráez-Bravo

    2016-10-01

    Full Text Available Abstract Background Sarcoptic mange is a contagious skin disease caused by the mite Sarcoptes scabiei, affecting different mammalian species worldwide including the Iberian ibex (Capra pyrenaica, in which mortalities over 90 % of the population have been reported. No efficient diagnostic methods are available for this disease, particularly when there are low mite numbers and mild or no clinical signs. In this study, three enzyme-linked immunosorbent assays (ELISA developed for dog (ELISA A, Cantabrian chamois (Rupicapra pyrenaica parva (ELISA B and Alpine chamois (Rupicapra rupicapra (ELISA C, were evaluated to detect specific antibodies (IgG to sarcoptic mange in Iberian ibex sera. Methods Serum samples from 131 Iberian ibexes (86 healthy and 45 scabietic were collected from 2005 to 2012 in the Sierra Nevada Natural and National Parks (southern Spain. Based on visual inspection, ibexes were classified into one of three categories, namely healthy (without scabietic compatible lesions, mildly affected (skin lesions over less than 50 % of the body surface and severely affected (skin lesions over more than 50 % of the body surface. The optimal cut-off point, specificity, sensitivity and the area under the curve (AUC were calculated, and the agreement between tests was determined. Moreover, differences in the optical density (OD related to scabies severity have been evaluated for the best test. Results ELISA C showed better performance than the two other tests, reaching higher values of sensitivity (93.0 % and specificity (93.5 % against the visual estimation of the percentage of affected skin, chosen as the gold standard. Significantly higher concentrations of specific antibodies were observed with this test in the mildly and severely infested ibexes than in healthy ones. Conclusions Our results revealed that ELISA C was an optimal test to diagnose sarcoptic mange in the Iberian ibex. Further studies characterizing immune response during the

  17. Enzyme-linked immunosorbent assay characterization of basal variation and heritability of systemic microfibrillar-associated protein 4.

    Directory of Open Access Journals (Sweden)

    Susanne Gjørup Sækmose

    Full Text Available BACKGROUND: Microfibrillar-associated protein 4 (MFAP4 is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM, and variation in systemic MFAP4 (sMFAP4 has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing the basal sMFAP4 variability and the genetic contribution to the basal variation. METHODS: The sandwich ELISA was based on two monoclonal anti-MFAP4 antibodies and was optimized and calibrated with a standard of recombinant MFAP4. The importance of pre-analytical sample handling was evaluated regarding sample tube type, time, and temperature conditions. The mean value structure and variance structure was determined in a twin cohort including 1,417 Danish twins (age 18-67 years by mixed-effect linear regression modeling. RESULTS: The practical working range of the sandwich ELISA was estimated to be 4-75 U/ml. The maximum intra- and inter-assay variation was estimated to be 8.7% and 6.6%, respectively. Sample handling and processing appeared to influence MFAP4 measurements only marginally. The average concentration of sMFAP4 in the serum was 18.9 ± 8.4 (SD U/ml in the twin cohort (95% CI: 18.5-19.4, median sMFAP4 17.3 U/ml. The mean structure model was demonstrated to include waist-hip ratio, age, and cigarette smoking status in interactions with gender. A relatively low heritability of h(2 = 0.24 was found after applying a model including additive genetic factors and shared and non-shared environmental factors. CONCLUSIONS: The described ELISA provides robust measures of the liver fibrosis marker sMFAP4. The low heritability and the relatively

  18. High seroprevalence of Mycoplasma pneumoniae IgM in acute Q fever by enzyme-linked immunosorbent assay (ELISA.

    Directory of Open Access Journals (Sweden)

    Chung-Hsu Lai

    Full Text Available Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA, a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001 and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001 of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5% and seroconversion rate (33.3% of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases with those who were negative (43 cases, the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255, sore throat (8.5% vs. 16.3%, p=0.351, cough (35.6% vs. 23.3%, p=0.199, and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258, were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Western blot profile in HIV infection

    Directory of Open Access Journals (Sweden)

    Sudha T

    2006-01-01

    Full Text Available Background: Although the overall sensitivity and specificity of the western blot (WB test for detection of antibodies to various viral proteins is high, there has been a substantial difference in the timing of the appearance of antibody bands and their intensities during different stages of HIV infection. Aims: Mapping different band patterns of Western blot results and correlating them with stages of HIV infection. Methods: We performed a retrospective study with 1,467 HIV-1 infected cases confirmed by WB test between January 2002 to July 2005, with the objective of mapping different band patterns of western blot results and determining whether the presence or absence of certain bands was associated with any specific stage of HIV infection. For the interpretation of the WB results in this study, the guidelines recommended by NACO, India were followed. Results: Reactivity with all the bands was the most commonly observed WB pattern, occurring in 92.91% (1363/1467 of cases, whereas the other 7.09% showed uncommon band patterns. Of all individual bands, p31 band was the most frequently missing one, absent in 7.09% cases. On classifying the WB reactive cases by the WHO clinical staging system, 38.45% (564/1467 were in Stage 1, 47.99% (704/1467 in stages 2 and 3 and 13.56% in stage 4. Correlation of CD4 cell counts with the various uncommon band patterns showed that only 5.56% (4/72 had counts in the 200-500 cells/µl range, whereas 45.83% and 48.61% had counts of < 200 and> 500 cells/µl respectively. Conclusion: Interpretation of the WB band pattern in combination with clinical features may be occasionally useful in predicting the stage of HIV infection.

  1. Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

    Directory of Open Access Journals (Sweden)

    Marco Tamba

    2010-01-01

    Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

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

    DEFF Research Database (Denmark)

    Barington, T; Sparholt, S; Juul, L

    1992-01-01

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

  3. Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring

    Directory of Open Access Journals (Sweden)

    Miroslav Fojta

    2008-01-01

    Full Text Available Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE, followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

  4. Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked Immunosorbent Assay That Uses a Specific Monoclonal Antibody for Sensitive Detection of Ralstonia solanacearum in Asymptomatic Potato Tubers

    Science.gov (United States)

    Caruso, Paola; Gorris, María Teresa; Cambra, Mariano; Palomo, José Luis; Collar, Jesús; López, María M.

    2002-01-01

    Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29°C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods. PMID:12089053

  5. Performance and reliability of five commercial enzyme-linked immunosorbent assay kits in screening for anti-human immunodeficiency virus antibody in high-risk subjects.

    OpenAIRE

    Ozanne, G; Fauvel, M

    1988-01-01

    Anti-human immunodeficiency virus enzyme-linked immunosorbent assay kits marketed by Electro-Nucleonics Inc. (ENI), Genetic Systems Corp. (GSC), Organon Teknika Inc. (OTI), Ortho Diagnostic Systems Inc. (ODSI), and Wellcome Diagnostics (WD) were evaluated by using 289 randomly selected serum samples from a high-risk population and 53 serum samples likely to produce false-positive results. The radioimmunoprecipitation assay was used as the reference test. Sensitivities ranged from 96.51% (ODSI...

  6. Evaluation of an enzyme-linked immunosorbent assay based on crude leishmania histone proteins for serodiagnosis of human infantile visceral leishmaniasis.

    OpenAIRE

    Lakhal, Sami; Mekki, Salima; Ben-Abda, Imène; Mousli, Mohamed; Amri, Fethi; Aoun, Karim; Bouratbine, Aïda

    2012-01-01

    International audience; Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crude Leishmania histone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were ...

  7. Rapid Detection of Campylobacter coli, C. jejuni, and Salmonella enterica on Poultry Carcasses by Using PCR-Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Hong, Yang; Berrang, Mark E.; Liu, Tongrui; Hofacre, Charles L.; Sanchez, Susan; Wang, Lihua; Maurer, John J.

    2003-01-01

    Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobac...

  8. Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

    Science.gov (United States)

    Sackstein, Robert; Fuhlbrigge, Robert

    2015-01-01

    Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex

  9. An alternative strategy to western blot as a confirmatory diagnostic test for HIV infection.

    Science.gov (United States)

    Feng, Xia; Wang, Jibao; Gao, Zhiyun; Tian, Yu; Zhang, Ling; Chen, Huichao; Zhang, Tong; Xiao, Lin; Yao, Jun; Xing, Wenge; Qiu, Maofeng; Jiang, Yan

    2017-03-01

    In China, western blot (WB) is the recommended procedure for the diagnosis of HIV infection. However, this technique is time consuming and labor intensive, and its complexity restricts wide application in resource-limited regions. The aim of this study was to evaluate the efficacy of a dry blood spots (DBS)-urine paired enzyme-linked immunosorbent assay (ELISA) test, instead of WB, for HIV antibody detection. Plasma, DBS, and urine samples were collected from 1213 subjects from different populations. Two diagnostic testing strategies were conducted in parallel. The equivalence of the paired ELISA and WB strategies was assessed. A diagnosis of HIV was determined in 250 subjects according to the paired ELISA test, and in 249 according to the WB strategy. The discordant case was judged HIV-positive during follow-up. In total, 18 subjects were diagnosed with possible HIV using the paired ELISA test, among whom, 11 subjects tested negative with WB, and one was confirmed to be HIV-positive during follow-up. For the remaining 945 subjects, both strategies indicated a negative result. The kappa test indicated good conformity (kappa=0.954) between the two diagnostic strategies. The DBS-urine paired ELISA could be applied as an alternative to WB in HIV diagnosis, which would be valuable in resource-limited regions owing to the associated affordability and ease of use. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Detection of IgM antibodies against canine distemper virus in dog and mink sera employing enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Blixenkrone-Møller, M; Pedersen, I R; Appel, M J; Griot, C

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against canine distemper virus (CDV) in canine and mink serum is described. The diagnostic potential of this technique was evaluated by analyzing sera from natural or experimental infections in dog and mink and negative control sera. These results were compared with results obtained in the developed CDV IgG ELISA and in the virus neutralization test. The IgM test, which requires only a single serum specimen, is a useful method for diagnosing current or recent CDV infections in dog and mink.

  11. Detection of single nucleotide polymorphisms in p53 mutation hotspots and expression of mutant p53 in human cell lines using an enzyme-linked electrochemical assay

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Šimková, Eva; Vychodilová, Zdenka; Brázdová, Marie; Fojta, Miroslav

    2009-01-01

    Roč. 21, č. 15 (2009), s. 1723-1729 ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA203/07/1195; GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : enzyme-linked electrochemical assay * SNP typing * p53 mutation Subject RIV: AQ - Safety, Health Protection, Human - Machine Impact factor: 2.630, year: 2009

  12. Quantification of urinary 5-hydroxyindoleacetic acid by in-house nitrosonaphthol reaction compared with nitrosonaphthol micro column chromatography and enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Joyce Matie Kinoshita da Silva

    2014-06-01

    Full Text Available The aim of this study was to compare the colorimetric "kit" and enzyme-linked immunosorbent assay (ELISA methods to quantify urinary 5-hydroxyindoleacetic acid through the Goldenberg's technique, exploring the potential of replacing it. 24-hour urine samples were tested by Goldenberg's assay and compared with kits. The agreement was almost perfect for the comparison of Goldenberg's assay with both colorimetric kit, and with ELISA kit, considering ≤ 7.5 mg/24h normal cutoff value. Therefore, both "kits" would be good alternatives to Goldenberg's technique due to practicality and agreement between values.

  13. Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

    International Nuclear Information System (INIS)

    Booth, J.C.; Hannington, G.; Bakir, T.M.F.; Stern, H.; Kangro, H.; Griffiths, P.D.; Heath, R.B.

    1982-01-01

    The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems. (author)

  14. Protein purification and analysis: next generation Western blotting techniques.

    Science.gov (United States)

    Mishra, Manish; Tiwari, Shuchita; Gomes, Aldrin V

    2017-11-01

    Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

  15. Immunoblot Patterns of Taenia asiatica Taeniasis

    OpenAIRE

    Jeon, Hyeong-Kyu; Eom, Keeseon S.

    2009-01-01

    Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97 kDa and 21.5 kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and ...

  16. A rapid Western blotting protocol for the Xenopus oocyte.

    Science.gov (United States)

    Lin-Moshier, Yaping; Marchant, Jonathan S

    2013-03-01

    Often experimentalists require a quantitative assessment of the levels of heterologously expressed proteins to best interpret changed Ca(2+) signaling patterns. Here, we detail a rapid and convenient western blotting method for individual Xenopus oocytes. The method exploits recently introduced rapid blotting systems, commercially available from Invitrogen (iBlot) or Bio-Rad (Trans-Blot Turbo). The key advantage is speed: from live cell to transferred membrane in western blotting to assess relative expression levels, even after a long day of Ca(2+) imaging experiments.

  17. Measurement of antibody to Dermatophilus congolensis in sera from cattle in the west of Scotland by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Lloyd, D H

    1981-11-07

    Serum antibody titres to Dermatophilus congolensis demonstrated by an enzyme-linked immunosorbent assay (ELISA) in young steers and in adult cows from an Ayrshire herd showed a bimodal distribution and provided evidence of subclinical infection. Very high titres detected in sera from crossbred Galloway steers were indicative of recent or existing infection which may have been masked by concurrent ringworm. The ELISA is a sensitive and technically simple method which enables sera to be screened for evidence of infection by D congolensis which may otherwise pass unrecognised. Such infections may be of importance not only in the epidemiology of the disease in farm animals but also as a potential source of infection for man and his domestic pets.

  18. Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP) - Assessment of corresponding epitopes

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Larsen, D.V.; Zhang, C.

    2010-01-01

    Objectives: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. Methods: Monoclonal antibodies were raised against...... corresponding rat and human PINP sequences and competitive assays were developed for each species. They were evaluated in relevant pre-clinical or clinical studies. Results: The antibody characterizations indicated that PINP indeed was recognized. Technical robust assays were obtained. Rat PINP and tALP showed...... similar patterns in the gold standard osteoporosis rat ovariectomized (OVX) model. No liver contribution was observed in the liver fibrosis rat bile duct ligation model (BDL). In an osteoporosis study, the human serum PINP levels were significantly decreased after ibandronate treatment compared to placebo...

  19. DETECTION OF OXYTETRACYCLINE IN BROILER CHICKEN MEAT MARKETED IN SEVERAL CITIES IN JAVA ISLAND USING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA METHOD

    Directory of Open Access Journals (Sweden)

    R. Widiastuti

    2015-09-01

    Full Text Available Oxytetracycline (OTC is one of the tetracycline (TCs broad-spectrum antibiotics widely used inthe chicken industry. However, improper use of OTC with excessive doses potentially leads to residueformation in animal products that can be harmful to consumers in the form of allergic reaction orresistance. This study aimed to detect OTC residues in broiler chicken meat, marketed in traditionalmarkets and supermarkets in Depok, Bekasi, Bandung, Cilegon, Surakarta and Yogyakarta using indirectcompetitive enzyme-linked immunosorbent assay (icELISA method. The analyses of 67 broiler meatsamples showed only 1 (1.5% sample was positive for the OTC residue at 86.1 ng/g which meant belowthe maximum residue limits of permissible OTC (100 ng/g. Nevertheless, a stricter regulation for theuse of OTC in the poultry industry and the monitoring of its residue in chicken products prior tomarketing is still necessary to avoid the adverse effects of the residue present in animal products.

  20. Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b

    International Nuclear Information System (INIS)

    Kristensen, K.; Weis Bentzon, M.

    1992-01-01

    The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was indepenedent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines. (au)

  1. Development of an enzyme-linked immunosorbent assay to determine the numbers of chemolithotrophic bacteria at acid-mine-drainage sites. Technical report (Final)

    Energy Technology Data Exchange (ETDEWEB)

    Blake, R.C.; Revis, N.W.; Holdsworth, G.

    1990-09-01

    Thiobacillus ferrooxidans is a prominent member of a group of chemo-lithotrophic bacteria that bear principal responsibility for the formation of acid mine drainage. A prototype enzyme-linked immunosorbent assay (ELISA) for enumerating and qualifying T. ferrooxidans was assembled and characterized. The immunoassay protocol consisted of sequential incubations of the sample with (i) the primary antibody, (ii) the enzyme-labeled secondary antibody, and (iii) a chromogenic substrate specific for the enzyme lable. The necessary reagents comprised primary polyclonal rabbit antibodies directed against T. ferrooxidans ATCC 23270, alkaline phosphatase-copled goat anti-rabbit polyclonal antibodies, and phenolphrhalein monophosphate. The ELISA developed herein correctly identified whether iron-oxidizing bacteria were present in each of 4 samples supplied and analyzed by an independent laboratory. Sufficient preliminary data was obtained to warrant further research and development activities.

  2. Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b

    Energy Technology Data Exchange (ETDEWEB)

    Kristensen, K. (Streptococcus Department, Statens Seruminstitut, Copenhagen (Denmark)); Weis Bentzon, M. (Department of Biostatistics, Statens Seruminstitut, Copenhagen (Denmark))

    1992-01-01

    The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was indepenedent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines. (au).

  3. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

    Science.gov (United States)

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  4. Bilirubin oxidase label for an enzyme-linked affinity assay with O2 as substrate in a neutral pH NACL solution.

    Science.gov (United States)

    Kim, Hyug-Han; Zhang, Yongchao; Heller, Adam

    2004-04-15

    Laccase, a copper enzyme catalyzing the four-electron reduction of O(2) to water, has been shown by others to be a useful label in enzyme-linked immunoassays, in which the substrate is ambient O(2) instead of an added chemical, such as hydrogen peroxide, or a phosphate ester of a phenol. Laccase-catalyzed O(2) reduction is, however, inhibited by halides, which complex the enzyme's copper ions. Replacement of laccase by bilirubin oxidase, a copper enzyme retaining its maximal activity at high chloride concentrations and at pH 7.2, allows enzyme-amplified affinity assays with O(2) as the substrate in neutral-pH chloride solutions, exemplified here by the assay of DNA, the duplexes of which are unstable at low ionic strength but are stable in strong NaCl solutions.

  5. Seroprevalence study of Equine rhinitis B virus (ERBV) in Australian weanling horses using serotype-specific ERBV enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Horsington, Jacquelyn; Hartley, Carol A; Gilkerson, James R

    2013-09-01

    Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50-80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74-86%); ERBV-3 antibodies were most prevalent (58-62%) and ERBV-2 antibodies were least prevalent (10-16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.

  6. Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Oviedo, J M; Valiño, F; Plasencia, I

    2001-01-01

    We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...... used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein...... pronounced changes on the conformation of SP-B when the solvent was evaporated and dry lipid-protein films were formed, a necessary step to expose protein to antibodies in ELISA. Under these conditions, negatively charged lipids, but not zwitterionic ones, induced a marked decrease on the ellipticity of SP...

  7. Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S)

    DEFF Research Database (Denmark)

    Leeming, Diana J; Nielsen, Mette J; Dai, Yueqin

    2012-01-01

    Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P type IV collagen...... expression in BDL rats (r = 0.49, P serum assay specific for P4NP 7S was highly related to liver fibrosis...

  8. Development of a solid-phase extraction-enzyme-linked immunosorbent assay for the determination of 17beta-19-nortestosterone levels in antifatigue functional foods.

    Science.gov (United States)

    Zhang, Yan; Pan, Tao; Fang, Guozhen; Ma, Dongyue; Wang, Shuo

    2009-10-01

    17beta-19-nortestosterone (17beta-NT) has been illegally used in antifatigue functional foods to promote muscle growth and improve endurance. A rapid and sensitive solid-phase extraction-enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and successfully applied to analyze the levels of 17beta-NT in antifatigue functional foods. A polyclonal antibody against 17beta-NT was produced from rabbits immunized with the 17beta-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17beta-NT. The concentration causing 50% inhibition (IC(50)) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL, respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL. C(18) cartridges were investigated for use in removing the effects of matrix in foods, and the sample purification protocol was optimized. Using the developed SPE-ELISA method, recoveries of functional food samples were obtained in the range of 71% to 91.5%. Moreover, 2 kinds of antifatigue functional foods were analyzed using the established ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2 methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method proved that it is a specific, sensitive, and precise tool that can be used for the practical detection of 17beta-NT in various antifatigue functional food samples.

  9. Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot.

    Science.gov (United States)

    Putra, Sulistyo Emantoko Dwi; Tsuprykov, Oleg; Von Websky, Karoline; Ritter, Teresa; Reichetzeder, Christoph; Hocher, Berthold

    2014-01-01

    Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04-5.71%) as assessed by coefficient of variation. ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.

  10. The Design of a Quantitative Western Blot Experiment

    Directory of Open Access Journals (Sweden)

    Sean C. Taylor

    2014-01-01

    Full Text Available Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013 and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  11. The design of a quantitative western blot experiment.

    Science.gov (United States)

    Taylor, Sean C; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

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

    Science.gov (United States)

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

    2017-11-01

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

  13. Human immune response to botulinum pentavalent (ABCDE) toxoid determined by a neutralization test and by an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Siegel, L S

    1988-11-01

    To determine the immune status of persons receiving botulinum pentavalent (ABCDE) toxoid and to evaluate the effectiveness of the vaccine, we surveyed immunized individuals for neutralizing antibodies to type A and to type B botulinum toxins. After the primary series of three immunizations administered at 0, 2, and 12 weeks, 21 of 23 persons tested (91%) had a titer for type A that was greater than or equal to 0.08 international units (IU)/ml, and 18 (78%) had a titer for type B of greater than or equal to 0.02 IU/ml. (One international unit is defined as the amount of antibody neutralizing 10,000 mouse 50% lethal doses of type A or B botulinum toxin). Just before the first annual booster, 10 of 21 (48%) and 14 of 21 (67%) people lacked a detectable titer for type A and for type B, respectively. After the first booster, all individuals tested had a demonstrable titer to both types A and B. Of 77 persons who had previously received from one to eight boosts of the toxoid, 74 (96%) had an A titer of greater than or equal to 0.25 IU/ml and would not require an additional booster, according to the recommendations of the Centers for disease Control. However, only 44 of 77 (57%) had a B titer of greater than or equal to 0.25 IU/ml. In each group by booster number, even the group having had eight boosts, at least one person would require reimmunization on the basis of B titer. There was a wide range of antibody levels among individuals at the same point in the immunization scheme. Results from an enzyme linked immunosorbent assay, with purified type A or type B neurotoxin as the capture antigen, were compared with neutralization test results on 186 serum samples for type A and 168 samples for type B. Statistically, the correlation coefficients for results from the two assays were high (r = 0.69, P < 0.0001, for type A and r = 0.77, P < 0.0001, for type B). However, due to the wide dispersion of values obtained, using enzyme-linked immunosorbent assay results to predict

  14. Reliability of Blotting Techniques to Assess Contact Lens Water Content.

    Science.gov (United States)

    Cañadas, Pilar; López-Miguel, Alberto; Gómez, Alba; López-de la Rosa, Alberto; Fernández, Itziar; González-García, María J

    2018-02-15

    To determine the reliability of wet and modified dry blotting techniques used in the gravimetric method to assess contact lens (CL) water content (WC), the accuracy of both techniques in comparison with the nominal WC, and also their agreement. We evaluated hydrated and dry CL mass values and WC using the gravimetric method in 440 daily disposable CLs. Samples assessed corresponded to Dailies Total 1, Dailies AquaComfort Plus, 1-Day Acuvue TruEye, and Biotrue ONEday. Back vertex power ranged from +3.00 diopters (D) to -6.00 D. Within-subject coefficient of variation (CVw) and intraclass correlation coefficients were calculated. Bland-Altman analysis was also performed. The modified dry blotting technique yielded significantly (P≤0.0001) higher hydrated CL mass values. The wet blotting technique provided significantly (P≤0.04) better consistency than the modified dry one. Values of CVw for wet and modified dry blotting techniques ranged from 1.2% to 2.1% and from 3.7% to 5.4%, respectively. As for dry CL mass values, CVw values were not significantly different (P≥0.05) between wet (range: 1.1%-1.9%) and dry (range: 1.0%-5.1%) blotting techniques, except for Dailies AquaComfort Plus (P=0.03). Bland-Altman analysis showed poor agreement between the techniques. The wet blotting technique yielded WC values close (around 1%) to nominal ones, in contrast to modified dry blotting technique (≥2.5%). The wet blotting technique is not only more reliable than the modified dry one when obtaining hydrated CL mass but also provides more accurate nominal WC measurements. Agreement between the techniques was poor.

  15. Multistrip Western blotting to increase quantitative data output

    OpenAIRE

    Kiyatkin, Anatoly; Aksamitiene, Edita

    2009-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single mem...

  16. Western Blotting Analysis of CCN Proteins in Calcified Tissues.

    Science.gov (United States)

    Kawaki, Harumi; Kubota, Satoshi; Takigawa, Masaharu

    2017-01-01

    Western blotting is widely used for protein analysis. We routinely perform such analysis for evaluating the production levels of CCN family proteins in a variety of cells under various conditions. In this chapter, we describe our Western blotting protocol to estimate protein production profiles of CCN family members after having assessed the specificity of the antibodies against each CCN member protein to ensure no cross-reaction with other CCN member proteins.

  17. The Design of a Quantitative Western Blot Experiment

    OpenAIRE

    Taylor, Sean C.; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in...

  18. Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells

    Science.gov (United States)

    Wang, Jian; She, Yongxin; Wang, Miao; Jin, Maojun; Li, Yongfei; Wang, Jing; Liu, Yuan

    2015-01-01

    A novel enzyme-linked receptor assay (ELRA) based on β2-adrenergic receptor (β2-AR) has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists) in urine. Human embryonic kidney cells (HEK293) were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine. PMID:26422475

  19. Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

    Directory of Open Access Journals (Sweden)

    Jian Wang

    Full Text Available A novel enzyme-linked receptor assay (ELRA based on β2-adrenergic receptor (β2-AR has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists in urine. Human embryonic kidney cells (HEK293 were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

  20. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    2009-01-01

    Full Text Available To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver, we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2>0.95, which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

  1. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    Science.gov (United States)

    Zhang, Yan; Wang, FengXia; Fang, Li; Wang, Shuo; Fang, GuoZhen

    2009-01-01

    To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2 > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods. PMID:19826637

  2. Rapid and sensitive detection of redspotted grouper nervous necrosis virus (RGNNV) infection by aptamer-coat protein-aptamer sandwich enzyme-linked apta-sorbent assay (ELASA).

    Science.gov (United States)

    Zhou, L; Li, P; Ni, S; Yu, Y; Yang, M; Wei, S; Qin, Q

    2017-12-01

    Redspotted grouper nervous necrosis virus (RGNNV) is one of the most devastating pathogens in the aquaculture of the grouper, Epinephlus sp., worldwide. The early and rapid diagnosis of RGNNV is important for the prevention of RGNNV infection. In this study, an aptamer (A10)-based sandwich enzyme-linked apta-sorbent assay (ELASA) was developed for RGNNV diagnosis. This sandwich ELASA showed high specificity for the RGNNV coat protein (CP) and virions in virus-infected cells and tissues. At the optimized working concentration of 200 nM of aptamer, the ELASA could detect RGNNV in the lysates of as few as 4 × 10 3 RGNNV-infected GB cells. Incubation for 10 min was sufficient to produce accurate results. The sandwich ELASA was most stable at incubation temperatures of 4-25°C, but could still distinguish RGNNV-infected samples from the controls at 37°C. It could detect RGNNV infection in brain lysates diluted 1/10, with results consistent with those of reverse transcription PCR, although with 10% less sensitivity. The main equipment required includes dissection tools, a water bath, Pierce™ Streptavidin Coated Plates and a microplate reader. The sandwich ELASA has great potential utility for the rapid and sensitive diagnosis of RGNNV in its early stages by fish farmers. © 2017 John Wiley & Sons Ltd.

  3. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    Science.gov (United States)

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases. PMID:22815145

  4. Assessment of a 27-kDa antigen in enzyme-linked immunosorbent assay for the diagnosis of fasciolosis in Vietnamese patients.

    Science.gov (United States)

    Nguyen, T G T; Le, T H; De, N V; Doan, T T; Dao, T H T; Vercruysse, J; Dorny, P

    2010-04-01

    Fasciolosis has emerged as an important zoonotic disease in many parts of the world. In recent years, an increasing number of human cases were reported in Vietnam. In this study, the 27-kDa component protein from the excretory/secretory production of adult Fasciola gigantica, purified by high performance liquid chromatography, was assessed in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola spp. for diagnosis of human fasciolosis. The ELISA showed a high sensitivity (100%) and specificity (97.67%) when tested on patients with fasciolosis, other parasitic infections, cholangiocarcinoma and on healthy controls. The assay was applied for diagnosis on 143 patients in the Viet Duc-Hanoi hospital who presented with clinical signs of liver disease and lesions in their livers as shown by imaging techniques. Antibodies were found in 37 (25.9%) of these patients, of whom only 3 shed Fasciola eggs in their stools (2.1%). The excellent response to triclabendazole treatment of 37 sero-positive patients confirmed the diagnosis of fasciolosis. This study demonstrated the diagnostic potential for human fasciolosis of the 27-kDa antigen ELISA. Fasciolosis should be considered in the differential diagnosis of hepatic disease in Vietnam.

  5. Development of a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sunset Yellow FCF in food samples.

    Science.gov (United States)

    Xing, Yue; Meng, Meng; Xue, Huyin; Zhang, Taichang; Yin, Yongmei; Xi, Rimo

    2012-09-15

    Sunset Yellow FCF is widely used as food additives to make foods more attractive. Due to its abuse and potential risk to human health, Sunset Yellow FCF is precisely limited to use in food. To monitor the illegal use of Sunset Yellow FCF, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with satisfactory sensitivity and specificity was developed. A carboxyl group was introduced to Sunset Yellow FCF, then the modified hapten was coupled with carrier proteins to synthesize the immunogen and coating antigen. The IC(50) value of 0.52 ng mL(-1) and detection limit of 25 pg mL(-1) (in buffer) were achieved by this method. The cross-reactivity values of the antibodies with six structurally related colorants were less than 1.5%, indicating the high selectivity. Three kinds of food samples (beverage, dried beancurd, braised pork) and serum were chosen to evaluate the application of the immunoassay in real systems. The limits of detection (LOD) in the above three food samples were 0.12, 0.04 and 1.11, respectively (mean+3SD). The recovery (94%-106%), intra-assay (foods and serum samples were also acceptable. The results suggest that this ELISA method is a specific, sensitive and simple method for the determination of Sunset Yellow FCF additives. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    1998-03-15

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

  7. A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles

    Science.gov (United States)

    2014-01-01

    This paper presents a functional nanoparticle-enhanced enzyme-linked immunosorbent assay (FNP-ELISA) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Immunomagnetic nanoparticles (IMMPs) conjugated with monoclonal anti-O157:H7 antibody were used to capture E. coli O157:H7. Beacon gold nanoparticles (B-GNPs) coated with polyclonal anti-O157:H7 and biotin single-stranded DNA (B-DNA) were then subjective to immunoreaction with E. coli O157:H7, which was followed by streptavidin-horseradish peroxidase (Strep-HRP) conjugated with B-GNPs based on a biotin-avidin system. The solutions containing E. coli O157:H7, IMMPs, B-GNPs, and Strep-HRP were collected for detecting color change. The signal was significantly amplified with detection limits of 68 CFU mL-1 in PBS and 6.8 × 102 to 6.8 × 103 CFU mL-1 in the food samples. The FNP-ELISA method developed in this study was two orders of magnitude more sensitive than immunomagnetic separation ELISA (IMS-ELISA) and four orders of magnitude more sensitive than C-ELISA. The entire detection process of E. coli O157:H7 lasted only 3 h, and thus FNP-ELISA is considered as a time-saving method. PMID:24864164

  8. Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies.

    Science.gov (United States)

    Bruno, John G; Richarte, Alicia M; Phillips, Taylor; Savage, Alissa A; Sivils, Jeffrey C; Greis, Alex; Mayo, Michael W

    2014-01-01

    A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼ 100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.

  9. Development of a nano-SiO2based enzyme-linked ligand binding assay for the determination of ibuprofen in human urine.

    Science.gov (United States)

    Wang, Qian-Long; Xie, Jing; Li, Xing-De; Ding, Li-Sheng; Liang, Jian; Luo, Pei; Qing, Lin-Sen

    2017-05-15

    The application domains of classic enzyme-linked ligand binding assay (ELBA) is relatively narrow due to the high cost and hardly available binding receptor. In here, we described for the first time the possibility of developing a new ELBA based on silica nanoparticles (nano-SiO 2 ) to assess the ibuprofen in human urine. Nano-SiO 2 with a large surface area was introduced as stationary phase to improve the analytical performance. In the experiment, a competitively binding procedure with human serum albumin (HSA) was performed between the ibuprofen presented in sample and horseradish peroxidase labeled ibuprofen (HRP-ibuprofen) subsequently added. After centrifugal separation, the HRP/ibuprofen/nano-SiO 2 composite catalyzed the substrate solution (TMB/H 2 O 2 ) with a color change from colorless to yellow for quantitative measurement via an ultraviolet spectrophotometer. As a validation of the new principle, the developed nano-ELBA method was applied in the determination of ibuprofen excreted in human urine with excellent performance. This detection range only depends on the solubility of ligand and sensitivity of UV spectrophotometer. Our results indicate that this new method demonstrated to be able to rapidly and adequately determine the concentration of components in biological samples and advocate its effectiveness for various applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Anti-Toxoplasma gondii antibody detection in serum and urine samples by enzyme-linked immunosorbent assay in HIV-infected patients

    Directory of Open Access Journals (Sweden)

    Sayan Bhattacharyya

    2013-01-01

    Full Text Available Background: Toxoplasmosis is a common parasitic infection of man, and reactivation of latent disease in HIV-infected patients can cause fatal encephalitis. Diagnosis depends on demonstration of parasite-specific antibodies in serum. In HIV-infected patients, IgM is often undetectable, whereas IgG remains detectable in the majority. Urine sample is very easily available and has not been evaluated for immunodiagnosis of toxoplasmosis. Aim: The study was an effort to find whether urine sample can be used in place of serum for immunodiagnosis of toxoplasmosis. Materials and Methods: Enzyme-linked immunosorbent assay (ELISA was carried out in serum and urine samples collected from 100 HIV-infected patients to detect anti-toxoplasma IgG and IgM antibodies and whether positivity correlated with the CD4 T-cell counts of patients. Results: In this study, we observed that there was no significant difference in positivity of anti-toxoplasma IgM and IgG between serum and urine samples of HIV-infected patients by ELISA. There was a negative correlation between CD4 count and seropositivity. Conclusion: Urine sample can be satisfactorily used in place of serum for immunodiagnosis of toxoplasmosis.

  11. Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust

    Directory of Open Access Journals (Sweden)

    Melanie Sanders

    2016-04-01

    Full Text Available A sample preparation method was developed for the screening of deoxynivalenol (DON in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA was compared to the sensor-based techniques of surface plasmon resonance (SPR and biolayer interferometry (BLI in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889 was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg.

  12. Removal of Lipid from Serum Increases Coherence between Brucellosis Rapid Agglutination Test and Enzyme-linked Immunosorbent Assay in Bears in Alaska, USA.

    Science.gov (United States)

    Godfroid, Jacques; Beckmen, Kimberlee; Helena Nymo, Ingebjørg

    2016-10-01

    In cases of chronic Brucella spp. infection, results of the rose bengal plate test (RBPT) and indirect enzyme-linked immunosorbent assay (ELISA) should be coherent, as reported in controlled conditions in the literature. We compared RBPT and ELISA results in 58 Alaska grizzly bears ( Ursus arctos horribilis), eight Kodiak brown bears ( Ursus arctos middendorffi), and six Alaska Peninsula brown bears ( Ursus arctos gyas). Of the 72 bears tested, 42 (58%) were ELISA positive and 53 (73%) were RBPT positive. However, the coherence between the tests was only fair (K=0.37, SE=0.11), suggesting that either the serologic results were not compatible with Brucella spp. infection or that there was a technical problem with the tests. To address a potential technical problem, we performed a 30-min chloroform/centrifugation cleanup. Following cleanup, the ELISA identified 43 positives (59%) and the RBPT identified 47 (65%), and the coherence between the tests was much improved (K=0.80, SE=0.07). We recommend cleaning wildlife sera with a high lipid content before performing RBPT and performing RBPT and ELISA in parallel to assess coherence. Our results suggest that Alaskan brown bears have been exposed to Brucella spp.

  13. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red

    International Nuclear Information System (INIS)

    Ju Chunmei; Tang Yong; Fan Huiying; Chen Jinding

    2008-01-01

    To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01 ng mL -1 in phosphate-buffered saline (PBS) buffer and 0.5 ng g -1 in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  15. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    Directory of Open Access Journals (Sweden)

    Kathleen Ingenhoven

    2017-07-01

    Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Widiastuti R

    2013-06-01

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

  18. Novel biotechnological platform based on baculovirus occlusion bodies carrying Babesia bovis small antigenic peptides for the design of a diagnostic enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    López, M G; Pallarés, H M; Alfonso, V; Carmona, S J; Farber, M; Taboga, O; Wilkowsky, S E

    2018-01-01

    Baculoviruses are large DNA virus of insects principally employed in recombinant protein expression. Its ability to form occlusion bodies (OBs), which are composed mainly of polyhedrin protein (POLH), makes them biotechnologically attractive, as these crystals (polyhedra) can incorporate foreign peptides and can be easily isolated. On the other hand, peptide microarrays allow rapid and inexpensive high-throughput serological screening of new candidates to be incorporated to OBs. To integrate these 2 biotechnological approaches, we worked on Babesia bovis, one of the causative agents of bovine babesiosis. Current molecular diagnosis of infection with B. bovis includes enzyme-linked immunosorbent assay (ELISA) techniques, which use merozoite lysate obtained from infected bovine erythrocytes. However, it is important to produce recombinant antigens that replace the use of crude antigens. Here, we describe a new biotechnological platform for the design of indirect ELISAs based on 5 antigenic peptides of 15 amino acid residues of B. bovis (ApBb), selected from a peptide microarray and expressed as a fusion to POLH. An Sf9POLH E44G packaging cell line infected with recombinant baculoviruses carrying POLH-ApBb fusions yielded higher levels of chimeric polyhedra, highlighting the advantage of a trans-contribution of a mutant copy of polyhedrin. Finally, the use of dissolved recombinant polyhedra as antigens was successful in an ELISA assay, as B. bovis-positive sera recognized the fusion POLH-ApBb. Thus, the use of this platform resulted in a promising alternative for molecular diagnosis of relevant infectious diseases.

  19. Comparative study of direct and indirect immunofluorescence and of bullous pemphigoid 180 and 230 enzyme-linked immunosorbent assays for diagnosis of bullous pemphigoid.

    Science.gov (United States)

    Sárdy, Miklós; Kostaki, Dimitra; Varga, Rita; Peris, Ketty; Ruzicka, Thomas

    2013-11-01

    Direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA) are used for the laboratory diagnosis of bullous pemphigoid (BP). The diagnostic value of DIF and IIF on rabbit and monkey esophagus or human salt-split skin and commercial ELISAs was assessed. This was a single-center retrospective study where 313 patients with BP were compared with 488 control subjects. DIF was the most sensitive test (90.8%) whereas sensitivities for IIF on rabbit esophagus, IIF on monkey esophagus, IIF on salt-split skin, BP180 ELISA, and BP230 ELISA were 76.0%, 73.2%, 73.3%, 72.0%, and 59.0%, respectively. The sensitivity of the serologic tests was 88.8% altogether. The specificities for DIF, IIF on rabbit esophagus, IIF on monkey esophagus, IIF on salt-split skin, BP180 ELISA, and BP230 ELISA were 98%, 96.5%, 97.1%, 100%, 94.1%, and 99.2%, respectively. The retrospective nature of study was a limitation. Correlation of diagnostic data with clinical manifestations or disease course was not possible. In suspected BP, both serologic tests and DIF have to be performed because of a sensitivity issue. Although the ELISAs had a relatively low sensitivity, the serologic tests altogether almost reached the level of sensitivity of DIF. The specificities of all assays were excellent. Copyright © 2013 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

  20. Relative efficiency of polymerase chain reaction and enzyme-linked immunosorbant assay in determination of viral etiology in congenital cataract in infants

    Directory of Open Access Journals (Sweden)

    Shyamala G

    2008-01-01

    Full Text Available Background: Perinatal viral infections of fetus are among the leading causes of congenital cataract and identifying the viral etiology is important. Objectives: To detect the presence of Rubella virus (RV, herpes simplex virus (HSV and cytomegalovirus (CMV in lens aspirate specimens obtained from patients with congenital cataract and relate the results with serology. Setting and Design: Prospective study carried out in tertiary care hospital. Materials and Methods: Fifty lens aspirates from 50 infants with congenital cataract were subjected to HSV, RV isolation and polymerase chain reaction (PCR for detection of HSV and CMV. Reverse transcription polymerase chain reaction (RT-PCR was applied for RV detection. Peripheral blood specimens were screened for anti-HSV, RV and CMV antibodies by enzyme-linked immunosorbant assay (ELISA. Results: Rubella virus was detected in nine (18% lens aspirates, by nRT-PCR which includes six positive by culture. HSV-2 DNA was detected in nine other lens aspirates, while CMV was not detected by PCR. Serological results did not correlate with the presence of viruses in the lens aspirates. This is the first report of detection of HSV-2 DNA in cases of congenital cataract. Conclusions: Cytomegalovirus may not be playing a significant role in causation of congenital cataract. The role of serology in identifying causative viral infection for congenital cataract needs to be re-evaluated.

  1. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

  2. Establishment of the enzyme-linked immunosorbent assay system to detect the amino terminal secretory form of rat Erc/Mesothelin.

    Science.gov (United States)

    Nakaishi, Masayuki; Kajino, Kazunori; Ikesue, Masahiro; Hagiwara, Yoshiaki; Kuwahara, Maki; Mitani, Hiroaki; Horikoshi-Sakuraba, Yuko; Segawa, Tatsuya; Kon, Shigeyuki; Maeda, Masahiro; Wang, Tegexibaiyin; Abe, Masaaki; Yokoyama, Masayoshi; Hino, Okio

    2007-05-01

    By representational difference analysis, we previously identified the rat Erc (Expressed in renal carcinoma) gene that was more abundantly expressed in the renal carcinoma tissues of Eker rats than in the rat normal kidney. In this study, we raised antibodies against the amino-terminal portion of the rat Erc, and demonstrated the existence of a approximately 30-kDa secretory form in the supernatant of cultured cells derived from rat renal carcinoma. The enzyme-linked immunosorbent assay (ELISA) system using these antibodies detected high concentrations of this form in the sera of Eker rats bearing renal carcinomas, and in the sera of rats transplanted with mesothelioma cells. Mesothelin, a human homolog of the rat Erc, was recently reported to be a serum marker of malignant mesothelioma. The prognosis of mesothelioma is poor and there is no effective treatment at present. There are several rat model systems of mesothelioma that may be promising tools in the development of an antimesothelioma treatment. We hope our ELISA to detect the soluble form of rat Erc/Mesothelin is useful in the rat model system to exploit the antimesothelioma therapy to be used in human cases.

  3. In-house validation and quality control of commercial enzyme-linked immunosorbnet assays for screening of nitrofuran metabolites in food of animal origin

    Directory of Open Access Journals (Sweden)

    Dimitrieska-Stojkovic Elizabeta

    2012-01-01

    Full Text Available Application of nitrofuran antimicrobials at food production animals was prohibited by Commission Regulation 2003/181/EC because of their potential carcinogenic and mutagenic effects on humans. Main protein-bound metabolites of nitofurans are 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ, 1-aminohydantoin (AHD, semicarbazide (SEM and 3-amino-2-oxazolidinone (AOZ. Since then numerous costly liquid chromatography with tandem mass spectrometry (LC/MS/MS methods have been developed for screening and confirmation of nitrofuran metabolites in line with the EU requirements for performing official controls. As an inexpensive and less time consuming alternative, enzyme-immunoassay methods were developed for screening of the respective compounds. In this study validation and evaluation of four commercial enzyme-linked immunosorbent assay (ELISA has been performed. According to the requirements of Commission Decision 2002/657/EC, different performance characteristics (specificity, detection capability, precision for various matrices (liver, eggs, honey have been determined for each kit. The validation study has confirmed that the methods studied possess suitable characteristics: detectionlimits between 0.126 and 0.240 μg/kg, detection capabilities ≤1.0 μg/kg and the inter-day precision in the range from 16.20% to 22.11 %. The validation study was finalized by participation in FAPAS Proficiency testing scheme in 2011, and the obtained results have confirmed the capability of applied methods for unambiguous discrimination between negative and positive sample.

  4. Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay.

    Directory of Open Access Journals (Sweden)

    Marua Prevato

    Full Text Available Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA and neuraminidase (NA are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1 and avian A/turkey/Turkey/01/2005 (H5N1 influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA.

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

    Science.gov (United States)

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

    2004-01-01

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

  6. Field estimation of the flock-level diagnostic specificity of an enzyme-linked immunosorbent assay for Avian metapneumovirus antibodies in turkeys.

    Science.gov (United States)

    Muñoz-Zanzi, Claudia; Trampel, Darrell; Hanson, Tim; Harrison, Kristen; Goyal, Sagar; Cortinas, Roberto; Lauer, Dale

    2009-03-01

    Routine serologic testing for Avian metapneumovirus (AMPV) infection of turkey flocks at slaughter is currently being used to monitor changes in the occurrence of AMPV infection in endemic areas and can also be used to detect the emergence of infection in currently unaffected areas. Because of the costs associated with false-positive results, particularly in areas that are free of AMPV infection, there is a need to obtain improved estimates of flock-level specificity (SP). The objective of this study was to estimate flock-level SP of a program to monitor AMPV infection in turkey flocks at processing using a standard enzyme-linked immunosorbent assay (ELISA). A study was carried out in which 37 AMPV-free flocks from 7 Midwest operations were followed serologically. Six percent, 3%, and 0.2% of total samples tested AMPV positive at 8 weeks, 12 weeks, and at processing, respectively. Overall, flock-level SP increased as the cutoff increased and as age increased. Flock-level SP at processing was 97%, if a cutoff of 1 was used (the flock was classified as positive if at least 1 sample tested positive), and 100%, if any other cutoff was used. Administration of antibiotics (P = 0.02) and vaccination for Bordetella avium (P = 0.08) were positively associated with the probability of (false) positive test results. These findings suggest possible cross-reactions with other infections and highlight the need to consider variable diagnostic performance depending on farm conditions.

  7. The External Quality Assurance Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay.

    Science.gov (United States)

    Sanchez, Ana M; Rountree, Wes; Berrong, Mark; Garcia, Ambrosia; Schuetz, Alexandra; Cox, Josephine; Frahm, Nicole; Manak, Mark; Sarzotti-Kelsoe, Marcella; D'Souza, M Patricia; Denny, Thomas; Ferrari, Guido

    2014-07-01

    The interferon-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay has been developed and used as an end-point assay in clinical trials for infectious diseases and cancer to detect the magnitude of antigen-specific immune responses. The ability to compare data generated by different laboratories across organizations is pivotal to understand the relative potency of different therapeutic and vaccine strategies. We developed an external proficiency program for the IFN-γ ELISpot assay that evaluates laboratory performance based on five parameters: timeliness for data reporting; ability to handle cellular samples; detection of background (non-specific) responses; accuracy to consensus of the results; and precision of the measurements. Points are awarded for each criterion, and the sum of the points is used to determine a numeric and adjectival performance rating. Importantly, the evaluation of the accuracy to the consensus mean for the detection of antigen-specific responses using laboratory-specific procedures informs each laboratory and its sponsor on the degree of concordance of its results with those obtained by other laboratories. This study will ultimately provide the scientific community with information on how to organize and implement an external proficiency program to evaluate longitudinally the performance of the participating laboratories and, therefore, fulfill the requirements of the GCLP guidelines for laboratories performing end-point IFN-γ ELISpot assay for clinical trials. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Development and validation of an indirect competitive enzyme linked-immunosorbent assay for the determination of potentially allergenic sesame (Sesamum indicum) in food.

    Science.gov (United States)

    Husain, Fatima Tazeen; Bretbacher, Ines Elisabeth; Nemes, Albert; Cichna-Markl, Margit

    2010-02-10

    This study was designed to develop an indirect competitive enzyme linked-immunosorbent assay (ELISA) to detect traces of sesame in food. Antibodies against sesame were prepared by immunizing a hen with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 12 of 13 food ingredients tested, only for chocolate was a low cross-reactivity of 0.7% observed. To eliminate matrix effects, sesame protein standard solutions were prepared by diluting the sesame extract with blank food matrix (1:20 diluted with PBS). Recovery of sesame protein in food samples (crisp toasts, snacks, and rolls) spiked with different sesame protein concentrations ranged from 85% to 120%, with the exception of multigrain crisp toast, resulting in too high recoveries (117%-160%) and whole grain bread, yielding too low recoveries (70%-85%). In crisp bread, cracker, cereals, and snacks the limit of detection (LOD) was found to be 5 microg of sesame protein/g of food, in fresh breads and rolls, the LOD was 11 microg of sesame protein/g of food.

  9. A single serum dilution enzyme-linked immunosorbent assay for determining anti-human papillomavirus (HPV) antibody titres in humans immunised with prophylactic HPV vaccines.

    Science.gov (United States)

    Jin, Yingji; Kim, Hyoung Jin; Yim, Ga Won; Kim, Young Tae; Chang, Don Yong; Kim, Hong-Jin

    2012-07-01

    Two types of prophylactic human papillomavirus (HPV) vaccines are currently available. However, there is no simple monitoring system for assessing acquired immunity that can cope simultaneously with large numbers of serum samples. Approximately 30% of women with normal cytology are known to be seropositive for HPV types 16 and 18 because of the high prevalence of these HPV types. Therefore, to be useful the monitoring system has to discriminate clearly between vaccine recipients and other serology groups. However, there has never been any focus on developing a method to satisfy this condition. In this study, we developed a high-throughput single-serum-dilution enzyme-linked immunoassay (ELISA) system for determining anti-HPV antibody titres following vaccination. We optimised the conditions for each ELISA step to increase its accuracy and precision and to avoid the high background of non-specific reactions that is a major problem for serology assays. The new ELISA system has superior linearity, accuracy and reproducibility. Moreover, it clearly discriminated between antibody levels in vaccine recipients and those in other serology groups such as individuals with normal cervical cytology and those with cervical cancer. Therefore, this single-serum-dilution ELISA should be very useful for assessing the acquired immunity of HPV vaccine recipients. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Development of an enzyme-linked immunosorbent assay to identify host-feeding preferences of Phlebotomus species (Diptera: Psychodidae) in endemic foci of visceral leishmaniasis in Nepal.

    Science.gov (United States)

    Burniston, Ian; Roy, Lalita; Picado, Albert; Das, Murari; Rijal, Suman; Rogers, Matthew; Coosemans, Marc; Boelaert, Marleen; Davies, Clive; Cameron, Mary

    2010-09-01

    Anthroponotic visceral leishmaniasis, transmitted by Phlebotomus argentipes Annandale & Brunetti (Diptera: Psychodidae) sand flies, is regarded as a major problem of public health importance in the Indian subcontinent. Understanding the feeding behavior of the vector can be used to investigate changes in human-vector contact during intervention programs. An enzyme-linked immunosorbent assay (ELISA) was modified to make it suitable to identify the origin of P. argentipes and Phlebotomus papatasi Scopoli (Diptera: Psychodidae) blood meals. The sensitivity and specificity of the precipitin ring test and ELISA were compared, as well as the stability of the tests across different stages of blood meal digestion. The ELISA was more sensitive and specific than the precipitin test for identifying the sources of blood meals. When using the ELISA method with a plate reader, it was possible to obtain 100% sensitivity and specificity. When comparing the techniques across digestion stages, it was found that there was a drop in sensitivity, 48 and 72 h postblood meal for precipitin and visually read ELISA, respectively. However, the sensitivity of the ELISA using a plate reader was not altered by the digestion time. The feeding habits of P. argentipes and P. papatasi from the Terai region of Nepal, determined by the ELISA developed, showed P. papatasi to be highly anthropophilic, and P. argentipes appeared to feed both on humans and animals, in particular bovines.

  11. Quantitative Analysis of Free 15-F2t-Isoprostane from Plasma of Obstructive Sleep Apnea Patients Using Enzyme Linked Immunosorbe

    Directory of Open Access Journals (Sweden)

    Bertha Rusdi

    2012-03-01

    Full Text Available 15-F2t-isoprostane is a biomarker in assessment of oxidative stress status that due to its relatively low concentration in biological fluid and also has many isomers, the 15-F2t-isoprostane sample need to be extracted prior to the quantifying processes. Extraction techniques commonly used to extract 15-F2t-isoprostane are solid phase extraction (SPE and immunoaffinity extraction. Improvements to the SPE and immunoaffinity extraction techniques had been conducted, and the recovery results was then compared. The quantification of 15-F2t-isoprostane then was conducted using Enzyme Linked Immunosorbent Assay (ELISA method. Then followed by the examination of the plasma recovery results. Extraction technique which had the highest recovery then was used to quantify 15-F2t-isoprostane from plasma of Obstructive Sleep Apnea (OSA patients. Immunoaffinity extraction technique has a good recovery result. OSA patients have the tendency to have high 15-F2t-isoprostane concentrations in the plasma, therefore have a potential risk to get diseases related to the biological activities of 15-F2t-isoprostane, such as arteriosclerosis.

  12. Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

    2010-05-26

    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

  13. A polymerase chain reaction and enzyme linked immunosorbent assay based approach for diagnosis and differentiation between vaccinated and infected cattle with Mycobacterium bovis

    Science.gov (United States)

    Sabry, Mohamed; Elkerdasy, Ahmed

    2014-01-01

    Background: In most African and Arabic countries tuberculosis (TB) causes great economic losses in bovine species and constitutes serious zoonotic problem. As the traditional diagnostic method delay the research because of low sensitivity and specificity, a rapid method of diagnosis is of outmost importance. Aim: The study was designed to evaluate the two rapid diagnostic methods of TB in cattle, further to differentiate between infected and bacillus Calmette-Guerin (BCG) vaccinated animals. Materials and Methods: Intradermal tuberculin test was applied to 300 cattle. Of these cattle, 15 cattle were vaccinated from cattle negative to tuberculin test with BCG. Blood samples were taken for lymphocyte separation to apply polymerase chain reaction (PCR) upon and for serum preparation for the enzyme-linked immunosorbent assay (ELISA) application, this blood collected from 65 cattle classified into three groups, viz. positive tuberculin test (35 animals), negative tuberculin test (15 animals), and vaccinated cow with BCG (15 animals). From blood samples lymphocytes were separated and the isolated lymphocytes were subjected to PCR and serum for ELISA application. Blood samples, specimens from lymph nodes and specific tissues were taken for PCR and for cultivation and isolation of Mycobacterium bovis. Results and Conclusions: The results of this study revealed that PCR can be used as rapid efficient and accurate diagnostic test in detection of ruminant TB. Moreover, cattle's ELISA reading showed higher sensitivity in positive tuberculin animals. However, the differentiations between vaccinated and infected animals not clear by using a single antigen only. PMID:24741280

  14. Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Preechakasedkit, Pattarachaya; Siangproh, Weena; Khongchareonporn, Nanthika; Ngamrojanavanich, Nattaya; Chailapakul, Orawon

    2018-04-15

    In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL -1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1-100ngmL -1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA. Copyright © 2017. Published by Elsevier B.V.

  15. Prospective evaluation of nonstructural 1 enzyme-linked immunosorbent assay and rapid immunochromatographic tests to detect dengue virus in patients with acute febrile illness.

    Science.gov (United States)

    Najioullah, Fatiha; Combet, Emilie; Paturel, Laure; Martial, Jenny; Koulmann, Laurence; Thomas, Laurent; Hatchuel, Yves; Cabié, André; Cesaire, Raymond

    2011-02-01

    We prospectively evaluated the Bio-Rad nonstructural 1 (NS1) enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatographic assay (LFIA) in comparison to an in-place reverse transcription-polymerase chain reaction for dengue diagnosis. Among 537 consecutive samples from patients with acute febrile disease, 264 (49.2%) tested positive in reverse transcription-polymerase chain reaction (RT-PCR), 156 (29.1%) in NS1-antigen (Ag) ELISA, and 125 (23.3%) in NS1-Ag LFIA. Compared to the RT-PCR status, the specificity was 100% for the NS1-Ag ELISA and LFIA, but their respective sensitivities were 61.2% [95% confidence interval (CI), 55.2-67.2] and 49.4% (95% CI, 43.2-55.6), with nadirs of 37.9% and 24.1% on day 6 of illness. The NS1-Ag ELISA and LFIA were positive, respectively, for 48.0% and 40.7% of the secondary infections versus 85.0% and 66.7% of the primary infections. For patients LFIA reached respective sensitivities of 100% and 90.5%. Reports of results of dengue NS1-Ag assays should specify that negativity does not preclude DENV infection, and require further investigations in the case of severe disease. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

    Science.gov (United States)

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-01-01

    Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta. PMID:8705668

  17. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows.

    Science.gov (United States)

    Walsh, Robert B; Kelton, David F; Hietala, Sharon K; Duffield, Todd F

    2013-04-01

    Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.

  18. Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

    Science.gov (United States)

    do Brasil, Pedro Emmanuel Alvarenga Americano; Castro, Rodolfo; de Castro, Liane

    2016-01-01

    Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects. PMID:26814640

  19. [Evaluation of usefulness of commercial recomwell Campylobacter enzyme--linked immunosorbent assays for routine serodiagnosis of Campylobacter jejuni and Campylobacter coli infections].

    Science.gov (United States)

    Rokosz, Natalia; Rastawicki, Waldemar; Jagielski, Marek

    2008-01-01

    The commercially available enzyme-linked immunosorbent assays (ELISA recomWell Campylobacter) from Mikrogen was evaluated for the diagnosis of Campylobacter jejuni and Campylobacter coli infections. Serum samples from 20 healthy controls, 44 persons with symptoms of primary Campylobacter infection and 24 serum samples from patients with Yersinia enterocolitica or Salmonella infections were tested. This ELISA assay detects IgA and IgG antibodies against three recombinant antigens of the Campylobacter jejuni and Campylobacter coli: OMP 18 (18 kDa), PEB4 (31 kDa) and P39 (39 kDa). The healthy controls showed significantly lower antibody titers in all two immunoglobulin classes. The IgA antibodies were diagnosed only in 2 (18.2%) serum samples obtained from patients with bacteriologically confirmed campylobacteriosis. The presence of IgG antibodies was confirmed in 82% of serum samples. Furthermore, we showed that 66.7% of the 33 serum samples obtained from the patients suspected for campylobacteriosis not confirmed by isolation, were positive for IgG and 15.2% for IgA antibodies. We observed also not specific reactions in ELISA recom Well Campylobacter with sera obtained form patients with yersiniosis and salmonelosis. This study demonstrates the usefulness of commercially available assay for the routine diagnosis of Campylobacter infection but with some limitations.

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

    Science.gov (United States)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    2011-12-01

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

  2. Mixed lubrication after rewetting of blotted pleural mesothelium.

    Science.gov (United States)

    Bodega, Francesca; Sironi, Chiara; Porta, Cristina; Pecchiari, Matteo; Zocchi, Luciano; Agostoni, Emilio

    2013-01-15

    Coefficient of kinetic friction (μ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that μ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, μ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan μ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

    Science.gov (United States)

    Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

    2012-04-30

    Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva

    2003-07-01

    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  5. Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

    Directory of Open Access Journals (Sweden)

    NUNES Cáris Maroni

    1997-01-01

    Full Text Available Visceral larva migrans (VLM is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA using the larval excretory-secretory antigen of T. canis (TES, the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa. Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

  6. Application of the Ceditest FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda

    DEFF Research Database (Denmark)

    Ayebazibwe, Chrisostom; Mwiine, Frank Norbert; Balinda, Sheila Nina

    2012-01-01

    Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest FMDV type...

  7. Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein e(rns) for serologic diagnosis of pestivirus infections in swine

    NARCIS (Netherlands)

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

    2001-01-01

    Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure

  8. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

    Science.gov (United States)

    Rojas-Espinosa, O; Silva-Miranda, M; Wek-Rodriguez, K; Arce-Paredes, P

    2006-01-01

    We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

  9. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    Science.gov (United States)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  10. Western blotting via proximity ligation for high performance protein analysis.

    Science.gov (United States)

    Liu, Yanling; Gu, Jijuan; Hagner-McWhirter, Åsa; Sathiyanarayanan, Poojahrau; Gullberg, Mats; Söderberg, Ola; Johansson, Johan; Hammond, Maria; Ivansson, Daniel; Landegren, Ulf

    2011-11-01

    Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

  11. Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

    International Nuclear Information System (INIS)

    Bodkin, D.K.

    1985-01-01

    RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

  12. Enzyme-linked immunosorbent assay gliadin assessment in processed food products available for persons with celiac disease: a feasibility study for developing a gluten-free food database.

    Science.gov (United States)

    Agakidis, Charalampos; Karagiozoglou-Lampoudi, Thomais; Kalaitsidou, Marina; Papadopoulos, Theodoros; Savvidou, Afroditi; Daskalou, Efstratia; Dimitrios, Triantafyllou

    2011-12-01

    Inappropriate food labeling and unwillingness of food companies to officially register their own gluten-free products in the Greek National Food Intolerance Database (NFID) result in a limited range of processed food products available for persons with celiac disease (CDP). The objective of the study was to evaluate the feasibility of developing a gluten-free food product database based on the assessment of the gluten content in processed foods available for CDP. Gluten was assessed in 41 processed food products available for CDP. Group A consisted of 26 products for CDP included in the NFID, and group B contained 15 food products for CDP not registered in the NFID but listed in the safe lists of the local Celiac Association (CA). High-sensitivity ω-gliadin enzyme-linked immunosorbent assay (ELISA) was used for analysis. Gluten was lower than 20 ppm in 37 of 41 analyzed products (90.2%): in 24 of 26 (92.3%) products in group A and in 13 of 15 (86.7%) products in group B (P = .61). No significant difference was found between the 2 groups regarding gluten content. No product in either group contained gluten in excess of 100 ppm. Most of the analyzed products included in the Greek NFID or listed in the lists of the local CA, even those not officially labeled "gluten free," can be safely consumed by CDP. The use of commercially available ω-gliadin ELISA is able to identify those products that contain inappropriate levels of gluten, making feasible it to develop an integrated gluten-free processed food database.

  13. Diagnostic accuracy of the anti-glutamic acid decarboxylase antibody in type 1 diabetes mellitus: Comparison between radioimmunoassay and enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Murata, Takashi; Tsuzaki, Kokoro; Nirengi, Shinsuke; Watanabe, Tomokazu; Mizutani, Yukako; Okada, Hayami; Tsukamoto, Masami; Odori, Shinji; Nakagawachi, Reiko; Kawaguchi, Yaeko; Yoshioka, Fumi; Yamada, Kazunori; Shimatsu, Akira; Kotani, Kazuhiko; Satoh-Asahara, Noriko; Sakane, Naoki

    2017-07-01

    The distributer of the anti-glutamic acid decarboxylase antibody assay kit using radioimmunoassay (RIA) recently announced its discontinuation, and proposed an alternative kit using enzyme-linked immunosorbent assay (ELISA). The aim of the present study was to investigate the diagnostic values of the anti-glutamic acid decarboxylase antibody by RIA and ELISA among type 1 diabetes mellitus patients and control participants. A total of 79 type 1 diabetes mellitus patients and 79 age-matched controls were enrolled and assessed using RIA and ELISA. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for cut-off values (RIA = 1.5 U/mL and ELISA = 5.0 U/mL, respectively). Kappa coefficients were used to test for agreements between the RIA and ELISA methods regarding the diagnosis of type 1 diabetes mellitus. The sensitivity, specificity, positive predictive values, and negative predictive values for diagnosing type 1 diabetes mellitus were 57.0, 97.5, 95.7, and 69.4% by RIA, and 60.8, 100.0, 100.0 and 71.8% by ELISA, respectively. The diagnosis of type 1 diabetes mellitus using the RIA and ELISA methods showed substantial agreement with the kappa values of 0.74 for all participants, and of 0.64 for the acute type; however, there was moderate agreement with the kappa value of 0.56 for the slowly progressive type. The present study suggests that both anti-glutamic acid decarboxylase antibody by RIA and ELISA was useful for diagnosing type 1 diabetes mellitus. However, in the slowly progressive type, the degree of agreement of these two kits was poorer compared with those in all participants or in the acute type. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

  14. Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

    2017-10-01

    A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers. © 2017 Institute of Food Technologists®.

  15. 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.

  16. Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

    Science.gov (United States)

    Peng, Dapeng; Ye, Shengqiang; Wang, Yulian; Chen, Dongmei; Tao, Yanfei; Huang, Lingli; Liu, Zhenli; Dai, Menghong; Wang, Xiaoqing; Yuan, Zonghui

    2012-01-11

    Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

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

    Science.gov (United States)

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

    2010-01-01

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

  18. Detection of serum antimüllerian hormone in women approaching menopause using sensitive antimüllerian hormone enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Robertson, David M; Kumar, Ajay; Kalra, Bhanu; Shah, Shivani; Pruysers, Enid; Vanden Brink, Heidi; Chizen, Donna; Visser, Jenny A; Themmen, Axel P; Baerwald, Angela

    2014-12-01

    Current antimüllerian hormone (AMH) immunoassays are insufficiently sensitive to detect circulating AMH levels in ovulatory women approaching menopause. The aim of this study was to detect serum AMH levels across the menstrual cycle with age, using two new AMH enzyme-linked immunosorbent assay (ELISA) kits with increased sensitivity and differing specificity. Serum AMH levels were determined every 2 to 3 days across the interovulatory interval of menstrual cycles among women of early-mid reproductive age (18-35 y; n = 10) and late reproductive age (45-55 y; n = 17). Two highly sensitive AMH ELISAs (designated 24/32 and 24/37) with differing sensitivities were developed and applied to sera using a recombinant human pro-mature AMH preparation as reference. A third AMH ELISA (Gen II AMH ELISA kit; Beckman Coulter, Brea, CA) used was directed on mature-pro regions of AMH. AMH levels in all cycles were detectable with the 24/32 and 24/37 AMH ELISAs. AMH levels across the menstrual cycle were highly correlated (r = 0.98) between the 24/32 and 24/37 AMH ELISAs and the Gen II AMH ELISA (r = 0.94), but with large intracycle variations observed in older women. In late reproductive age, more than 95% of AMH values were detectable with the 24/32 and 24/37 AMH ELISAs, whereas only 36% of AMH values were detectable with the Gen II AMH ELISA. AMH levels were detected in cycles with lower antral follicle count and at a later age using the 24/32 and 24/37 AMH ELISAs compared with the Gen II AMH ELISA. AMH level correlated with antral follicle count in younger women, but not in older women. The new 24/32 and 24/37 AMH ELISAs have the sensitivity to monitor ovarian follicle profiles in late reproductive age.

  19. Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.

    Science.gov (United States)

    Thålin, Charlotte; Daleskog, Maud; Göransson, Sophie Paues; Schatzberg, Daphne; Lasselin, Julie; Laska, Ann-Charlotte; Kallner, Anders; Helleday, Thomas; Wallén, Håkan; Demers, Mélanie

    2017-06-01

    There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

  20. A biolayer interferometry-based enzyme-linked aptamer sorbent assay for real-time and highly sensitive detection of PDGF-BB.

    Science.gov (United States)

    Gao, Shunxiang; Zheng, Xin; Wu, Jihong

    2018-04-15

    Accurate, fast and sensitive detection of disease-specific protein biomarkers, especially in blood, urine, or other bodily fluids, is an important approach to achieve early disease diagnosis. Platelet-derived growth factor-BB (PDGF-BB), a widely used biomarker, is involved in a substantial number of serious diseases, such as hepatic fibrosis, atherosclerosis, age-related macular degeneration and diabetic eye disease and is often over-expressed in human malignant tumors. Therefore, the development of sensitive and specific detection methods for PDGF-BB is of great importance for the early diagnosis of disease and assessments of patient recovery. In the current study, a biolayer interferometry-based enzyme-linked aptamer sorbent assay (BLI-ELASA) was successfully established for rapid (20-25min), high-throughput (8 or 16 samples) and real-time monitoring of PDGF-BB in clinical samples. The method exhibited a broad detection range from 0.5 to 1000ng/mL of PDGF-BB (good linear range from 0.5 to 10ng/mL), with a low detection limit of 0.08ng/mL. Moreover, BLI-ELASA was applied to the detection of PDGF-BB in spiked serum and urine samples and showed a high degree of selectivity for PDGF-BB, good reproducibility, and stability. We believe that the methodology in this work can be easily adapted to detect other biomolecules in clinical samples, including viruses, pathogens and toxins, in a rapid, sensitive, high-throughput and real-time manner. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. The usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals.

    Science.gov (United States)

    Kornacka, Aleksandra; Cybulska, Aleksandra; Bień, Justyna; Goździk, Katarzyna; Moskwa, Bożena

    2016-09-15

    The aim of the study was to compare the usefulness of two antibody-based methods, the direct agglutination test (DAT) and enzyme linked immuosorbent assay (ELISA), with that of the polymerase chain reaction (PCR) for detecting anti-Toxoplasma gondii in samples derived from naturally-infected wild animals. Antibodies against T. gondii were detected in meat juice samples collected from 129 free- living carnivores and omnivores. T. gondii seroprevalence was confirmed in 73,6% of examined samples when DAT and ELISA were used separately, but in only 88,4% samples when both immunological tests were used in parallel. PCR results confirmed the presence of DNA of the parasite in 24 of all the 129 samples. Sixteen samples were classified as positive when all three tests were used. A moderate degree of agreement was found between DAT and ELISA (κ=0.55). However, no agreement was found between the molecular and serological tests: κ=-1.75 for DAT versus PCR; κ=-1.67 ELISA versus PCR. By using both serological tests, antibodies against T. gondii were found in 77.5% of red foxes, 12.5% of badgers, 40% of martens and 8.3% of raccoon dogs. Antibodies against the parasite were detected also in one mink, but not in the sample derived from a polecat. T.gondii DNA was found in the brain tissue of 20 red foxes, three badgers and one raccoon dog. Our studies confirm that ELISA and DAT are suitable and reliable techniques for T. gondii antibody detection in meat juice from wild animals when serum samples are unavailable. Positive results obtained by immunological tests do not always reflect that the host was infected by T. gondii. They indicate only a contact with parasite. PCR should be used to confirm te presence of DNA from T. gondii. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. 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.

  3. Use of heavy water (D2O) in developing thermostable recombinant p26 protein based enzyme-linked immunosorbent assay for serodiagnosis of equine infectious anemia virus infection.

    Science.gov (United States)

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Singh, Raj K

    2014-01-01

    Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4 °C, 37 °C, 42 °C, and 45 °C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37 °C < 42 °C < 45 °C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45 °C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

  4. Use of Heavy Water (D2O in Developing Thermostable Recombinant p26 Protein Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Equine Infectious Anemia Virus Infection

    Directory of Open Access Journals (Sweden)

    Harisankar Singha

    2014-01-01

    Full Text Available Thermostabilizing effect of heavy water (D2O or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA for serodiagnosis of equine infectious anemia virus (EIAV infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4°C, 37°C, 42°C, and 45°C over a storage time from 2 weeks to 10 months. A set of positive serum (n=12 consisting of strong, medium, and weak titer strength (4 samples in each category and negative serum (n=30 were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37°C < 42°C < 45°C was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45°C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

  5. Performance Assessment and Comparability of a Commercial Enzyme-Linked Immunosorbent Assay Kit with Liquid Chromatography-Tandem Mass Spectrometry for Chloramphenicol Residues in Crab and Shrimp.

    Science.gov (United States)

    Jester, Edward L E; Loader, Jared I; El Said, Kathleen R; Abraham, Ann; Flores Quintana, Harold A; Plakas, Steven M

    2016-01-01

    Monitoring for chloramphenicol (CAP) in aquaculture products is primarily performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which requires expensive equipment and specialized training. Many laboratories prefer to screen samples with facile and high-throughput enzyme-linked immunosorbent assay (ELISA) kits for CAP residues before submitting samples for LC-MS/MS quantification and confirmation. We evaluated the performance of a Ridascreen (R-Biopharm) ELISA kit for CAP in spiked and incurred crab and shrimp muscle at levels bracketing the minimum required performance level for analysis (0.3 ng/g). The Ridascreen ELISA kit incorporates antibody directed against CAP. Incurred CAP levels in crab and shrimp muscle were verified using LC-MS/MS. We found good repeatability (relative standard deviation) of the ELISA in spiked and incurred crab and shrimp muscle samples, with values ranging from 6.8 to 21.7%. Recoveries of CAP from tissues spiked at 0.15 to 0.60 ng/g ranged from 102 to 107%. Minimal cross-reactivity with blank crab and shrimp muscle matrix components was observed. ELISA data were highly correlated with those of LC-MS/MS for CAP in incurred muscle tissue. We believe this study to be the first evaluation of the performance and comparability of a CAP ELISA kit and LC-MS/MS for determination of CAP residues, as well as their elimination, in crab muscle. Our findings support the use of this ELISA kit for screening purposes and, when used in conjunction with validated instrumental methods, for regulatory monitoring of CAP in these species.

  6. A sensitive double-antibody enzyme-linked immunosorbant assay for bovine myeloperoxidase and its application to serum and neutrophil extracts.

    Science.gov (United States)

    Cooray, R; Petersson, C G; Moberg, L; Båge, R

    1995-10-01

    The heme enzyme myeloperoxidase (MPO), with a spectral A430/A280 ratio > 0.78, was purified from isolated bovine neutrophils. Using highly specific anti-MPO monoclonal and anti-MPO polyclonal antibodies raised against MPO, a specific and sensitive double-antibody enzyme-linked immunosorbant assay (ELISA) was developed to measure bovine MPO in serum and neutrophil extracts. The ELISA shows good precision and accuracy, with intra- and interassay coefficients of variation of < 10% for MPO concentrations ranging from 0.5 to 50 ng/ml. The accuracy of the ELISA for measuring MPO in bovine serum was further confirmed by the similarity between the standard curve and curves obtained with successive dilutions of MPO-rich serum samples. The mean analytical recovery of MPO from serum was approximately 90%. Long delays between blood sampling and serum preparation were found to affect the level of MPO in the serum. Mean MPO values in the serum of healthy adult cows were 6.5 ng/ml, with a range of 3.5-15.3 ng/ml. In dairy cows with acute mastitis, mean serum MPO values were approximately 30 ng/ml, with a range of 6.0-59.6 ng/ml, and the elevation was markedly higher than the normal values (P = 0.0001). In isolated neutrophils from healthy cattle, MPO concentrations were found to be 7 x 10(-4) ng, with a range of 6.5-8.3 x 10(-4) ng/neutrophil. The ELISA was used to study the distribution of MPO in the bovine neutrophil granules; it was found to be localized to one distinct compartment.

  7. Development of an enzyme-linked immunosorbent assay for seven sulfonamide residues and investigation of matrix effects from different food samples.

    Science.gov (United States)

    Zhang, Hongyan; Wang, Lei; Zhang, Yan; Fang, Guozhen; Zheng, Wenjie; Wang, Shuo

    2007-03-21

    Direct competitive enzyme-linked immunosorbent assays (ELISA) were developed to detect a broad range of sulfonamides in various matrices. Screening for this class of antibiotics in pig muscle, chicken muscle, fish, and egg extracts was accomplished by simple, rapid extraction methods carried out with only phosphate-buffered saline (PBS) buffer. Twenty milliliters of extract solution was added to 4 g of sample to extract the sulfonamide residues, and sample extracts diluted with assay buffer were directly analyzed by ELISA; matrix effects could be avoided with 1:5 dilution of pig muscle, chicken muscle, and egg extracts with PBS and 1:5 dilution of fish extract with 1% bovine serum albumin (BSA)-PBS. For liver sample, the extraction method was a little more complicated; 2 g of sample was added to 20 mL of ethanol, mixed, and then centrifuged. The solvent of 10 mL of the upper liquid was removed, and the residues were dissolved in 10 mL of PBS and then filtered; the filtrate was diluted two-fold with 0.5% BSA-PBS for ELISA. These common methods were able to detect seven sulfonamide residues such as sulfisozole, sulfathiazole, sufameter, sulfamethoxypyridazine, sulfapyridine, sulfamethizole, and sulfachlorpyridazine in pig muscle, liver, chicken muscle, egg, and fish. The assay's detection limits for these compounds were less than 100 microg kg-1. Various extraction methods were tested, and the average recovery (n=3) of 100 microg kg-1 for the matrices was found to range from 77.3 to 123.7%.

  8. Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Xue, Sheng; Li, He-Ping; Zhang, Jing-Bo; Liu, Jin-Long; Hu, Zu-Quan; Gong, An-Dong; Huang, Tao; Liao, Yu-Cai

    2013-11-19

    A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) μg/mL, 1000-fold more sensitive than that reported previously (1 μg/mL). The fusion protein was able to detect fungal concentrations below 1 μg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 μg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

  9. A protein A/G indirect enzyme-linked immunosorbent assay for the detection of anti-Brucella antibodies in Arctic wildlife.

    Science.gov (United States)

    Nymo, Ingebjørg H; Godfroid, Jacques; Åsbakk, Kjetil; Larsen, Anett K; das Neves, Carlos G; Rødven, Rolf; Tryland, Morten

    2013-05-01

    A species-independent indirect enzyme-linked immunosorbent assay (iELISA) based on chimeric protein A/G was established for the detection of anti-Brucella antibodies in Arctic wildlife species and compared to previously established brucellosis serological tests for hooded seals (Cystophora cristata), minke whales (Balaenoptera acutorostrata), sei whales (Balaenoptera borealis), fin whales (Balaenoptera physalus), and polar bears (Ursus maritimus), as well as bacteriology results for reindeer and caribou (Rangifer tarandus sp.). The protein A/G iELISA results were consistent with the other serological tests with Cohen kappa values between 0.47 and 0.92, and the protein A/G iELISA can thus offer a technically simple method for these species yielding results consistent with established brucellosis serological tests. Receiver operator characteristics analysis proved that the reindeer and caribou protein A/G iELISA results were consistent with the bacteriological gold standard with an area under the curve of 0.99, and the protein A/G iELISA was thus validated as a sensitive and specific serological method for the detection of anti-Brucella antibodies in reindeer and caribou. The binding of the antibodies from the respective species to protein A and G were also evaluated in the iELISA. The antibodies from hooded seals and polar bears reacted stronger to protein A than to G. The sei whale, fin whale, reindeer, and caribou antibodies reacted stronger to protein G than to A. The minke whale antibodies reacted to both protein A and G. There was a strong correlation (r s = 0.88-0.98) between the optical density results obtained with the iELISA with protein A/G and protein A or G, showing that protein A/G is as well suited as protein A or G for the detection of anti-Brucella antibodies in these species with the iELISA.

  10. Effects of thermal processing on the enzyme-linked immunosorbent assay (ELISA) detection of milk residues in a model food matrix.

    Science.gov (United States)

    Downs, Melanie L; Taylor, Steve L

    2010-09-22

    Food products and ingredients are frequently tested for the presence of undeclared allergenic food residues (including milk) using commercial enzyme-linked immunosorbent assays (ELISAs). However, little is understood about the efficacy of these kits with thermally processed foods. This study evaluated the performance of three milk ELISA kits with a model food processed by several methods. A model food (pastry dough squares) was spiked with nonfat dry milk at several concentrations. The pastry squares were processed by boiling (100 °C for 2 min), baking (190 °C for 30 min), frying (190 °C for 2 min), and retorting (121 °C for 20 min with 17 psi overpressure). Samples were analyzed with three commercial ELISA kits: Neogen Veratox Total Milk, ELISA Systems β-lactoglobulin, and ELISA Systems casein. The detection of milk residues depended upon the type of processing applied to the food and the specific milk analyte targeted by the ELISA kit. Poor recoveries were obtained in all processed samples (2-10% of expected values) using the β-lactoglobulin kit. Better recoveries were obtained in boiled samples (44 and 59%, respectively) using the total milk and casein kits. However, these kits performed poorly with baked (9 and 21%) and fried (7 and 18%) samples. Moderate recoveries were observed in retorted samples (23 and 28%). The decreased detection in processed samples is likely due to protein modifications, including aggregation and Maillard reactions, which affect the solubility and immunoreactivity of the antigens detected by the ELISA methods. The observed decreases in ELISA detection of milk are dramatic enough to affect risk-assessment decisions. However, a lower detection of milk residues does not necessarily indicate decreased allergenicity. These ELISA kits are not acceptable for all applications, and users should understand the strengths and limitations of each method.

  11. Development and use of an indirect enzyme-linked immunosorbent assay for detection of iridovirus exposure in gopher tortoises (Gopherus polyphemus) and eastern box turtles (Terrapene carolina carolina).

    Science.gov (United States)

    Johnson, April J; Wendland, Lori; Norton, Terry M; Belzer, Bill; Jacobson, Elliott R

    2010-05-19

    Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (G. polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only 1 turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response. Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence. Copyright 2009 Elsevier B.V. All rights reserved.

  12. Inhibitory effect of aqueous extract of different parts of Gossypium herbaceum on key enzymes linked with type 2 diabetes and oxidative stress in rat pancreas in vitro

    Directory of Open Access Journals (Sweden)

    Ayodeji Augustine Olabiyi

    2016-06-01

    Full Text Available This study sought to determine the inhibitory effect of aqueous extract of different parts (bark, leaf, and flower of cotton plant (Gossypium herbaceum on key enzymes linked with type 2 diabetes and oxidative stress in rat pancreas in vitro. The aqueous extract (1:10 w/v of Gossypium herbaceum was prepared and the ability of the extract to inhibit the activity of α-amylase and α-glucosidase as well as activities of pro-oxidant Fe2+-induced lipid peroxidation was determined spectrophotometrically. The results revealed that the three varieties were able to inhibit the activity of α-amylase and α-glucosidase in rat's pancreas in a dose dependent manner (0–88.8 mg/ml. Also, the incubation of pancreas tissue homogenate in the presence of Fe2+ caused a significant increase (233.3% in the malondialdehyde (MDA content of pancreas homogenate, nevertheless, the introduction of the aqueous extract inhibited MDA production dose dependently (0–33.33 mg/ml and also exhibited further antioxidant properties as represented by their high radical scavenging and Fe2+ chelating abilities. Inhibition of α-amylase and α-glucosidase activities has been the primary treatment for the management/prevention of type 2 diabetes. Therefore, the α-amylase and α-glucosidase inhibitory activities of aqueous extracts of different parts of Gossypium herbaceum in rat pancreas and prevention of lipid peroxidation in the tissue may be attributed to the presence of polyphenol content of the plant.

  13. Application of enzyme-linked immunosorbent assay for measurement of polychlorinated biphenyls from hydrophobic solutions: Extracts of fish and dialysates of semipermeable membrane devices: Chapter 26

    Science.gov (United States)

    Zajicek, James L.; Tillitt, Donald E.; Huckins, James N.; Petty, Jimmie D.; Potts, Michael E.; Nardone, David A.

    1996-01-01

    Determination of PCBs in biological tissue extracts by enzyme-linked immunosorbent assays (ELISAs) can be problematic, since the hydrophobic solvents used for their extraction and isolation from interfering biochemicals have limited compatibility with the polar solvents (e.g. methanol/water) and the immunochemical reagents used in ELISA. Our studies of these solvent effects indicate that significant errors can occur when microliter volumes of PCB containing extracts, in hydrophobic solvents, are diluted directly into methanol/water diluents. Errors include low recovery and excess variability among sub-samples taken from the same sample dilution. These errors are associated with inhomogeneity of the dilution, which is readily visualized by the use of a hydrophobic dye, Solvent Blue 35. Solvent Blue 35 is also used to visualize the evaporative removal of hydrophobic solvent and the dissolution of the resulting PCB/dye residue by pure methanol and 50% (v/v) methanol/water, typical ELISA diluents. Evaporative removal of isooctane by an ambient temperature nitrogen purge with subsequent dissolution in 100% methanol gives near quantitative recovery of model PCB congeners. We also compare concentrations of total PCBs from ELISA (ePCB) to their corresponding concentrations determined from capillary gas chromatography (GC) in selected fish sample extracts and dialysates of semipermeable membrane device (SPMD) passive samplers using an optimized solvent exchange procedure. Based on Aroclor 1254 calibrations, ePCBs (ng/mL) determined in fish extracts are positively correlated with total PCB concentrations (ng/mL) determined by GC: ePCB = 1.16 * total-cPCB - 5.92. Measured ePCBs (ng/3 SPMDs) were also positively correlated (r2 = 0.999) with PCB totals (ng/3 SPMDs) measured by GC for dialysates of SPMDs: ePCB = 1.52 * total PCB - 212. Therefore, this ELISA system for PCBs can be a rapid alternative to traditional GC analyses for determination of PCBs in extracts of biota or in

  14. Kinetic determination of vitellogenin induction in the epidermis of cyprinid and perciform fishes: Evaluation of sensitive enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Allner, Bernhard; Hennies, Mark; Lerche, Cristiano F; Schmidt, Thomas; Schneider, Klaus; Willner, Marco; Stahlschmidt-Allner, Petra

    2016-12-01

    Induction of vitellogenin (VTG) in male and immature fish is a standardized endpoint in endocrine-disruption testing. To establish a nondestructive swab sampling method, VTG induction in the epidermis of Cypriniformes and Perciformes species was investigated. Both VTG and estrogen receptor genes are expressed in epidermal cells. Immunoaffinity and mass fingerprint analyses show induction of identical VTG peptides in liver and epidermis. Induction of VTG by estradiol (E2) and bisphenol A (BPA) in the epidermis was quantified with homolog enzyme-linked immunosorbent assays. Initial values in juveniles and males were below 1 ng VTG/mL extraction buffer. Exposure to E2 led to values between 200 ng/mL and 4600 ng/mL in cyprinids and between 10 ng/mL and 81 ng/mL in perciforms. Exposure to BPA increased VTG amounts to 250 ng/mL in fathead minnows, 1360 ng/mL in goldfish, 100 ng/mL in zebrafish, and 12 ng/mL in bluegills. Serum VTG contents demonstrated a similar dose-response pattern in the epidermis and the blood. These results show that VTG induction may be reliably assessed in the skin mucus of fishes, demonstrating the suitability of this biological sample for investigating estrogenic activity in compliance with Organisation for Economic Co-operation and Development standard protocols. This broadens the perspectives in toxicological screening and environmental monitoring, reducing the number of tested animals and minimizing harmful effects for animals, allowing for follow-up of individual induction profiles. Environ Toxicol Chem 2016;35:2916-2930. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

  15. Inhibitory effect of some tropical green leafy vegetables on key enzymes linked to Alzheimer's disease and some pro-oxidant induced lipid peroxidation in rats' brain.

    Science.gov (United States)

    Oboh, Ganiyu; Akinyemi, Ayodele Jacobson; Ademiluyi, Adedayo Oluwaseun; Bello, Fatai Olumide

    2014-05-01

    This study sought to investigate the inhibitory effect of some commonly consumed Nigerian green leafy vegetables (raw and blanched) on acetylcholinesterase and butyrylcholinesterase (key enzyme linked to Alzheimer's disease) activities and some pro-oxidants (FeSO4, Sodium nitroprusside and Quinolinic acid) induced lipid peroxidation in rat brain in vitro. Three commonly consumed green leafy vegetables in Nigeria [Amarantus cruentus (Arowojeja), Struchium sparganophora (Ewuro-odo) and Telfairia occidentalis (Ugwu] were blanched in hot water for 10 min, and the extracts of the raw and blanched vegetables were prepared and used for subsequent analysis. The result revealed that all the vegetables inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity as well as the pro-oxidants induced lipid peroxidation in rat brain in a dose dependent manner; however, Amarantus cruentus extract (EC50 = 97.9 μg/ml) had the highest inhibitory effect on acetylcholinesterase activity while Telfairia occidentalis extract (EC50 = 52.7 μg/ml) had the highest inhibitory effect on butyrylcholinesterase activity. However, blanching of the vegetables caused a significant (P vegetables on AChE activities while it enhanced the inhibition of the pro-oxidants induced lipid peroxidation in rat brain in vitro. Therefore, some of the possible mechanism by which green leafy vegetables exert their neuroprotective activities could be through the inhibition of acetylcholinesterase and butyrylcholinesterase activities and prevention of lipid peroxidation in the brain. However, blanching of the vegetables could reduce their ability to inhibit acetylcholinesterase and butyrylcholinesterase activity.

  16. Lateral flow immunoassay and enzyme linked immunosorbent assay as effective immunomethods for the detection of synthetic cannabinoid JWH-200 based on the newly synthesized hapten.

    Science.gov (United States)

    Fojtíková, Lucie; Šuláková, Anna; Blažková, Martina; Holubová, Barbora; Kuchař, Martin; Mikšátková, Petra; Lapčík, Oldřich; Fukal, Ladislav

    2018-01-01

    In recent years, the use of synthetic cannabinoids (SCs) as drugs of abuse has greatly increased. SCs are associated with a risk of severe poisoning or even death. Therefore, more rapid, cost effective and reliable methods are needed, especially for the screening of drivers after traffic accidents and for detailed toxicological analysis in forensic laboratories. In this study, we developed a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of JWH-200 in oral fluids. For this purpose a new hapten was prepared using a ten-step synthetic route. The developed immuno methods are based on antibodies obtained from rabbit immunized with synthesized hapten conjugated to carrier protein. The proposed methods are highly sensitive (LOD LFIA  = 0.08 ± 0.04 ng mL -1 ; LOD ELISA  = 0.04 ± 0.02 ng mL -1 ). They were applied to the quantification of JHW-200 in spiked oral fluids. The recoveries ranged from 82 to 134% for both methods. The results correlated excellently with results obtained using UHPLC-MS/MS (R 2 LFIA  = 0.99; R 2 ELISA  = 0.99). Our developed methods could be an important tool for analyses of JWH-200 in human oral fluids. The one-step LFIA is particularly suitable for roadside and on-site monitoring due to the rapid qualitative results it delivers, while the ELISA is especially useful for laboratory quantitative analyses of positive samples captured by LFIA.

  17. Enzyme-linked immunosorbent assays based on Neospora caninum dense granule protein 7 and profilin for estimating the stage of neosporosis.

    Science.gov (United States)

    Hiasa, Jun; Nishimura, Maki; Itamoto, Kazuhito; Xuan, Xuenan; Inokuma, Hisashi; Nishikawa, Yoshifumi

    2012-03-01

    Neospora caninum is an intracellular protozoan parasite that causes bovine and canine neosporosis, characterized by fetal abortion and neonatal mortality and by neuromuscular paralysis, respectively. Although many diagnostic methods to detect parasite-specific antibodies or parasite DNA have been reported, to date no effective serodiagnostic techniques for estimating pathological status have been described. Our study aimed to elucidate the relationship between the parasite-specific antibody response, parasite activation, and neurological symptoms caused by N. caninum infection by using a recombinant antigen-based enzyme-linked immunosorbent assay. Among experimentally infected mice, anti-N. caninum profilin (NcPF) antibody was only detected in neurologically symptomatic animals. Parasite numbers within the brains of the symptomatic mice were significantly higher than those in asymptomatic animals. In addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody-positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in the neurologically symptomatic dogs were significantly higher than those in the non-neurologically symptomatic animals. Our results suggested that the levels of anti-NcGRA7 and anti-NcPF antibodies reflect parasite activation and neurological symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have potential as suitable indicators for estimating the pathological status of neosporosis.

  18. Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for Chikungunya Virus IgM Using Samples from Deceased Organ and Tissue Donors.

    Science.gov (United States)

    Prince, Harry E; Altrich, Michelle L; Nowicki, Marek J

    2016-10-01

    The identification of nearly 3,500 cases of chikungunya virus (CHIKV) infection in U.S. residents returning in 2014 and 2015 from areas in which it is endemic has raised concerns within the transplant community that, should recently infected individuals become organ and/or tissue donors, CHIKV would be transmitted to transplant recipients. Thus, tests designed to detect recent CHIKV infection among U.S. organ and tissue donors may become necessary in the future. Accordingly, we evaluated 2 enzyme-linked immunosorbent assays (ELISAs) for CHIKV IgM readily available in the United States using 1,000 deidentified serum or plasma specimens collected from donors between November 2014 and March 2015. The Euroimmun indirect ELISA identified 38 reactive specimens; however, all 38 were negative for CHIKV IgG and IgM in immunofluorescence assays (IFAs) conducted at a reference laboratory and, thus, were falsely reactive in the Euroimmun CHIKV IgM assay. The InBios IgM-capture ELISA identified 26 reactive samples, and one was still reactive (index ≥ 1.00) when retested using the InBios kit with a background subtraction modification to identify false reactivity. This reactive specimen was CHIKV IgM negative but IgG positive by IFAs at two reference laboratories; plaque reduction neutralization testing (PRNT) demonstrated CHIKV-specific reactivity. The IgG and PRNT findings strongly suggest that the InBios CHIKV IgM-reactive result represents true reactivity, even though the IgM IFA result was negative. If testing organ/tissue donors for CHIKV IgM becomes necessary, the limitations of the currently available CHIKV IgM ELISAs and options for their optimization must be understood to avoid organ/tissue wastage due to falsely reactive results. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

    2011-01-01

    Background Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (William's) tissues. Results The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. Conclusions The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide. PMID:21970317

  20. Influence of pigments and protein aging on protein identification in historically representative casein-based paints using enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ren, Fang; Atlasevich, Natalya; Baade, Brian; Loike, John; Arslanoglu, Julie

    2016-01-01

    A systematic study on the influence of pigments and sample aging on casein identification was performed on 30 reconstructed paints. The protein in all the paints was extracted into solution for analysis. The amount of protein that can be retrieved for solution-based analysis in each of the reconstructed paints was studied with a well-developed NanoOrange method before and after artificial aging. The results showed that in the paints with calcium phosphate (in bone black) and copper carbonate, hydroxide, or acetate (in verdigris and azurite), the amount of protein that can be retrieved for liquid-phase analysis is much smaller than the other paints, indicating that the protein degradation was accelerated significantly in those paints. Carbon (in vine black), calcium carbonate (in natural chalk), and calcium sulfate (terra alba gypsum and ground alabaster) did not affect much the amount of protein that can be retrieved in the paints compared to non-pigmented binder, meaning that the protein degradation rate was not affected much by those pigments. Artificial aging was observed to decrease the amount of retrievable protein in all the reconstructed paints that were studied. The enzyme-linked immunosorbent assay (ELISA) method was applied to the 28 reconstructed paints (except two verdigris paints) to assess the protein identification. The ELISA responses from the different paints were compared at fixed protein concentrations. Natural chalk, bone black, raw sienna, stack lead white, and cochineal red-violet lake had the lowest ELISA signal in this study, which indicated that the binding sites (epitopes) on the target protein in these paints are likely to deteriorate more than those in the other paints. Artificial aging did not influence the ELISA response as much as the pigments when the protein concentration was kept the same for the paints that were studied.

  1. Impact of grey zone sample testing by enzyme-linked immunosorbent assay in enhancing blood safety: Experience at a tertiary care hospital in North India.

    Science.gov (United States)

    Solanki, Archana; Singh, Abhay; Chaudhary, Rajendra

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) used for screening blood donors for transfusion transmitted infections (TTIs) can sometimes fail to detect blood donors who are recently infected or possessing the low strength of pathogen. Estimation of a grey zone in ELISA testing and repeat testing of grey zone samples can further help in reducing the risks of TTI in countries where nucleic acid amplification testing for TTIs is not feasible. Grey zone samples with optical density (OD) lying between cut-off OD and 10% below the cut-off OD (cut-off OD × 0.9) were identified during routine ELISA testing. On performing repeat ELISA testing on grey zone samples in duplicate, the samples showing both OD value below grey zone were marked nonreactive, and samples showing one or both OD value in the grey zone were marked indeterminate. The samples on repeat testing showing one or both OD above cut-off value were marked positive. About 119 samples (77 for hepatitis B virus [HBV], 23 for human immunodeficiency virus [HIV], and 19 for hepatitis C virus [HCV]) were found to be in grey zone. On repeat testing of these samples in duplicate, 70 (58.8%) samples (45 for HBV, 12 for HIV, and 13 for HCV) were found to be reactive. Six (5%) samples (four for HBV, one for HIV, and one for HCV) were found to be indeterminate. Seventy donors initially screened negative, were found out to be potentially infectious on repeat grey zone testing. Thus, estimation of grey zone samples with repeat testing can further enhance the safety of blood transfusion.

  2. Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples.

    Science.gov (United States)

    Stefan, A; Scaramagli, S; Bergami, R; Mazzini, C; Barbanera, M; Perelle, S; Fach, P

    2007-03-01

    This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157 for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.

  3. Western Blotting Using PVDF Membranes and Its Downstream Applications.

    Science.gov (United States)

    Komatsu, Setsuko

    2015-01-01

    Western blotting using polyvinylidene difluoride (PVDF) membranes is one of the most popular techniques for detection and characterization of proteins. If this technique is combined with immunodetection, the behavior of a particular protein can be clarified. On the other hand, if it is combined with Edman sequencing, the primary structure of the protein can be determined. A protein sample is transferred from an SDS-polyacrylamide gel electrophoresis (PAGE) gel onto a PVDF membrane by electroblotting. The membrane carrying the protein is either used for immunodetection or protein sequencing. SDS-PAGE followed by Western blotting combined with immunodetection using antibodies can easily detect protein behavior in crude protein mixtures. Furthermore, two-dimensional PAGE followed by Western blotting and Edman sequencing allows effective sequence determination of crude protein mixtures that may not be easily purified by conventional column chromatography.

  4. Bioconjugation of quantum dot luminescent probes for Western blot analysis.

    Science.gov (United States)

    Makrides, Savvas C; Gasbarro, Christina; Bello, Job M

    2005-10-01

    Western blot analysis is a widely used technique for protein immunodetection. Its current format, however is unsuitable for multiplex detection of proteins, primarily due to intrinsic limitations of standard organic dyes employed as probes. Quantum dot (QD) semiconductor nanoparticles exhibit significant advantages over organic dyes, including their broad absorption bands, narrow, tunable, and symmetric emission spectra, large Stokes shifts, and excellent photostability. Here we describe a novel method for the functionalization of streptavidin-coated QDs with an in vivo biotinylated peptide (head-to-tail dimerized Z domain derived from protein A) that permits the facile conjugation of antibodies to QDs. In this study, we demonstrate the simultaneous detection of two different types of protein in a Western blot. The bioconjugation of QDs described here makes it possible to achieve multiplex detection of proteins in Western blot analysis in a more straightforward manner.

  5. Blame it on Southern, but it's a western blot.

    Science.gov (United States)

    Klionsky, Daniel J

    2017-01-02

    Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the "Southern blot" (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.

  6. Improved semiquantitative Western blot technique with increased quantification range.

    Science.gov (United States)

    Heidebrecht, F; Heidebrecht, A; Schulz, I; Behrens, S-E; Bader, A

    2009-06-30

    With the development of new interdisciplinary fields such as systems biology, the quantitative analysis of protein expression in biological samples gains more and more importance. Although the most common method for this is ELISA, Western blot also has advantages: The separation of proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative analysis and proposes a mathematical model of the reaction kinetics. Using this model, a semiquantitative Western blot method for simultaneous quantification of different proteins using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays.

  7. Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera Avaliação de testes sorológicos para a detecção de anticorpos anti-HIV em painéis de soros de brasileiros

    Directory of Open Access Journals (Sweden)

    Jairo Ivo-Dos-Santos

    1990-04-01

    Full Text Available Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA, as well as of four alternative assays, namely indirect immunofluorescence (IIF, passive hemagglutination (PHA, dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA to 84.2% (PHA and the specificities ranged from 99.3% (IIF to 80.2% (PHA. The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.Os soros de 472 brasileiros, confirmados como sendo positivos ou negativos em relação à presença de anticorpos anti-HIV e compreendendo todo o espectro clínico da infecção, foram utilizados na avaliação de seis ensaios imunoenzimáticos comerciais (ELISA, bem como de quatro testes alternativos tais como imunofluorescência indireta (IFI, hemaglutinação passiva (HP, dot blot e Karpas AIDS cell test. As sensibilidades variaram de 100% (ELISA Abbott e Roche a 84,2% (HP e as especificidades variaram de 99,3% (IFI a 80,2% (HP. A sensibilidade e especificidade da HP e a sensibilidade do Karpas AIDS cell test foram significativamente menores que os outros ensaios. Embora a IFI e o dot blot tivessem apresentado uma boa sensibilidade e especificidade, os ensaios imunoenzimáticos (ELISA foram mais adequados para serem utilizados em triagem quando outros parâmetros tais como facilidade de leitura e interpretação dos resultados e duração dos ensaios foram considerados.

  8. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible

  9. Nonstripping "Rainbow" and Multiple Antigen Detection (MAD) Western Blotting.

    Science.gov (United States)

    Krajewski, Stan Stanislaw; Tsukamoto, Michelle M; Huang, Xianshu; Krajewski, Sebastian B

    2015-01-01

    A variation of immunoblotting method (the "Rainbow Western"), permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping off prior antibodies. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple reprobings of the same membrane with different primary antibodies (≥12), retaining strong signal intensities for all sequential antibody probings. The procedure utilizes horseradish peroxidase (HRPase)-based detection with both a chemiluminescent and colorimetric substrate. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. In the "Rainbow Western," four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same membrane. By allowing large amounts of data to be obtained from a single blot, the MAD-immunoblotting and Rainbow Western methods have the potential for researchers to compare the expression of several proteins within a single biological sample. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples is minute or only available on a one-time basis.

  10. Phenolic constituents and modulatory effects of Raffia palm leaf (Raphia hookeri extract on carbohydrate hydrolyzing enzymes linked to type-2 diabetes

    Directory of Open Access Journals (Sweden)

    Felix A. Dada

    2017-10-01

    Full Text Available This study sought to investigate the effects of Raffia palm (Raphia hookeri leaf extract on enzymes linked to type-2 diabetes mellitus (T2DM and pro-oxidant induced oxidative stress in rat pancreas. The extract was prepared and its α-amylase and α-glucosidase inhibitory effects were determined. Radical [2,2-diphenyl-1-picrylhydrazyl (DPPH] scavenging and Fe2+-chelating abilities, and inhibition of Fe2+-induced lipid peroxidation in rat pancreas homogenate were assessed. Furthermore, total phenol and flavonoid contents, reducing property, and high performance liquid chromatography diode array detector (HPLC-DAD fingerprint of the extract were also determined. Our results revealed that the extract inhibited α-amylase (IC50 = 110.4 μg/mL and α-glucosidase (IC50 = 99.96 μg/mL activities in concentration dependent manners which were lower to the effect of acarbose (amylase: IC50 = 18.30 μg/mL; glucosidase: IC50 = 20.31 μg/mL. The extract also scavenged DPPH radical, chelated Fe2+ and inhibited Fe2+-induced lipid peroxidation in rat pancreas all in concentration dependent manners with IC50 values of 402.9 μg/mL, 108.9 μg/mL and 367.0 μg/mL respectively. The total phenol and flavonoid contents were 39.73 mg GAE/g and 21.88 mg QAE/g respectively, while the reducing property was 25.62 mg AAE/g. The HPLC analysis revealed the presence of chlorogenic acid (4.17 mg/g and rutin (5.11 mg/g as the major phenolic compounds in the extract. Therefore, the ability of the extract to inhibit carbohydrate hydrolyzing enzymes and protect against pancreatic oxidative damage may be an important mechanisms supporting its antidiabetic properties and could make Raffia palm leaf useful in complementary/alternative therapy for management of T2DM. However, further studies such as in vivo should be carried out.

  11. A New Enzyme-Linked Immunosorbent Assay for a Total Anti-T Lymphocyte Globulin Determination: Development, Analytical Validation, and Clinical Applications.

    Science.gov (United States)

    Montagna, Michela; La Nasa, Giorgio; Bernardo, Maria E; Piras, Eugenia; Avanzini, Maria A; Regazzi, Mario; Locatelli, Franco

    2017-06-01

    Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological posttransplant complications, in particular rejection and graft-versus-host disease, after allogeneic hematopoietic stem cell transplantation (HSCT). We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with β-Thalassemia, receiving allogeneic HSCT. Diluted serum samples were incubated with Goat-anti-Rabbit IgG antibody coated on a microtiter plate and then, with Goat-anti-Human IgG labeled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492 nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200-0.095 ng/mL), prepared by diluting a known amount of ATLG in phosphate-buffered saline (PBS). Low, medium, and high-quality control concentrations were 1.56, 6.25, and 25 ng/mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095 ng/mL. We analyzed serum samples from 14 children with β-Thalassemia who received ATLG (Grafalon) at a dose of 10 mg/kg administered as intravenous (IV) infusion on days -5, -4, and -3 before HSCT (day 0). Blood sampling for PK evaluation was performed on days -5, -4, and -3 before and after drug infusion; and then from day -2 to +56. The median total ATLG levels pre-IVand post-IV were 0 and 118 mcg/mL on day -5; 85.9 and 199.2 mcg/mL on day -4; 153 and 270.9 mcg/mL on day -3, respectively. The median PK values of CL was 0.0029 (range: 0.0028-0.0057) L·kg·d, Vd was 0.088 (range: 0.025-0.448) L/kg and t1/2 was 20.2 (range: 5.8-50.2) days. These data suggest that given the marked interindividual variability of total ATLG disposition, the development of

  12. Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its evaluation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis.

    Science.gov (United States)

    Assana, E; Kanobana, K; Tume, C B; Zoli, P A; Nguekam; Geerts, S; Berkvens, D; Dorny, P

    2007-06-01

    A fraction with a major band of 14kDa was obtained from crude cyst fluid of Taenia solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration (Sephacryl S-300 high resolution) was purified using an anion exchange column (Mono Q HR 5/5) on high performance liquid chromatography. Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody enzyme linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense and Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with metacestode cyst fluid of T. hydatigena and T. solium. Antibody titres of lightly or heavily infected pigs differed in their kinetics. However, the increase in F3-specific antibodies could not be related to the infection level. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using cyst fluid as antigen, but the F3 antigen fraction was not recognized by rabbit hyper-immune serum samples to T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric receiver operating characteristic (ROC) analysis using 66 serum samples from Zambian village pigs. The total number of cysticerci of these pigs was determined by dissection (28 pigs harboured T. solium cysticerci and 38 were negative at dissection). In addition, 58 serum samples from Cameroonian pigs (28 pigs from cysticercosis-free farms and 30 pigs with cysticerci at tongue inspection) were used in a separate ROC analysis. The results from the ROC analysis yielded a low

  13. Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Branaa, P; Naar, J; Chinain, M; Pauillac, S

    1999-01-01

    With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate

  14. Enzyme-linked immunosorbent assays for detection of anti-transcriptional intermediary factor-1 gamma and anti-Mi-2 autoantibodies in dermatomyositis.

    Science.gov (United States)

    Fujimoto, Manabu; Murakami, Akihiro; Kurei, Shunsuke; Okiyama, Naoko; Kawakami, Atsushi; Mishima, Michiaki; Sato, Shinji; Seishima, Mariko; Suda, Takafumi; Mimori, Tsuneyo; Takehara, Kazuhiko; Kuwana, Masataka

    2016-12-01

    Autoantibodies against transcriptional intermediary factor 1 (TIF1) and Mi-2 are selectively detected in patients with dermatomyositis (DM). To measure these antibodies readily, the development of reliable ELISA systems has been needed. This study aimed to establish enzyme-linked immunosorbent assays (ELISAs) for anti-TIF1γ and anti-Mi-2β antibodies (Abs) and to assess their utility. Serum samples were obtained from 104 patients with classic DM, 68 with clinically amyopathic DM (CADM) and 70 with polymyositis, who were followed up at 8 medical centers across Japan. Serum samples from 190 patients with other connective tissue diseases (CTDs) and 123 healthy individuals were also assessed. Serum antibody levels were examined by ELISAs coated with full-length TIF1γ or Mi-2β proteins produced by a baculovirus expression system. To assess the cross-reactivity, partial-length Mi-2β proteins with or without mutations were produced and examined for reactivity. When compared with immunoprecipitation assay, anti-TIF1γ Ab ELISA showed 100% sensitivity and 100% specificity, while anti-Mi-2β Ab ELISA showed 100% sensitivity and 99.6% specificity. Anti-TIF1γ Ab was positive in 30 (28.8%) with classic DM and 4 (5.9%) with CADM, whereas 14 (13.5%) with classic DM, but none with CADM, were positive for anti-Mi-2β Ab. Of 30 anti-TIF1γ Ab-positive DM patients, 23 (67.6%) had malignancy. Anti-Mi-2β Ab-positive serum samples exhibited modest cross-reactivity with the TIF1γ protein due to the homologous amino acid sequence containing cysteines in their plant homeodomains. The current study demonstrates the utility of newly established ELISAs for anti-TIF1γ and anti-Mi-2β Abs, which can serve as easier detection systems for routine testing. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  15. Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Maiolini, Elisabetta; Ferri, Elida; Pitasi, Agata Laura; Montoya, Angel; Di Giovanni, Manuela; Errani, Ermanno; Girotti, Stefano

    2014-01-07

    Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 μg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 μg mL(-1)) and by the Directive 2004/19/EC (0.6 μg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one

  16. Microchamber Western blotting using poly-L-lysine conjugated polyacrylamide gel for blotting of sodium dodecyl sulfate coated proteins.

    Science.gov (United States)

    Chung, Minsub; Kim, Dohyun; Herr, Amy E

    2013-08-20

    We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time.

  17. Western Blotting Against Tagged Virulence Determinants to Study Bacterial Pathogenicity.

    Science.gov (United States)

    Aviv, Gili; Gal-Mor, Ohad

    2018-01-01

    Western blotting is a common approach to detect the presence of a target protein in biological samples or proteins mixture using specific antibodies. This method is also useful to study regulation of virulence determinants by analyzing changes in protein expression between different genetic backgrounds or under varying environmental conditions. To avoid the need to raise specific antibodies for each studied protein, commercial antibody against commonly used peptidic epitopes can be utilized if the right target tagged version is constructed. Here we describe a C-terminal fusion between a protein of interest and the two hemagglutinin A (2HA) tag. The tagged protein is cloned into a low-copy number vector and expressed under its native promoter in Salmonella enterica. Then, the expression of the tagged protein can be analyzed by Western blotting and commercially available anti-2HA antibodies.

  18. Sensitive detection of fluorescence in western blotting by merging images.

    Science.gov (United States)

    Kondo, Yukari; Higa, Shinichiro; Iwasaki, Takeshi; Matsumoto, Tomoya; Maehara, Kazumitsu; Harada, Akihito; Baba, Yoshihiro; Fujita, Masatoshi; Ohkawa, Yasuyuki

    2018-01-01

    The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

  19. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    OpenAIRE

    Tanaka, Hiroyuki; Putalun, Waraporn; Shoyama, Yukihiro

    2012-01-01

    We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb). Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was...

  20. Extracellular Vesicle Isolation and Analysis by Western Blotting.

    Science.gov (United States)

    Kowal, Emma J K; Ter-Ovanesyan, Dmitry; Regev, Aviv; Church, George M

    2017-01-01

    Extracellular vesicles (EVs) are released by mammalian cells and are thought to be important mediators of intercellular communication. There are many methods for isolating EVs from cell culture media, but the most popular methods involve purification based on ultracentrifugation . Here, we provide a detailed protocol for isolating EVs by differential ultracentrifugation and analyzing EV proteins (such as the tetraspanins CD9 , CD63 and CD81 ) by western blotting.

  1. Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

    Science.gov (United States)

    Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

    2016-01-01

    Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of

  2. Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and Western-blot

    Directory of Open Access Journals (Sweden)

    Rosana MARTINS

    1997-09-01

    Full Text Available Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8 and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P. brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot and 84% (ELISA of the patients presented elevated IgG anti-P. brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (5l.9% and 5l.8%: Dot-blot; 16.5 and 36%: ELISA, respectively but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodiesVinte e sete pacientes portadores de paracoccidioidomicose (PCM foram tratados com itraconazole (100-200 mg/dia no primeiro mês e 100 mg/dia até 6-8 meses e avaliados sob o ponto de vista clínico e sorológico, até 3 e meio anos após o início do tratamento, utilizando-se os testes de Dot-blot e ELISA para medir os títulos de anticorpos IgG, IgA e IgM anti-P. brasiliensis, e Western-blot

  3. Western blotting revisited: critical perusal of underappreciated technical issues.

    Science.gov (United States)

    Gorr, Thomas A; Vogel, Johannes

    2015-04-01

    The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

    Directory of Open Access Journals (Sweden)

    A Halajian

    2009-05-01

    Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

  5. Characterization of Acute-Phase Humoral Immunity to Monkeypox: Use of Immunoglobulin M Enzyme-Linked Immunosorbent Assay for Detection of Monkeypox Infection during the 2003 North American Outbreak

    OpenAIRE

    Karem, Kevin L.; Reynolds, Mary; Braden, Zach; Lou, Gin; Bernard, Nikeva; Patton, Joanne; Damon, Inger K.

    2005-01-01

    A monkeypox outbreak occurred in the United States in 2003. Patient's sera were sent to the Centers for Disease Control and Prevention as a part of outbreak response measures. Clinical and epidemiologic information was abstracted from the case investigation forms. Serum samples from patients were tested by using an immunoglobulin M (IgM)-capture and an IgG enzyme-linked immunosorbent assay ELISA against Orthopoxvirus antigen. The detection of antiviral IgG and IgM antibodies and the kinetics ...

  6. A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; He, Y.; Veidal, S. S.

    2011-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL......) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung-and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically...

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    . Positive reactions were found in sera from fattening pigs and sows 16 weeks and 3.5 years postoutbreak, respectively. There was, however, no positive reaction in sows with at least 10 vaccinations. Maternally derived antibodies to the 3AB antigen persisted in piglets up to 13 weeks of age. A high......Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...

  8. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonia; Pearce, Richard

    2005-01-01

    . However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...... the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt...

  9. Comparison of two 3ABC enzyme-linked immunosorbent assays for diagnosis of multiple-serotype foot-and-mouth disease in a cattle population in an area of endemicity

    DEFF Research Database (Denmark)

    Bronsvoort, B.M.D.; Sørensen, K.J.; Anderson, J.

    2004-01-01

    . The nonstructural polyprotein 3ABC has recently been proposed as such an antigen, and a number of diagnostic tests are being developed. This paper evaluates the performance of two FMDV tests for antibodies to nonstructural proteins in an unvaccinated cattle population from a region of Cameroon with endemic multiple......-serotype FMD. The CHEKIT-FMD-3ABC bo-ov (CHEKIT) enzyme-linked immunosorbent assay (ELISA) (Bommeli Diagnostics/Intervet) is a commercially available test that was compared with a competitive 3ABC ELISA (C-ELISA) developed in Denmark. The tests were compared with the virus neutralization test as the "gold...

  10. Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

    Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

  11. Investigation of Yeast Mitophagy with Fluorescence Microscopy and Western Blotting.

    Science.gov (United States)

    Nagumo, Sachiyo; Okamoto, Koji

    2017-03-24

    Selective clearance of superfluous or dysfunctional mitochondria is a fundamental process that depends on the autophagic membrane trafficking pathways found in many cell types. This catabolic event, called mitophagy, is conserved from yeast to humans and serves to control mitochondrial quality and quantity. In budding yeast, degradation of mitochondria occurs under various physiological conditions, such as respiration at stationary phase, or starvation in a prolonged period. During these events, the transmembrane protein Atg32 localizes to the mitochondrial surface and plays a specific and essential role in yeast mitophagy. In this chapter, we describe methods to observe transport of mitochondria to the vacuole, a lytic compartment in yeast, using fluorescence microscopy, and semi-quantify the progression of Atg32-mediated mitophagy by Western blotting.

  12. Defining thermostability of membrane proteins by western blotting.

    Science.gov (United States)

    Ashok, Y; Nanekar, R; Jaakola, V-P

    2015-12-01

    Membrane proteins are relatively challenging targets for structural and other biophysical studies. Insufficient expression in various expression systems, inherent flexibility, and instability in the detergents that are required for membrane extraction are the main reasons for this limited success. Therefore, identification of suitable conditions and membrane protein variants that can help stabilize functional protein for extended periods of time is critical for structural studies. Here, we describe a western blot-based assay that simplifies identification of thermostabilizing conditions for membrane proteins. We show successful testing of a variety of parameters such as additive lipids, ligands and detergents. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Identification and validation of rice reference proteins for western blotting.

    Science.gov (United States)

    Li, Xiaoming; Bai, Hui; Wang, Xianyun; Li, Liyun; Cao, Yinghao; Wei, Jian; Liu, Yumeng; Liu, Lijuan; Gong, Xiaodong; Wu, Lin; Liu, Siqi; Liu, Guozhen

    2011-10-01

    Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen-antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.

  14. Serological Diagnosis of Paracoccidioidomycosis through a Western Blot Technique

    Science.gov (United States)

    Perenha-Viana, M. C. Z.; Gonzales, I. A. A.; Brockelt, S. R.; Machado, L. N. C.

    2012-01-01

    Paracoccidioidomycosis (PCM) is a serious infectious disease that progresses toward death if untreated. Its confirmatory diagnosis is made by the detection of the fungus Paracoccidioides brasiliensis in a direct mycological examination or by histopathology. However, these techniques are of low sensitivity. Serological tests seem to be more promising. The objective of this study was to test Western blot (WB) analysis using sera from patients suspected of PCM to determine whether it represents a safe and sensitive serological technique for a rapid and effective diagnosis for this disease. Sera from 517 patients were analyzed through WB analysis and double-immunodiffusion (DID) techniques using a crude exoantigen of P. brasiliensis 339. DID gave positive reactions for 140 sera (27%) and WB for 250 sera (48.4%). All sera that had a positive reaction by DID also had a positive result with a 43-kDa glycoprotein by WB analysis. Among the 377 samples that were negative by DID, 29.1% were reactive in WB analysis. For the cutoff dilution used (1:400), a positive reaction was not observed with any of the 102 sera from patients with other diseases in regions where such diseases are endemic and 30 healthy individuals tested as negative controls. These results prove WB analysis to be a sensitive technique and suggest its inclusion among routine laboratory assays as a safe method for PCM diagnosis. PMID:22301695

  15. Western blotting by thin-film direct coating.

    Science.gov (United States)

    Yen, Yi-Kuang; Jiang, Yi-Wei; Chang, Shih-Chung; Wang, An-Bang

    2014-05-20

    A novel thin-film direct coating (TDC) technique was developed to markedly reduce the amount of antibody required for Western blotting (WB). Automatic application of the technique for a few seconds easily and homogeneously coats the specific primary antibody on the polyvinylidene fluoride (PVDF) membrane. While conventional WB requires 0.4 μg of the primary antibody, the proposed technique only uses 4 × 10(-2) μg, which can be reduced further to 4 × 10(-5) μg by reducing the coater width. Moreover, the proposed process reduces antibody probing times from 60 to 10 min. The quantification capability of TDC WB showed high linearity within a 4-log2 dynamic range for detecting target antigen glutathione-S-transferase. Furthermore, TDC WB can specifically detect the extrinsic glutathione-S-transferase added in the Escherichia coli or 293T cell lysate with better staining sensitivity than conventional WB. TDC WB can also clearly probe the intrinsic β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase, which are usually used as control proteins in biological experiments. This novel technique has been shown to not only have valuable potential for increasing WB efficiency but also for providing significant material savings for future biomedical applications.

  16. Serological diagnosis of paracoccidioidomycosis through a Western blot technique.

    Science.gov (United States)

    Perenha-Viana, M C Z; Gonzales, I A A; Brockelt, S R; Machado, L N C; Svidzinski, T I E

    2012-04-01

    Paracoccidioidomycosis (PCM) is a serious infectious disease that progresses toward death if untreated. Its confirmatory diagnosis is made by the detection of the fungus Paracoccidioides brasiliensis in a direct mycological examination or by histopathology. However, these techniques are of low sensitivity. Serological tests seem to be more promising. The objective of this study was to test Western blot (WB) analysis using sera from patients suspected of PCM to determine whether it represents a safe and sensitive serological technique for a rapid and effective diagnosis for this disease. Sera from 517 patients were analyzed through WB analysis and double-immunodiffusion (DID) techniques using a crude exoantigen of P. brasiliensis 339. DID gave positive reactions for 140 sera (27%) and WB for 250 sera (48.4%). All sera that had a positive reaction by DID also had a positive result with a 43-kDa glycoprotein by WB analysis. Among the 377 samples that were negative by DID, 29.1% were reactive in WB analysis. For the cutoff dilution used (1:400), a positive reaction was not observed with any of the 102 sera from patients with other diseases in regions where such diseases are endemic and 30 healthy individuals tested as negative controls. These results prove WB analysis to be a sensitive technique and suggest its inclusion among routine laboratory assays as a safe method for PCM diagnosis.

  17. Routine Western blot to check autophagic flux: cautions and recommendations.

    Science.gov (United States)

    Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M S; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M; Fuentes, José M; González-Polo, Rosa A

    2015-05-15

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    J.J.N. Ngeranwa

    2008-09-01

    Full Text Available The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra and domestic animal (cattle, sheep and goat populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA, using a recombinant antigen (MSP-5 from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA, as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

  19. The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens, followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period

    DEFF Research Database (Denmark)

    Skov, M.N.; Feld, Niels Christian; Carstensen, B.

    2002-01-01

    uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups...... at the onset of egg production. For both S. typhimurium and S. enteritidis, positive bacteriologic cultures were obtained by sampling from internal organs at the end of the experiment, more than 1 yr from the time of infection. At the age of 6-7 wk, 50% of the chickens in the two infected groups showed...... a measurable serologic response in serum samples. The response persisted throughout the study in both serum and egg yell, samples. The inclusion of serologic methods is a valuable additional tool in the detection of salmonella in poultry, but serology should be used in conjunction with bacteriologic methods...

  20. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Moller, T

    1999-01-01

    The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L....... donovani GBP (GBPP) in the diagnosis of L. donovani infections in Sudan. The sensitivity of the rGBP ELISA in diagnosing visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) was 92% and 93%, respectively. In contrast, the sensitivity of the GBPP ELISA was 55% for VL and 63% for PKDL...... for malaria but free of leishmaniasis was negative in both assays....

  1. Inhibition of key enzymes linked to type 2 diabetes and sodium nitroprusside-induced lipid peroxidation in rat pancreas by water-extractable phytochemicals from unripe pawpaw fruit (Carica papaya).

    Science.gov (United States)

    Oboh, Ganiyu; Olabiyi, Ayodeji A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

    2014-02-01

    Various parts of unripe pawpaw (Carica papaya Linn) fruit have been reportedly used for the management or treatment of diabetes mellitus in folklore medicine. Therefore, the present study sought to investigate the inhibitory effects of the aqueous extract of different parts of unripe pawpaw fruit on key enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and sodium nitroprusside (SNP)-induced lipid peroxidation in rat pancreas in vitro. The aqueous extracts of the unripe pawpaw (C. papaya) fruit parts were prepared (1:20 w/v) and the ability of the extracts to inhibit α-amylase, α-glucosidase and SNP-induced lipid peroxidation in rat pancreas in vitro was investigated. The results revealed that all the extracts inhibited α-amylase (IC50=0.87-1.11 mg/mL), α-glucosidase (IC50=1.76-2.64 mg/mL) and SNP-induced lipid peroxidation (IC50=1.99-2.42 mg/mL) in a dose-dependent manner. However, combination of the flesh, seed and peel in equal amounts had the highest inhibitory effect on α-amylase and α-glucosidase activities. Strong inhibitory activities of the unripe pawpaw fruit against key enzymes linked to type 2 diabetes and SNP-induced lipid peroxidation in rat pancreas could be part of the mechanism by which unripe pawpaw is used in the management/prevention of diabetes mellitus in folk medicine. However, combining the unripe pawpaw fruit parts in equal amounts exhibited synergistic properties on α-amylase and α-glucosidase inhibitory activities.

  2. The Combined Utility of Ex vivo IFN-γ Release Enzyme-Linked ImmunoSpot Assay and In vivo Skin Testing in Patients With Antibiotic-Associated Severe Cutaneous Adverse Reactions.

    Science.gov (United States)

    Trubiano, Jason A; Strautins, Kaija; Redwood, Alec J; Pavlos, Rebecca; Konvinse, Katherine C; Aung, Ar Kar; Slavin, Monica A; Thursky, Karin A; Grayson, M Lindsay; Phillips, Elizabeth J

    2017-10-31

    For severe cutaneous adverse reactions (SCARs) associated with multiple antibiotics dosed concurrently, clinical causality is challenging and diagnostic approaches are limited, leading to constricted future antibiotic choices. To examine the combined utility of in vivo and ex vivo diagnostic approaches at assigning drug causality in a cohort of patients with antibiotic-associated (AA)-SCARs. Patients with AA-SCARs were prospectively recruited between April 2015 and February 2017. In vivo testing (patch testing or delayed intradermal testing) was performed to the implicated antibiotic(s) at the highest nonirritating concentration and read at 24 hours through 1 week. Ex vivo testing used patient peripheral blood mononuclear cells (PBMCs) stimulated with a range of pharmacologically relevant concentrations of implicated antibiotics to measure dose-dependent IFN-γ release from CD4+ and CD8+ T cells via an enzyme-linked immunoSpot assay. In 19 patients with AA-SCARs, combined in vivo and ex vivo testing assigned antibiotic causality in 15 (79%) patients. Ten patients (53%) with AA-SCARs were positive on IFN-γ release enzyme-linked immunoSpot assay, with an overall reported sensitivity of 52% (95% CI, 29-76) and specificity of 100% (95% CI, 79-100), with improved sensitivity noted in acute (within 1 day to 6 weeks after SCAR onset) testing (75%) and in patients with higher phenotypic scores (59%). There was increased use of narrow-spectrum beta-lactams and antibiotics from within the implicated class following testing in patients with a positive ex vivo or in vivo test result. We demonstrate the potential utility of combined in vivo and ex vivo testing in patients with AA-SCARs to assign drug causality with high specificity. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  3. Northern blot analysis to investigate the abundance of microorganisms

    International Nuclear Information System (INIS)

    Krause, D.O.

    2005-01-01

    areas known as hyper-variable regions which have a high degree of sequence variation. As a result of this structure, it is possible to design signature oligonucleotide probes varying in length from about 15 to 30 nucleotides that are diagnostic of microorganisms at the kingdom, domain, genus and even species level. These signature sequences can be used in a variety of applications such as PCR analysis, construction of clone libraries or direct probing of bulk rRNA. In this chapter, I provide detailed protocols for the analysis of extracted rRNA and give detailed procedures that must be followed to do northern blot analysis of bulk RNA extracted from the rumen

  4. Conductive carbon nanoparticles-based electrochemical immunosensor with enhanced sensitivity for alpha-fetoprotein using irregular-shaped gold nanoparticles-labeled enzyme-linked antibodies as signal improvement.

    Science.gov (United States)

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

    2010-08-15

    A new electrochemical immunoassay protocol for sensitive detection of alpha-fetoprotein (AFP, as a model) is designed using carbon nanoparticles (CNPs)-functionalized biomimetic interface as immunosensing probe and irregular-shaped gold nanoparticles (ISNGs)-labeled horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-ISNG) as trace label. The low-toxic and high-conductive CNPs provided a high capacity nanoparticulate immobilization surface and a facile pathway for electron transfer. In comparison with conventional label methods, i.e. spherical gold nanoparticles-labeled HRP-anti-AFP and HRP-labeled anti-AFP, the electrochemical immunosensor using HRP-anti-AFP-ISNGs as trace labels exhibited high bioelectrocatalytic response toward enzyme substrate and a wide dynamic range from 0.02 to 4.0 ng/mL with a low detection limit of 10 pg/mL toward AFP (at 3sigma). The developed immunoassay method showed good selectivity and acceptable reproducibility. Clinical serum samples with various AFP concentrations were evaluated by using the electrochemical immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA), respectively, and received in good accordance with results obtained from these two methods. Copyright 2010 Elsevier B.V. All rights reserved.

  5. Validation according to OIE criteria of a monoclonal, recombinant p26-based, serologic competitive enzyme-linked immunosorbent assay as screening method in surveillance programs for the detection of Equine infectious anemia virus antibodies.

    Science.gov (United States)

    Nardini, Roberto; Autorino, Gian Luca; Ricci, Ida; Frontoso, Raffaele; Rosone, Francesca; Simula, Massimiliano; Scicluna, Maria Teresa

    2016-03-01

    The Italian National Reference Center for equine infectious anemia (CRAIE; Rome, Italy) developed and validated a monoclonal, recombinant p26-based competitive enzyme-linked immunosorbent assay (cELISA) for the detection of EIA virus antibodies employing the 2010 criteria of the World Organization for Animal Health (OIE). The following parameters were evaluated: cutoff values, repeatability, reproducibility, concordance, analytical sensitivity (Se), absolute analytical specificity (Sp), and diagnostic Se and Sp. Positive and negative predictive values were also defined in relation to the estimated prevalence. When the cELISA was used as a screening test for 96,468 samples in the Italian EIA surveillance program, 17% more EIA cases were detected than by the agar gel immunodiffusion test, and the apparent diagnostic Sp estimated from these samples was 99.8%, which was more than the diagnostic Sp (80.2%) estimated from validation. The high Se and Sp of the cELISA confirm its fit for purpose as a screening test. © 2015 The Author(s).

  6. Expression of Leptin and Visfatin in Gingival Tissues of Chronic Periodontitis With and Without Type 2 Diabetes Mellitus: A Study Using Enzyme-Linked Immunosorbent Assay and Real-Time Polymerase Chain Reaction.

    Science.gov (United States)

    Ghallab, Noha A; Amr, Eman M; Shaker, Olfat G

    2015-07-01

    The aim of this study is to investigate the protein and gene expression of leptin and visfatin in gingival tissue from patients with chronic periodontitis (CP), patients with CP and type 2 diabetes mellitus (T2DM), and healthy individuals. The study includes 50 individuals: 10 healthy individuals, 20 patients with CP, and 20 patients with CP and T2DM. Plaque index, gingival index, probing depth, and clinical attachment loss were measured, and gingival biopsies were obtained. Leptin and visfatin protein expression in gingival tissues was determined using enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was measured via real-time polymerase chain reaction. The highest leptin mRNA and protein expression was observed in the control group and was significantly (P ≤0.05) different from the CP and CP+T2DM groups. Gingival tissues from patients with CP and T2DM had a significant increase in visfatin and a decrease in leptin gene and protein expression (P <0.05) compared with both controls and patients with CP. Expression of leptin and visfatin in the gingival tissues suggests a possible role for these adipokines in the pathogenesis of CP and T2DM.

  7. Fabrication of paper devices via laser-heating-wax-printing for high-tech enzyme-linked immunosorbent assays with low-tech pen-type pH meter readout.

    Science.gov (United States)

    Le, Shangwang; Zhou, Hui; Nie, Jinfang; Cao, Chaohong; Yang, Jiani; Pan, Hongcheng; Li, Jianping; Zhang, Yun

    2017-01-26

    In this work, a new method named laser-heating-wax-printing (LHWP) is described to fabricate paper devices for developing sensitive, affordable, user-friendly paper-based enzyme-linked immunosorbent assays (P-ELISAs) that initially use common pen-type pH meters for portable, quantitative readout. The LHWP enables a rapid patterning of wax in paper via one step of heating the wax layer coated on the paper surface using a mini-type CO 2 laser machine. Wax-patterned paper microzones created in this way are utilized to conduct the pen-type pH meter-based P-ELISAs with enzyme-loaded SiO 2 microbeads for highly efficient signal amplification of each antibody-antigen binding event. The results show that this new P-ELISA system is quantitatively sensitive to the concentrations of a model protein analyte in buffer samples ranging from 12.5 to 200 pg mL -1 , with a limit of detection of ca. 7.5 pg mL -1 (3σ). Moreover, the satisfactory recovery results of assaying several human serum samples validate its feasibility for practical applications.

  8. Analysis of matrix metalloproteinase-8 levels in gingival crevicular fluid and whole mouth fluid among smokers and nonsmokers using enzyme-linked immune-sorbent assay and a novel chair-side test.

    Science.gov (United States)

    Akbari, Ghousia; Prabhuji, Munivenkatappa Lakshmaiah Venkatesh; Karthikeyan, Bangalore Vardhan; Raghunatha, Kanugondappa; Narayanan, Roopalakshmi

    2015-01-01

    To indigenously prepare a chair-side test kit for investigating and comparing the matrix metalloproteinase (MMP)-8 levels in gingival crevicular fluid (GCF) and saliva in patients with healthy periodontium, gingivitis and chronic periodontitis in smokers and nonsmokers. To validate the diagnostic accuracy of indigenously prepared chair-side test against enzyme-linked immune-sorbent assay (ELISA). Furthermore, to assess the effect of nonsurgical periodontal therapy (NSPT) on the levels of MMP-8 in GCF and saliva among the test groups. GCF and saliva were collected from 250 subjects. The study population were divided into five groups; health periodontium-nonsmokers (Group 1; n = 50), chronic gingivitis-nonsmokers (Group 2; n = 50), chronic periodontitis-nonsmokers (Group 3; n = 50), chronic gingivitis-smokers (Group 4; n = 50), chronic periodontitis-smokers (Group 5; n = 50). A chair-side test kit was indigenously prepared using polyclonal antibodies (principle of immunochromatography) to detect the MMP-8 levels, and it was validated against ELISA at baseline and 3 months after NSPT. The chair-side test detected MMP-8 levels with a sensitivity and specificity in accordance with ELISA. MMP-8 levels at baseline were higher in Group 2 and Group 3 as compared to controls (P chair-side test detected MMP-8 levels accurately making it a viable chair side diagnostic tool. It was effective for early diagnosis of the periodontal disease among high-risk population such as smokers.

  9. Enzyme-Linked Immunosorbent Assay Based on Soluble Promastigote Antigen Detects Immunoglobulin M (IgM) and IgG Antibodies in Sera from Cases of Visceral and Cutaneous Leishmaniasis

    Science.gov (United States)

    Ryan, Jeffrey R.; Smithyman, Anthony M.; Rajasekariah, G-Halli; Hochberg, Lisa; Stiteler, John M.; Martin, Samuel K.

    2002-01-01

    Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity. PMID:11880434

  10. A reliable screening test to identify adult carriers of the (--SEA) alpha zero-thalassemia deletion. Detection of embryonic zeta-globin chains by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Lafferty, J D; Crowther, M A; Waye, J S; Chui, D H

    2000-12-01

    Homozygous (--SEA) alpha zero-thalassemia deletion, the cause of up to 80% of fetal hydrops in Southeast Asia, is encountered in many other countries. Heterozygous carrier rates of the deletion in Southeast Asian populations range from 4% to 14%. The laboratory screening for adult carriers of (--SEA) and other alpha zero-thalassemia deletions currently rests primarily with microscopic detection of hemoglobin H inclusion bodies within erythrocytes (Hb H screen). This test is laborious and observer dependent and has poor sensitivity. We assessed a colorimetric enzyme-linked immunosorbent assay (ELISA) to detect embryonic zeta-globin chains in adult hemolysates as an alternative to detect (--SEA) alpha zero-thalassemia deletion carriers. Blood samples from 221 adults with a mean corpuscular volume less than 80 micron 3 (80 fL) were studied prospectively by currently accepted hemoglobin screening tests and ELISA. Suspected cases of alpha-thalassemia were confirmed by DNA-based diagnostics. ELISA was highly sensitive (1.0) and specific (0.94) for the detection of adult carriers of (--SEA) alpha zero-thalassemia deletion. The hemoglobin H screen had a sensitivity of 0.47 and specificity of 0.99. The zeta-globin ELISA proved simple to perform, rapid, and applicable to high volume or population-based screening programs.

  11. Enzyme linked immunosorbent assay (ELISA for the detection of IgG, IgM, IgE and IgA against Cysticercus cellulosae in cerebrospinal fluid of patients with neurocysticercosis

    Directory of Open Access Journals (Sweden)

    Odashima Newton Satoru

    2002-01-01

    Full Text Available The objective of this study was to analyze different immunoglobulins classes (IgG, IgM, IgE and IgA against Cysticercus cellulosae in the cerebrospinal fluid (CSF, through enzyme linked immunosorbent assay (ELISA, correlating them to clinical and tomographic profiles in patients with neurocysticercosis (NCC. Eighty-five specimens of CSF were obtained from 43 cases with NCC (26 with the active form and 17 with the inactive form and from 42 patients with other neurological diseases. The inactive form of NCC presented a profile in CSF similar to the group without NCC. The active form of NCC presented elevation of specific immunoglobulins (IgG, IgM, IgE, and IgA in decreasing order, with the highest values being detected among the cases with intraventricular cysts, or with inflammation signs in CSF or in those with multiple clinical manifestations. The highest sensitivity and specificity were obtained with ELISA-IgG (88.5% and 93.2%, respectively. This study confirmed the importance of ELISA in the immunologic diagnosis of NCC.

  12. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Mehrangiz Rajaii

    2015-01-01

    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

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

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    2011-06-01

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

  14. Comparison of bacterial culture, polymerase chain reaction, and a mix-enzyme-linked immunosorbent assay for the detection of Salmonella status in grow-to-finish pigs in western Canada with a Bayesian approach.

    Science.gov (United States)

    Wilkins, Wendy; Waldner, Cheryl; Rajić, Andrijana; McFall, Margaret; Chow, Eva; Muckle, Anne

    2011-10-01

    Among grow-to-finish pigs from 10 herds in Alberta and Saskatchewan, 23 (16%) of 144 fecal samples were culture-positive and 40 (28%) of 144 pigs were seropositive for Salmonella. With a Bayesian model specifying dependence between the 2 tests, the sensitivity (Se) of culture and real-time polymerase chain reaction (RT-PCR) was 79% to 86%, depending on the cut-off value for the enzyme-linked immunosorbent assay (ELISA). Culture specificity (Sp) was assumed to be 100%; RT-PCR Sp was found to be 94%. The ELISA Se was 76% and 51% at optical density cut-off values ≥ 20% and ≥ 40%, respectively; the Sp was 94% at each cut-off value. The model showed some sensitivity to ELISA prior information, the ELISA Se being approximately 8% lower when informative prior information was specified in the model. When there was no adjustment for dependence between culture and RT-PCR, the posterior estimates for both culture and RT-PCR Se were 11% higher than with the conditional-dependence model and had considerably narrower probability intervals, which suggests that correlation between culture and PCR is important and should be adjusted for in future studies.

  15. Zinc blotting assay for detection of zinc binding prolamin in barley (Hordeum vulgare) grain

    DEFF Research Database (Denmark)

    Uddin, Mohammad Nasir; Nielsen, Ane Langkilde-Lauesen; Vincze, Eva

    2014-01-01

    zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

  16. Immunochemische detectiemethoden na western blotting van cytochroom P-450 iso-enzymen

    NARCIS (Netherlands)

    Laan CA; Jansen EHJM

    1992-01-01

    In this report a number of staining techniques on Western blots have been compared with respect to sensitivity, background staining, practical applicability and cost aspects. After electrophoresis of a rat microsomal liver sample followed by blotting, an incubation was performed of a primary

  17. Preparation of Cell Lysate from Mouse Oocytes for Western Blotting Analysis.

    Science.gov (United States)

    Marangos, Petros

    2016-01-01

    Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis.

  18. When less is more: a simple Western blotting amendment allowing data acquisition on human single fibers

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Richter, Erik

    2011-01-01

    This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies.......This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies....

  19. Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.

    Science.gov (United States)

    Hou, Chenlu; Herr, Amy E

    2013-01-07

    We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.

  20. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    Science.gov (United States)

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  1. The fastest Western in town: a contemporary twist on the classic Western blot analysis.

    Science.gov (United States)

    Silva, Jillian M; McMahon, Martin

    2014-02-05

    The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

  2. Sensitivity improvement of rapid Vibrio harveyi detection with an enhanced chemiluminescent-based dot blot.

    Science.gov (United States)

    Li, H; Xiao, J; Zhou, Y; Wang, Q; Zhang, Y

    2017-09-01

    Vibrio harveyi is an opportunistic pathogen in seawater and can cause severe vibriosis. It is prevalent in hatcheries worldwide and can lead to severe economic losses. Therefore, there is an urgent need to develop a rapid detection method for monitoring this pathogen. In this study, to increase the detection sensitivity of our assay with monoclonal antibodies (Mabs) against V. harveyi, the conditions of the dot blot assay were optimized, and enhanced chemiluminescent (ECL) substrate replaced the traditional tetramethylbenzidine (TMB) substrate. Based on the optimization results, an ECL-based novel dot blot assay was developed for the rapid and sensitive detection of V. harveyi. Compared with the traditional dot blot assay, the incubation time was shortened from 8 to 2 h. The limit of detection (LOD) for V. harveyi was 2 × 10 5  CFU per ml (10 3  CFU per spot) in pure bacterial suspension, which was 50-fold more sensitive than the traditional dot blot assay (1 × 10 7  CFU per ml). Furthermore, when compared with indirect ELISA, the dot blot assay showed approximately 1000-fold higher sensitivity (CFU/CFU). After the test sample was pre-enriched in turbot homogenates for 6 h before the dot blot analysis, the LOD for V. harveyi was 10 CFU per ml. Vibrio harveyi is one of the most opportunistic pathogens that can cause high mortality in hatcheries worldwide. To detect this pathogen, a novel dot blot based on enhanced chemiluminescent (ECL) has been established. This ECL-based dot blot was found to be more sensitive and rapid for V. harveyi detection than traditional dot blot, and this technology is recommended as an applied protocol for monitoring V. harveyi in seawater to reduce economic losses. © 2017 The Society for Applied Microbiology.

  3. SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma.

    Science.gov (United States)

    García-Solaesa, Virginia; Abad, Sara Ciria

    2016-01-01

    Western blotting is used to analyze proteins after being separated by electrophoresis and subsequently electro-transferred to a membrane. Once immobilized, a specific protein can be identified through its reaction with a labeled antibody or antigen. It is a methodology commonly used in biomedical research such as asthma studies, to assess the pathways of inflammatory mediators involved in the disease.Here, we describe an example of western blotting to determine the factors involved in asthma. In this chapter, the methodology of western blotting is reviewed, paying attention on potential problems and giving interesting recommendations.

  4. Method for resolution and western blotting of very large proteins using agarose electrophoresis.

    Science.gov (United States)

    Greaser, Marion L; Warren, Chad M

    2015-01-01

    Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000-3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins.

  5. Development of a highly sensitive and specific monoclonal antibody based enzyme-linked immunosorbent assay for the detection of a new β-agonist, phenylethanolamine A, in food samples.

    Science.gov (United States)

    Jiang, Danni; Cao, Biyun; Wang, Meiyu; Yang, Hong; Zhao, Kang; Li, Jianguo; Li, Mingxin; Sun, Lulu; Deng, Anping

    2017-02-01

    All β-agonists are banned as feed additives for growth promotion in animals due to toxic effects on humans after consuming the β-agonist contaminated meats. Phenylethanolamine A (PA) is a newly emerged β-agonist. Thus there is a need to develop highly sensitive and specific analytical methods for the detection of PA in food samples. In this study, the monoclonal antibody (mAb) against PA was produced by hybridoma technology and used for the development of enzyme-linked immunosorbent assay (ELISA). The IC 50 values and limits of detection (LODs) of the ELISA using homogeneous combination of coating antigen/antibody for PA were 0.16 ng mL -1 and 0.011 ng mL -1 , respectively. The cross-reactive (CR) values of the assay with 14 structurally related β-agonists were lower than 0.59%. Swine liver and meat samples were spiked with PA at different content and analysed by ELISA. Acceptable recovery rates of 91.40-105.51% and intra-assay coefficients of variation of 1.56-9.92% (n = 3) were obtained. The ELISA for seven spiked samples was confirmed by LC-MS/MS with a high correlation coefficient of 0.9881. The proposed mAb-based ELISA was highly sensitive and specific for PA and could be used as a quantitative/screening method for PA analysis in food samples. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Natural anti-insulin autoantibodies in cats: enzyme-linked immunosorbent assay for the determination of plasma anti-insulin IgG and its concentrations in domestic cats.

    Science.gov (United States)

    Takashima, Satoshi; Nishii, Naohito; Hachisu, Tatsuyuki; Kojima, Masaaki; Kigure-Hoshino, Megumi; Ogawa, Shizuko; Suzuki, Takafumi; Iwasawa, Atsushi; Ohba, Yasunori; Kitagawa, Hitoshi

    2013-12-01

    Anti-insulin immunoglobulin G (IgG) has been found in the sera of healthy cats. To determine the concentrations of these antibodies, an enzyme-linked immunosorbent assay (ELISA) for anti-insulin IgG was developed. ELISA maintained the linearity of a standard concentration line between 67.5 and 2160 ng/ml. The coefficients of variances (CVs) of intra-assays in two different plasma samples were 4.0% and 3.7%, respectively. The inter-assay CVs in two different plasma samples were 5.1% and 6.9%, respectively. The dilution curves of two samples were rectilinear. Anti-insulin IgG was detected in all 84 of the healthy cats that were tested. Plasma anti-insulin IgG concentrations ranged from 80 to 1578 μg/ml, with a median concentration of 221 μg/ml, and this value correlated positively with total plasma IgG concentrations (r=0.383, pglucose tolerance test, plasma anti-insulin IgG concentrations did not alter, even with changes in plasma glucose and insulin concentrations. The ELISA that was developed was able to determine plasma anti-insulin IgG in domestic cats, and confirmed that all healthy cats had plasma anti-insulin IgG. Determining the plasma concentrations of anti-insulin IgG in cats with various pathological conditions might clarify the role of anti-insulin IgG. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Enzyme-Linked Immunospot Assay as a Complementary Method to Assess and Monitor Cytomegalovirus Infection in Kidney Transplant Recipients on Pre-emptive Antiviral Therapy: A Single-Center Experience.

    Science.gov (United States)

    Favi, E; Santangelo, R; Iesari, S; Morandi, M; Marcovecchio, G E; Trecarichi, E M; Salerno, M P; Ferraresso, M; Citterio, F; Romagnoli, J

    2017-10-01

    Cytomegalovirus (CMV) disease represents a major cause of post-transplantation morbidity and mortality. To estimate the risk of infection and monitor response to antiviral therapy, current guidelines suggest combination of viral load monitoring with direct assessment of CMV-specific immune response. We used enzyme-linked immunospot (ELISpot) for the evaluation of CMV-specific T-cell response in kidney transplant recipients with CMV viremia and investigated how information gained could help manage CMV infection. Seventeen patients on pre-emptive antiviral therapy and CMV quantitative polymerase chain reaction (qPCR) ≥500 copies/mL (first episode after transplantation) were assessed using ELISpot and divided into Weak (9 patients with baseline ELISpot antiviral therapy were prospectively recorded and compared between groups at 1, 2, and 24 months of follow-up. Demographic and transplant characteristics of Weak and Strong Responders were similar. No episodes of CMV disease were observed. Weak Responders were more likely to experience CMV syndrome (56% vs 36.5%) and late virus reactivation (56% vs 25%) than Strong Responders. Weak Responders showed higher baseline median viral loads (19,700 vs 9265 copies/mL) and needed antiviral therapy for longer (179 vs 59.5 days). T-cell response showed 2 main patterns: early and delayed. ELISpot provides prognostic information about infection severity, risk of late reactivation, and response to therapy. Randomized trials, evaluating the need for antiviral therapy in kidney transplant recipients with asymptomatic infection and effective virus-specific T-cell immune response, are warranted. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Novicki, T J; Daly, J A; Mottice, S L; Carroll, K C

    2000-02-01

    Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.

  9. 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.

  10. Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

    Science.gov (United States)

    Pannhorst, Katrin; Fröhlich, Andreas; Staubach, Christoph; Meyer, Denise; Blome, Sandra; Becher, Paul

    2015-07-01

    Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals. © 2015 The Author(s).

  11. Detection and quantification of protein-protein interactions by far-western blotting.

    Science.gov (United States)

    Jadwin, Joshua A; Mayer, Bruce J; Machida, Kazuya

    2015-01-01

    Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.

  12. A duplex approach for immunochemical staining and typing of protein in western blots

    NARCIS (Netherlands)

    Kuczius, T.; Brandstädter, L.; Karch, H.; Langeveld, J.P.M.

    2011-01-01

    The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a

  13. Multistrip Western blotting: a tool for comparative quantitative analysis of multiple proteins.

    Science.gov (United States)

    Aksamitiene, Edita; Hoek, Jan B; Kiyatkin, Anatoly

    2015-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western blotting increases data output per single blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.

  14. Development and evaluation of a Western blot kit for diagnosis of schistosomiasis

    NARCIS (Netherlands)

    Sulahian, Annie; Garin, Yves Jean François; Izri, Arezki; Verret, Caroline; Delaunay, Pascal; van Gool, Tom; Derouin, Francis

    2005-01-01

    We evaluated the performance of Western blot (WB) analysis using commercially available antigen strips and compared the results with those of indirect hemagglutination (IHA) and indirect immunofluorescence (IFAT) for the serodiagnosis of human schistosomiasis. The antigen preparation was a crude

  15. CRITERIA OF POSITIVITY FOR Ig ANTIBODIES IN THE METHOD OF IMMUNE BLOTTING OF LYME DISEASE

    Directory of Open Access Journals (Sweden)

    V G Barskova

    2001-01-01

    Full Text Available There are currently no accepted criteria for positive Western blots in Russian patients with Lyme borreliosis. The purpose of the current study was to develop criteria for a positive IgG westem-blot to aid particularly in the diagnosis of patients with joint manifestation of the disorder. Patients: 97 with Lyme disease, 145 - control subjects. IgG antibody responses were determined to 3 species ofB.burgdorferi sensu lato by Western blotting, using blots prepared by manufacturer. The best discriminatory ability of test criteria was chained by requiring any 3 of 11 IgG bands, a definition that could be used with B. burgdorferi sensu stricto, B.garinii and B.afzelii strains. With these 3 antigen preparation, positive IgG blots were found in 0 to 18% of patients with localized erythema migrans of < 4 weeks duration, 23 to 39% of those with disseminated infection < 20 weeks duration, and in 39 to 46% of those with late arthritis/arthralgia of >6 months duration the specificity was 93 to 99%. Thus, IgG Western blotting may bring greater specificity to serologic testing in Lyme borreliosis, but the sensitivity is limited.

  16. Using a large area CMOS APS for direct chemiluminescence detection in Western blotting electrophoresis

    Science.gov (United States)

    Esposito, Michela; Newcombe, Jane; Anaxagoras, Thalis; Allinson, Nigel M.; Wells, Kevin

    2012-03-01

    Western blotting electrophoretic sequencing is an analytical technique widely used in Functional Proteomics to detect, recognize and quantify specific labelled proteins in biological samples. A commonly used label for western blotting is Enhanced ChemiLuminescence (ECL) reagents based on fluorescent light emission of Luminol at 425nm. Film emulsion is the conventional detection medium, but is characterized by non-linear response and limited dynamic range. Several western blotting digital imaging systems have being developed, mainly based on the use of cooled Charge Coupled Devices (CCDs) and single avalanche diodes that address these issues. Even so these systems present key drawbacks, such as a low frame rate and require operation at low temperature. Direct optical detection using Complementary Metal Oxide Semiconductor (CMOS) Active Pixel Sensors (APS)could represent a suitable digital alternative for this application. In this paper the authors demonstrate the viability of direct chemiluminescent light detection in western blotting electrophoresis using a CMOS APS at room temperature. Furthermore, in recent years, improvements in fabrication techniques have made available reliable processes for very large imagers, which can be now scaled up to wafer size, allowing direct contact imaging of full size western blotting samples. We propose using a novel wafer scale APS (12.8 cm×13.2 cm), with an array architecture using two different pixel geometries that can deliver an inherently low noise and high dynamic range image at the same time representing a dramatic improvement with respect to the current western blotting imaging systems.

  17. Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method.

    Science.gov (United States)

    Huang, Hongsheng; Soutyrine, Andrei; Rendulich, Jasmine; O'Rourke, Katherine; Balachandran, Aru

    2011-01-01

    Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

  18. Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis.

    Directory of Open Access Journals (Sweden)

    Priscila Zacarias de Azevedo

    Full Text Available The diagnosis of chronic pulmonary aspergillosis (CPA depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA and four chronic fibrosing pulmonary aspergillosis (CFPA; G2: 28 patients with pulmonary tuberculosis (TB; G3: 23 patients with histoplasmosis (HST; G4: 50 patients with paracoccidioidomycosis (PCM; G5: 20 patients with cryptococcosis (CRC; and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations--Aspergillus fumigatus (DID1, ELISA1 and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2. The Platélia Aspergillus Enzyme Immunoassay (EIA kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio-especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after

  19. Validation and evaluation of VapA-specific IgG and IgG subclass enzyme-linked immunosorbent assays (ELISAs) to identify foals with Rhodococcus equi pneumonia.

    Science.gov (United States)

    Sanz, M G; Oliveira, A F; Loynachan, A; Page, A; Svansson, V; Giguère, S; Horohov, D W

    2016-01-01

    Rhodococcus equi (Rhodococcus hoagii/Prescottella equi) is a common cause of foal pneumonia, but its diagnosis remains a challenge for equine veterinarians. While the VapA-specific (virulence-associated protein A) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) has low sensitivity and specificity for detecting pneumonic foals, little is known about VapA-specific IgG subclasses. To evaluate the performance of VapA-specific ELISA for IgG and its subclasses IgGa, IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi. Assay validation followed by assessment of diagnostic performance using archived samples from animals of known status. Serum samples from exposed (n = 125) and nonexposed adult horses (n = 10) and from experimentally challenged and naturally infected foals were used for ELISA validation. Post mortem and tissue culture records of the last 24 years from the Institute for Experimental Pathology at the University of Iceland in Keldur, Iceland laboratory were evaluated to confirm the absence of R. equi cases in Iceland. The diagnostic performance of VapA-specific IgG and its subclasses was evaluated using banked serum samples from pneumonic (n = 21) and healthy foals (n = 80). To evaluate each IgG assay, a cut-off value was selected based on receiver operating characteristic curve analysis and used to calculate sensitivity and specificity. The intra- and interassay coefficients of variation were calculated for each ELISA. Using sera from Iceland, where R. equi infection has not been reported, the VapA-specific IgG ELISA differentiated exposed from nonexposed horses. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity and specificity. Horses from Iceland are not exposed to VapA(+) R. equi and can serve as negative controls. VapA-specific IgG subclasses, with the exception of IgG(T), are poor predictors of disease. Further

  20. Development and application of an enzyme-linked immunosorbent assay for specific detection of mangiferin content in various cultivars of Mangifera indica leaves using anti-mangiferin polyclonal antibody.

    Science.gov (United States)

    Yusakul, Gorawit; Kitirattrakarn, Wongsathorn; Tanwanichkul, Narunat; Tanaka, Hiroyuki; Putalun, Waraporn

    2012-04-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for determining mangiferin content in plant samples using a polyclonal antibody (PAb) against mangiferin. The developed ELISA showed a full measurement range from 0.12 to 31.25 μg/mL mangiferin with a relative standard deviation (RSD) less than 6% for both intra- and interassay precision levels. The accuracy was determined by a percent recovery experiment at three concentration levels and it showed 97.8%-103.7% recovery in Mangifera indica leaf samples. The developed ELISA exhibited a high correlation value (R² = 0.992) with the standard high-performance liquid chromatography (HPLC) method in various mangiferin-containing plant samples. Our results suggest that the validated ELISA methodology using a PAb against mangiferin can be applied to determine mangiferin content with high specificity, rapidity and simplicity in various mangiferin-containing plant samples. The mangiferin content in the mature leaves of fifty M. indica cultivars were determined using the developed ELISA. The mangiferin contents ranged from 1.94 ± 0.13% to 13.79 ± 0.84% dry wt. The Thawai cultivar leaves contained the highest level of mangiferin (13.79 ± 0.84% dry wt), but it is a rare cultivar. The Namdokmai, which is more commonly cultivated in Thailand, contain 12.41 ± 0.60% dry wt mangiferin; therefore, this cultivar leaf was recommended as the source of raw material for the pharmaceutical, nutraceutical and cosmetic product industries. Currently, natural heath products are accepted worldwide for healthcare. Mangiferin-containing plants and products exhibit health benefits against oxidative stress-related diseases, such as cardiovascular diseases, cancer, dyslipidemia and diabetes. We have developed an ELISA with high specificity, rapidity and simplicity for the quality control of mangiferin-derived product production. Moreover, we found that the Namdokmai leaf, a Thai M. indica cultivar, was recommended as the source of raw

  1. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia.

    Science.gov (United States)

    Chaorattanakawee, Suwanna; Tyner, Stuart D; Lon, Chanthap; Yingyuen, Kritsanai; Ruttvisutinunt, Wiriya; Sundrakes, Siratchana; Sai-gnam, Piyaporn; Johnson, Jacob D; Walsh, Douglas S; Saunders, David L; Lanteri, Charlotte A

    2013-07-12

    Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of "immediate ex vivo" (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission. Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson's correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples. IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the MSF assay, when evaluating ex

  2. Serum concentration of 20K human growth hormone (20K hGH) measured by a specific enzyme-linked immunosorbent assay. Study Group of 20K hGH.

    Science.gov (United States)

    Tsushima, T; Katoh, Y; Miyachi, Y; Chihara, K; Teramoto, A; Irie, M; Hashimoto, Y

    1999-01-01

    Several GH isoforms have been identified in pituitary and serum, the most abundant of which is the 22K human GH (hGH) isoform. The 20K hGH isoform is produced by alternative splicing of GH messenger ribonucleic acid and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined, partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunosorbent assays (ELISAs) specific to 20K and 22K hGH. To determine whether regulation of 20K hGH secretion is the same as that for 22K hGH, we measured serum concentrations of both species of hGH in normal subjects and patients with a variety of endocrine disorders. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (n = 282) in normal women, significantly higher than that in normal men (64 +/- 170 pg/mL; n = 226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, mean +/- SD; n = 176) and women (6.3 +/- 2.1%; n = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight, or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or that a chronic excess of 22K hGH alters the MCR of 20K hGH. The values in patients with adult GH deficiency, hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests, such as the insulin tolerance test and the GHRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH. This new assay may provide a tool for understanding the physiological or pathophysiological role of the 20K hGH isoform.

  3. Application of Monoclonal Antibodies against Bioactive Natural Products: Eastern Blotting and Preparation of Knockout Extract

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available Matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was used for the confirmation of hapten number in synthesized antigen. As application of MAb, the MAbs against ginsenosides and glycyrrhizin have been prepared resulting in the development of two new techniques that we named the eastern blotting method and the knockout extract preparation. In eastern blotting technique, glycosides like ginsenosides and glycyrrhizin separated by silica gel TLC were blotted to PVDF membrane that was treated with a NaIO4 solution followed by BSA resulted in glycoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double staining of eastern blotting for ginsenosides using antiginsenoside Rb1 and Rg1 MAbs promoted complete identification of ginsenosides in Panax species. The immunoaffinity concentration of glycyrrhizin was determined by immunoaffinity column conjugated with antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was determined by the synergic effect with glycyrrhizin on NO production using the cell line.

  4. Western blotting in the diagnosis of duodenal-biliary and pancreaticobiliary refluxes in biliary diseases.

    Science.gov (United States)

    Xian, Guo-Zhe; Wu, Shuo-Dong; Chen, Chun-Chih; Su, Yang

    2009-12-01

    Currently adopted diagnostic methods for duodenal-biliary and pancreaticobiliary refluxes carry many flaws, so the incidence of the two refluxes demands further larger sample size studies. This study aimed to evaluate Western blotting for the diagnosis of refluxes in biliary diseases. An oral radionuclide 99mTc-DTPA test (radionuclide, RN) was conducted for the observation of duodenal-biliary reflux prior to measuring bile radioactivity and Western blotting for detecting bile enterokinase (EK). Pancreaticobiliary reflux was assessed by biochemical and Western blotting tests for biliary amylase activity and trypsin-1, respectively. In accordance with bile sample origin, our samples were classified into ductal bile and gall bile groups; based on each individual biliary disease, we further classified the ductal bile group into five sub-groups, and the gall bile group into four sub-groups. Western blotting was conducted to assess the two refluxes in biliary diseases. Consistencies were noted between EK and RN tests when diagnosing duodenal-biliary reflux (P0.05); in the common bile duct cyst group, the EK positive rate was significantly lower than the trypsin-1 positive rate (PWestern blotting can accurately reflect duodenal-biliary and pancreaticobiliary refluxes. EK has greater sensitivity than RN for duodenal-biliary reflux. The majority of biliary amylase and lipase comes from the pancreas in all biliary diseases; pancreaticobiliary reflux is the predominant source in the common bile duct cyst group and duodenal-biliary reflux is responsible for the ductal pigment stone group.

  5. Prolonged incubation and stacked film exposure improve sensitivity in western blotting.

    Science.gov (United States)

    Luo, Haitao; Rankin, Gary O; Straley, Shannon; Chen, Yi Charlie

    2011-01-01

    Western blotting is a basic technique for protein detection. For proteins of less abundance or antibodies of poorer quality, an increased sensitivity is often desired. Although it is commonly known that higher concentrations of antibodies and prolonged film exposure times will help improve sensitivity in western blots, both measures come with their own risks, and it is often unclear to which extent these measures should be applied. We conducted time-course studies to investigate protein-antibody interactions and primary antibody-secondary antibody interactions in western blotting. We also propose a protocol of stacked film exposure and have tested it in standard curves and cancer cell samples. Our study found that protein-primary antibody interactions and primary antibody-secondary antibody interactions could take a longer time than commonly used "one hour" or "overnight", and in some cases longer than 48h, to reach its maximum binding. We also show that the modified protocol of stacked film exposure works well for both standard curves and biological samples, reaching a maximum sensitivity in western blots without blurring target signals or increasing backgrounds. In addition to regular optimization of antibody concentrations and film exposure time, a prolonged incubation with antibodies and stacked film exposure will also help improve sensitivity and reduce background in western blotting. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.

    Science.gov (United States)

    Eaton, Samantha L; Hurtado, Maica Llavero; Oldknow, Karla J; Graham, Laura C; Marchant, Thomas W; Gillingwater, Thomas H; Farquharson, Colin; Wishart, Thomas M

    2014-11-20

    The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term "western blot" suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many "optimized" western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

  7. Validation of a Dot-Blot quantitative technique for large scale analysis of beef tenderness biomarkers.

    Science.gov (United States)

    Guillemin, N; Meunier, B; Jurie, C; Cassar-Malek, I; Hocquette, J-F; Leveziel, H; Picard, B

    2009-10-01

    Beef tenderness is a very complex and multifactorial sensorial meat quality trait, which depends partly on muscle characteristics. This tissue is very variable according to animal type (age, breed and sex) and rearing conditions. Consequently, beef tenderness exhibits a great variability. Different research programs have revealed several genes or proteins which could be good markers of beef tenderness. In order to validate the relation of these markers with beef tenderness on a large population of bovines, it is necessary to have a large-scale and trusty technique which can access different quantities of proteins related to tenderness. In this study we firstly compared Western-Blot and Dot-Blot. Secondly, we evaluated Dot-Blot technical and biological capabilities for the quantification of protein biomarkers. The results demonstrated that the Dot-Blot technique with fluorescence detection presents numerous interests. This technique allows a good reproducibility and permits the simultaneous analysis of a large number of samples. The Dot-Blot technique defined and validated in this study can be used for protein biomarkers analyses, notably to predict beef tenderness. Another major result of this study is that about 5 to 10 animals per group are required to detect large differences (>1.5) in biomarker expression between tender and tough beef, whereas much larger numbers of animals (10 to 30) are required to detect smaller differences (about 1.2 to 1.3) taking into account the biological variability of these markers.

  8. Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein-DNA Complexes.

    Science.gov (United States)

    Harbers, Matthias

    2015-01-01

    The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes: First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means.

  9. Western blotting using in-gel protein labeling as a normalization control: stain-free technology.

    Science.gov (United States)

    Gilda, Jennifer E; Gomes, Aldrin V

    2015-01-01

    Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on both gels and membranes. Here, we describe the theory and use of stain-free gels for total protein quantification and normalization of Western blots.

  10. Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

    Science.gov (United States)

    Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

    2007-08-01

    A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

  11. Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

    Science.gov (United States)

    Rossano, M G; Mansfield, L S; Kaneene, J B; Murphy, A J; Brown, C M; Schott, H C; Fox, J C

    2000-01-01

    Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (Pblot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

  12. Diagnostic performance of ELISA, IFAT and Western blot for the detection of anti-Leishmania infantum antibodies in cats using a Bayesian analysis without a gold standard.

    Science.gov (United States)

    Persichetti, Maria Flaminia; Solano-Gallego, Laia; Vullo, Angela; Masucci, Marisa; Marty, Pierre; Delaunay, Pascal; Vitale, Fabrizio; Pennisi, Maria Grazia

    2017-03-13

    Anti-Leishmania antibodies are increasingly investigated in cats for epidemiological studies or for the diagnosis of clinical feline leishmaniosis. The immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are the serological tests more frequently used. The aim of the present study was to assess diagnostic performance of IFAT, ELISA and WB to detect anti-L. infantum antibodies in feline serum samples obtained from endemic (n = 76) and non-endemic (n = 64) areas and from cats affected by feline leishmaniosis (n = 21) by a Bayesian approach without a gold standard. Cut-offs were set at 80 titre for IFAT and 40 ELISA units for ELISA. WB was considered positive in presence of at least a 18 KDa band. Statistical analysis was performed through a written routine with MATLAB software in the Bayesian framework. The latent data and observations from the joint posterior were simulated in the Bayesian approach by an iterative Markov Chain Monte Carlo technique using the Gibbs sampler for estimating sensitivity and specificity of the three tests. The median seroprevalence in the sample used for evaluating the performance of tests was estimated at 0.27 [credible interval (CI) = 0.20-0.34]. The median sensitivity of the three different methods was 0.97 (CI: 0.86-1.00), 0.75 (CI: 0.61-0.87) and 0.70 (CI: 0.56-0.83) for WB, IFAT and ELISA, respectively. Median specificity reached 0.99 (CI: 0.96-1.00) with WB, 0.97 (CI: 0.93-0.99) with IFAT and 0.98 (CI: 0.94-1.00) with ELISA. IFAT was more sensitive than ELISA (75 vs 70%) for the detection of subclinical infection while ELISA was better for diagnosing clinical leishmaniosis when compared with IFAT (98 vs 97%). The overall performance of all serological techniques was good and the most accurate test for anti-Leishmania antibody detection in feline serum samples was WB.

  13. Should we ignore western blots when selecting antibodies for other applications?

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

    and then heated to very high temperatures (normally >100 °C) in a procedure that is sometimes termed 'epitope retrieval'. Obviously, this procedure might influence the target protein differently than the procedure used to prepare proteins for a western blot, in which the sample is instead treated with a detergent...... (SDS) before the electrophoresis step. Thus, as concluded by the members of the IWGAV1, the results obtained for a given antibody in western blot applications cannot be used to predict the specificity of the antibody in another assay based on an entirely different epitope-retrieval method, such as IHC...

  14. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    Science.gov (United States)

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

  15. Better management of Western blotting results using professional photo management software.

    Science.gov (United States)

    Iorio-Morin, Christian; Germain, Pascale; Parent, Jean-Luc

    2013-04-01

    Western blotting is a proven technique essential to a significant proportion of molecular biology projects. However, as results accumulate over the years, managing data can become daunting. Recognizing that the needs of a scientist working with Western blotting results are conceptually the same as those of a professional photographer managing a summer's worth of wedding photos, we report here a new workflow for managing Western blotting results using professional photo management software. The workflow involves (i) scanning all film-based results; (ii) importing the scans into the software; (iii) processing the scans; (iv) tagging the files with metadata, and (v) creating appropriate "smart-albums." Advantages of this system include space savings (both on our hard drives and on our desks), safer archival, quicker access, and easier sharing of the results. In addition, metadata-based workflows improve cross-experiment discovery and enable questions like "show me all blots labelled with antibody X" or "show me all experiments featuring protein Y". As project size and breadth increase, workflows delegating results management to the computer will become more and more important so that scientists can keep focussing on science. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed...

  17. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five...

  18. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

    Science.gov (United States)

    Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

    2016-09-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

  19. Evaluation of an adaptive virtual laboratory environment using Western Blotting for diagnosis of disease.

    Science.gov (United States)

    Polly, Patsie; Marcus, Nadine; Maguire, Danni; Belinson, Zack; Velan, Gary M

    2014-10-20

    Providing large numbers of undergraduate students in scientific disciplines with engaging, authentic laboratory experiences is important, but challenging. Virtual laboratories (vLABs) are a potential means to enable interactive learning experiences. A vLAB focusing on Western Blotting was developed and implemented in a 3rd year undergraduate Pathology course for science students to facilitate learning of technical molecular laboratory skills that are linked to development of diagnostic skills. Such skills are important for undergraduates in building a conceptual understanding of translation of laboratory techniques to changes in human biology due to disease. The Western Blotting vLAB was developed and deployed using the Adaptive eLearning Platform (AeLP) developed by Smart Sparrow (https://www.smartsparrow.com/). The vLAB was evaluated to assess students' perceptions of their laboratory skills relevant to the diagnosis of Muscular Dystrophy. A blended learning rotation model was applied in which wet laboratory and vLAB environments for Western Blotting were both delivered to three consecutive cohorts of 3rd year science undergraduates undertaking a Muscle Diseases practical class. Evaluation questionnaires were administered at the completion of the practical classes. Students indicated in online questionnaires that the Western Blotting vLAB was at least equivalent to the real lab in their perceived development of concepts, laboratory skills and diagnosis of disease. vLABs have great potential for improving students' development of diagnostic skills. Further studies are required to determine the impact of vLABs on student learning.

  20. A western blot protocol for detection of proteins heterologously expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins...

  1. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    Science.gov (United States)

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  2. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    Science.gov (United States)

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  3. Western blotting as a method for studying cell-biomaterial interactions : The role of protein collection

    NARCIS (Netherlands)

    van Kooten, T.G.; Klein, CL; Kirkpatrick, CJ

    2001-01-01

    Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure

  4. Indeterminate human immunodeficiency virus Western blot profiles in ethiopians with discordant screening-assay results

    NARCIS (Netherlands)

    Meles, Hailu; Wolday, Dawit; Fontanet, Arnaud; Tsegaye, Aster; Tilahun, Tesfaye; Aklilu, Mathias; Sanders, Eduard; Rinke de Wit, Tobias F.

    2002-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB

  5. Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

    Directory of Open Access Journals (Sweden)

    Yadetie Fekadu

    2004-06-01

    Full Text Available Abstract Background RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. Results RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. Conclusion RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

  6. Two-dimensional gel-based protein standardization verified by western blot analysis.

    Science.gov (United States)

    Haniu, Hisao; Watanabe, Daisuke; Kawashima, Yusuke; Matsumoto, Hiroyuki

    2015-01-01

    In data presentation of biochemical investigation the amount of a target protein is shown in the y-axis against the x-axis representing time, concentrations of various agents, or other parameters. Western blot is a versatile and convenient tool in such an analysis to quantify and display the amount of proteins. In western blot, so-called housekeeping gene product(s), or "housekeeping proteins," are widely used as internal standards. The rationale of using housekeeping proteins for standardization of western blot is based on the assumption that the expression of chosen housekeeping gene is always constant, which could be false under certain physiological or pathological conditions. We have devised a two-dimensional gel-based standardization method in which the protein content of each sample is determined by scanning the total protein density of two-dimensional gels and the expression of each protein is quantified as the density ratio of each protein divided by the density of the total proteins on the two-dimensional gel. The advantage of this standardization method is that it is not based on any presumed "housekeeping proteins" that are supposed to be being expressed constantly under all physiological conditions. We will show that the total density of a two-dimensional gel can render a reliable protein standardization parameter by running western blot analysis on one of the proteins analyzed by two-dimensional gels.

  7. Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis

    Directory of Open Access Journals (Sweden)

    S. T. Beck

    Full Text Available Two recombinant antigens and a crude bacterial antigen of a wild M. tuberculosis strain were used to detect specific IgG antibodies in sera from 52 patients with pulmonary tuberculosis, confirmed by an acid-fast smear and serum culture of these patients and that of 25 contacts. The patients were not infected with HIV. We evaluated the sensitivity and specificity of ELISA, based on the recombinant TbF6® and TbF6/DPEP antigen and a search for reactivity patterns in the Western blot technique, using whole mycobacterium antigen. Serum samples from 22 healthy individuals and from 30 patients with lung diseases other than tuberculosis were used as controls. The best ELISA results were obtained with the TbF6/DPEP antigen combination, which gave 85% sensitivity and 91% specificity. ELISA sensitivity improved from 85% to 92% when the Western blot results were used. Western blot specificity was 100% when antibody reactivity with different antigenic bands was analyzed and associated. The association of TbF6/DPEP antigens used in ELISA with specific patterns of reactivity determined by Western blot can help make an identification when classic methods for the diagnosis of pulmonary tuberculosis are not sufficient.

  8. Use of Nonradioactive Detection Method for North- and South-Western Blot.

    Science.gov (United States)

    Franke, Claudia; Gräfe, Daniel; Bartsch, Holger; Bachmann, Michael P

    2015-01-01

    Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

  9. Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis.

    Science.gov (United States)

    Beck, Sandra Trevisan; Leite, O M; Arruda, R S; Ferreira, A W

    2005-02-01

    Two recombinant antigens and a crude bacterial antigen of a wild M. tuberculosis strain were used to detect specific IgG antibodies in sera from 52 patients with pulmonary tuberculosis, confirmed by an acid-fast smear and serum culture of these patients and that of 25 contacts. The patients were not infected with HIV. We evaluated the sensitivity and specificity of ELISA, based on the recombinant TbF6 and TbF6/DPEP antigen and a search for reactivity patterns in the Western blot technique, using whole mycobacterium antigen. Serum samples from 22 healthy individuals and from 30 patients with lung diseases other than tuberculosis were used as controls. The best ELISA results were obtained with the TbF6/DPEP antigen combination, which gave 85% sensitivity and 91% specificity. ELISA sensitivity improved from 85% to 92% when the Western blot results were used. Western blot specificity was 100% when antibody reactivity with different antigenic bands was analyzed and associated. The association of TbF6/DPEP antigens used in ELISA with specific patterns of reactivity determined by Western blot can help make an identification when classic methods for the diagnosis of pulmonary tuberculosis are not sufficient.

  10. [Clinical manifestation of Lyme borreliosis in children with positive and negatiwe western blot results].

    Science.gov (United States)

    Ołdak, Elzbieta; Rozkiewicz, Doroto; Sulik, Artur

    2008-01-01

    In the afforested area of North-Eastern Poland the risk of Borrelia burgdorferi infection seems to be higher compared to the other regions. Because of unspecific clinical manifestation of Lyme borreliosis in children the positive ELISA IgM results should be confirmed with Western blot IgM tests. Retrospective analysis of clinical signs and symptoms of Lyme borreliosis in children with positive ELISA IgM and positive Western blot IgM results and in children with positive ELISA IgM and negative Western blot IgM results. The study included 20 children reactive with ELISA IgM (Bellco Biomedica, Austria), hospitalized in Pediatric Infectious Diseases Clinic in 2007 due to probable diagnosis of Lyme disease. All children were tested with B. burgdorferi Western blot IgM and/or IgG assay (DRG, Diagnostics, Germany) as a second-step diagnosis. In 10 (50% females, 50% males) out of 20 children the results were positive (borreliosis) and in other 10 (80% females, 20% males) the results were negative (controls). In both groups of patients the retrospective analysis of signs and symptoms was done. The most often clinical manifestation of Lyme borreliosis in children was neuroborreliosis. Children presented Lyme meningitis (30%), facial nerve palsy (10%) and chronic or recurrent headaches (40%), associated with vertigo (20%), weakness (30%), fever (40%), and fatigue syndrome (30%). One patient presented Lyme arthritis. Children of control group presented with unspecific symptoms like isolated headaches (40%), arthralgias (70%), myalgias (10%) and abdomen pain (20%) (1) The most frequent clinical presentation of Lyme borreliosis in analyzed children was neuroborreliosis; (2) Isolated arthralgias in children reactive with B. burgdorferi ELISA IgM need to be confirmed with Western blot assay before implementing the antibiotic therapy.

  11. Diagnosis of Giardia duodenalis Infection using Dot Blot in Comparison with Microscopy.

    Science.gov (United States)

    Yazdani, Hajar; Sharafi, Seyedeh Maryam; Yousefi, Hoseynali; Hadipur, Mahbobeh; Sepahvand, Akram; Darani, Hossein Yousofi

    2016-01-01

    Giardia duodenalis is an intestinal flagellate parasite which spreads all over the world and is considered as a health problem in the most rural and low sanitation areas. Many diagnostic tests have been developed for the detection of Giardia parasite in stool samples but all of them have some disadvantages such as lack of sensitivity and specificity. In search for a simple and accurate test, diagnosis of Giardia infection using dot blot method has been investigated in this work. In this descriptive study, 30 stool samples which their infection with Giardia were confirmed by direct examination and formalin ether considered as case group. Thirty stool samples without Giardia infection according to formalin ether examination were also considered as a control group. Giardia cysts were isolated from the stool samples using sucrose method. In order to raise antiserum against Giardia cysts, the purified cysts were then sonicated and injected to a rabbit. A mono specific antiserum against the 66KDa band of Giardia cyst antigen was also prepared. The two antisera were used in the dot blot test. Finally, the sensitivity and specificity of the dot-blot method were estimated by considering formalin ether as the gold standard. When Poly specific antiserum was used, the sensitivity and specificity of the dot blot for detection of Giardia infection were 77% and 64% respectively. However the sensitivity and specificity of this assay were 97% and 64% respectively when monospecific antiserum was used. It seems that dot blot is an easy method for the diagnosis of Giardia especially in the rural areas. However more work is recommended for further development of this test.

  12. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  13. The Western blot is a highly sensitive and efficient technique in diagnosing allergy to wasp venom.

    Science.gov (United States)

    Zollner, T M; Spengler, K; Podda, M; Ergezinger, K; Kaufmann, R; Boehncke, W H

    2001-11-01

    Diagnosis of allergy to wasp venom and decision to perform immunotherapy are based on the patient's history, along with skin and in vitro tests. Given the high prevalence of specific IgE also in non-allergic individuals, we evaluated the sensitivity and specificity of Western blots as a possible alternative to serum analyses of venom-specific IgE. Skin prick and/or intracutaneous tests were performed in 30 patients with allergy to wasp venom (generalized reaction following sting) along with serum analysis of venom-specific IgE (AlaSTAT microplate) and Western blots. Western blots were subsequently scanned and evaluated qualitatively and semiquantitatively by means of densitometry. Bands were scored 'positive' in cases of signal intensities beyond the mean plus 3 standard deviations of control sera. Twenty newborns (age 2-7 days) and 30 adults without systemic or increased local reactions to hymenoptera stings served as controls. Western blot sensitivity reached 100% in the samples studied and was thus superior to the sensitivities of serum analysis of venom-specific IgE using AlaSTAT microplate assay (90%) and skin tests (87%). The sensitivity of detection of a phospholipase A1 and antigen 5-specific band was higher compared with a hyaluronidase-specific band (97%, 97% and 86%, respectively). Twenty-four out of twenty-nine (83%) patients exhibited specific IgE antibodies against at least three distinct allergens. With regard to the specificities, skin tests as well as AlaSTAT microplate assays were comparable (90% and 93%, respectively), whereas the specificity of the Western blots was 70% if the appearance of any single band was regarded as a positive result. However, when analysing the appearance of a specific band for antigen 5 or hyaluronidase the specificity and overall diagnostic value increased markedly, making it the most efficient test (specificity 97% and 100%, efficiency 96.8% and 93.2%, respectively). As allergy to wasp venom is a severe and potentially

  14. An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit.

    Science.gov (United States)

    Furuya, Hirokazu; Yamada, Takeshi; Ikezoe, Koji; Ohyagi, Yasumasa; Fukumaki, Yasuyuki; Fujii, Naoki

    2006-08-31

    An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.

  15. DISKRIMINASI KELAMIN PADA IKAN TUNA SIRIP KUNING, Yellowfin tuna MENGGUNAKAN ANALISIS DOT BLOT DAN ELISA

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2014-08-01

    Full Text Available Pemahaman tentang penentuan jenis kelamin dalam populasi induk merupakan hal yang sangat penting bagi keberhasilan program pembenihan. Pengukuran reaksi antibodi dan aktivitas hormon testosterone, serta estradiol adalah metode dengan potensi yang secara akurat dapat menentukan jenis kelamin ikan tanpa mematikan ikan. Tujuan penelitian ini adalah untuk mengetahui akurasi metode dot blot dan ELISA dengan 11-ketotestorsterone (11-KT yang tersedia secara komersial EIA-kit untuk membedakan jenis kelamin ikan tuna sirip kuning. Hasil analisis menunjukkan bahwa metode dot blot menghasilkan ekspresi vitelogenin tampak jelas pada individu betina dan efek plasma terlihat transparan, jika dibandingkan dengan individu jantan. Interpretasi dari metode ini memerlukan pengalaman dan keahlian dalam akurasi pembacaan hasil. Aktivitas hormon 11-KT dengan sampel klip sirip dan plasma memberikan hasil yang baik dengan aktivitas hormon terlihat jelas.

  16. Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

    Science.gov (United States)

    Thacker, Jonathan S; Yeung, Derrick H; Staines, W Richard; Mielke, John G

    2016-03-01

    Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

    Science.gov (United States)

    Carthew, James; Karakesisoglou, Iakowos

    2016-01-01

    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.

  18. Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting.

    Science.gov (United States)

    Moore, Jacen S; Scofield, R Hal

    2015-01-01

    Nanogold-conjugated immunodetection systems are now widely and commercially available for use in a number of research applications including electron microscopy, light microscopy, and western blotting. Nanogold clusters are small, uniform in size, and stable, unlike gold colloids historically used in protein detection. Covalent linkage of nanogold particles to secondary antibodies prevents dissociation of the gold particles during the staining process, making protein detection reliable, antigen specific, and highly sensitive. Nanogold labeling is extremely versatile and can be used in conjunction with other staining methodologies including Alexa Fluor immunofluorescence detection to perform coupled staining procedures. Silver enhancement increases the limits of sensitivity for nanogold staining, thus improving detection signals for antigens with reduced expression levels. Herein, we describe the use of nanogold-silver detection as an immunodetection system for standard western blotting of autoantigens.

  19. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada

    OpenAIRE

    Ogden, Nicholas H.; Arsenault, Julie; Hatchette, Todd F.; Mechai, Samir; Lindsay, L. Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographi...

  20. Use of a Western blot technique for the serodiagnosis of glanders

    OpenAIRE

    Elschner, Mandy C; Scholz, Holger C; Melzer, Falk; Saqib, Muhammad; Marten, Peggy; Rassbach, Astrid; Dietzsch, Michael; Schmoock, Gernot; de Assis Santana, Vania L; de Souza, Marcilia MA; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

    2011-01-01

    Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was va...

  1. A Fast and Inexpensive Western Blot Experiment for the Undergraduate Laboratory

    Science.gov (United States)

    Farrell, Shawn O.; Farrell, Lynn E.

    1995-08-01

    Western blotting is an important, modern technique for transferring proteins from a gel onto nitrocellulose or other suitable support and then detecting a protein of interest using antibodies. We have developed an experiment and optimized the conditions for the undergraduate laboratory. The experiment can be done quickly using an electrophoretic blotter or more cheaply using passive transfer. This experiment allows the student to learn valuable procedures currently used in biochemistry and other biological sciences.

  2. Evaluation of an immunodot blot technique for the detection of antibodies against Taenia solium larval antigens

    OpenAIRE

    Salazar-Anton, Fernando; Tellez, Aleyda; Lindh, Johan

    2012-01-01

    Immunodiagnostic tests represent an important tool for diagnosis of cysticercosis, the disease caused by cysticerci of Taenia solium. Accurate diagnosis of neurocysticercosis (NCC) requires costly neuroimaging techniques (magnetic resonance imaging and computed tomography), which are seldom affordable for people in endemic countries. Hence, new low-cost diagnostic methods offering good sensitivity and specificity are needed. Here, we studied four immunodiagnostic tests immunodot blot Tsol-p27...

  3. V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

    OpenAIRE

    Posch, Anton; Kohn, Jonathan; Oh, Kenneth; Hammond, Matt; Liu, Ning

    2013-01-01

    The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-fr...

  4. Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2013-01-01

    Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

  5. High-Resolution Northern Blot for a Reliable Analysis of MicroRNAs and Their Precursors

    Directory of Open Access Journals (Sweden)

    Edyta Koscianska

    2011-01-01

    Full Text Available This protocol describes how to perform northern blot analyses to detect microRNAs and their precursors with single-nucleotide resolution, which is crucial for analyzing individual length variants and for evaluating relative quantities of unique microRNAs in cells. Northern blot analysis consists of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization with radioactive probes. Earlier efforts to improve this method focused mainly on altering the sensitivity of short RNA detection. We have enhanced the resolution of the northern blot technique by optimizing the electrophoresis step. We have also investigated other steps of the procedure with the goal of enhancing the resolution of RNAs; herein, we present several recommendations to do so. Our protocol is applicable to analyses of all kinds of endogenous and exogenous RNAs, falling within length ranges of 20–30 and 50–70 nt, corresponding to microRNA and pre-microRNA lengths, respectively.

  6. Direct Blue 71 staining as a destaining-free alternative loading control method for Western blotting.

    Science.gov (United States)

    Zeng, Li; Guo, Jing; Xu, Hong-Bo; Huang, Rongzhong; Shao, Weihua; Yang, Liu; Wang, Mingju; Chen, Jianjun; Xie, Peng

    2013-08-01

    In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining-a novel, sensitive, dye-binding staining method compatible with immunodetection-may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5-40 μg of protein loaded. DB71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein-based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining-free alternative loading control method for Western blotting. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

    Directory of Open Access Journals (Sweden)

    Tappe D

    2009-09-01

    Full Text Available Abstract Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE and cystic echinococcosis (CE. Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect" and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively. In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band.

  8. Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein.

    Science.gov (United States)

    Correa, M M; Bedoya, A M; Guerrero, M P; Méndez, J; Restrepo, A; McEwen, J G

    2007-01-01

    A variety of immunological methods have proven useful for Paracoccidioidomycosis (PCM) diagnosis; however, they are often time consuming and many lack sensitivity and specificity, partially attributed to the use of crude antigens, which give cross reactivity. Until now, attempts to clone and express Paracoccidioides brasiliensis immunodominant antigens have presented difficulties of process and problems of cost. In an attempt to obtain a more rapid, sensitive, and specific test for PCM diagnosis, we subcloned the P. brasiliensis p27 gene and used the recombinant protein as the antigen in dot blot assays to evaluate its usefulness in paracoccidioidomicosis diagnosis. The development of an optimised procedure for p27 recombinant protein purification and production led to an easier and less expensive process than the one previously used in our laboratory and allowed the availability of enough purified protein for its evaluation as the antigen in the dot blot assays. In these assays, antibodies present in ten serum samples from seven patients with PCM recognised the recombinant protein showing a sensitivity of 100% with a specificity of 98%. These results confirm the value of the 27-kDa recombinant antigen in the serodiagnosis of paracoccidioidomycosis and that the dot blot format is an alternative to the immunoenzymatic assay procedure.

  9. Use of a Western blot technique for the serodiagnosis of glanders

    Directory of Open Access Journals (Sweden)

    de Souza Marcilia MA

    2011-01-01

    Full Text Available Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT. Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  10. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

    Directory of Open Access Journals (Sweden)

    Palacios Ricardo

    1999-01-01

    Full Text Available In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma, the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

  11. Use of a Western blot technique for the serodiagnosis of glanders.

    Science.gov (United States)

    Elschner, Mandy C; Scholz, Holger C; Melzer, Falk; Saqib, Muhammad; Marten, Peggy; Rassbach, Astrid; Dietzsch, Michael; Schmoock, Gernot; de Assis Santana, Vania L; de Souza, Marcilia M A; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

    2011-01-19

    The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  12. A no film slot blot for the detection of developing P. falciparum oocysts in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Bryan Grabias

    Full Text Available Non-microscopy-based assays for sensitive and rapid detection of Plasmodium infection in mosquitoes are needed to allow rapid and high throughput measurement of transmission intensity and malaria control program effectiveness. Here, we report on a modified enhanced chemiluminescence-based slot blot assay for detection of Plasmodium falciparum (Pf circumsporozite protein (PfCSP expressed on parasite oocysts developing inside the mosquito midgut. This modified assay has several novel features that include eliminating the need for exposure to autoradiography (AR film, as well as utilizing a novel high affinity anti-CSP antibody, and optimizing assay procedures resulting in significant reduction in the time required to perform the assay. The chemiluminescent signal for the detection of PfCSP in mosquito samples was captured digitally utilizing the C-Digit blot scanner that, allowed the detection of 0.01 pg of recombinant P. falciparum CSP and as few as 0.02 P. falciparum oocysts in a little over two hours. The earlier ECL-SB detected rCSP and oocysts and took approximately 5 h to perform. Whole mosquito lysates from both high and low prevalence-infected mosquito populations were prepared and evaluated for PfCSP detection on the ECL-SB by both AR film and digital data capture and analysis. There was a 100% agreement between the AR film and the C-Digit scanner methods for PfCSP detection in randomly sampled mosquitoes. This novel "No Film" Slot Blot assay obviates the need for AR film exposure and development and significantly reduces the assay time enabling widespread use in field settings.

  13. FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

    Directory of Open Access Journals (Sweden)

    D.V. Pilonetto

    2009-03-01

    Full Text Available Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L- was not detected in 77 patients (91.7%. In 2 patients (2.4%, there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L- and 5 patients (5.9% had both isoforms (FANCD2S+/FANCD2L+. This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84 and specificity of 100% (98/98. This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L- or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-, to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+ require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

  14. SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis.

    Science.gov (United States)

    Zaragoza, Concepción; Barrera, Rafael; Centeno, Francisco; Tapia, Jose A; Durán, Esther; González, Marta; Mañé, M Cinta

    2003-01-01

    Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.

  15. Subcellular dissemination of prothymosin alpha at normal physiology: immunohistochemical vis-a-vis western blotting perspective.

    Science.gov (United States)

    Kijogi, Caroline Mwendwa; Khayeka-Wandabwa, Christopher; Sasaki, Keita; Tanaka, Yoshimasa; Kurosu, Hiroshi; Matsunaga, Hayato; Ueda, Hiroshi

    2016-03-01

    The cell type, cell status and specific localization of Prothymosin α (PTMA) within cells seemingly determine its function. PTMA undergoes 2 types of protease proteolytic modifications that are useful in elucidating its interactions with other molecules; a factor that typifies its roles. Preferably a nuclear protein, PTMA has been shown to function in the cytoplasm and extracellularly with much evidence leaning on pathognomonic status. As such, determination of its cellular distribution under normal physiological context while utilizing varied techniques is key to illuminating prospective validation of its distinct functions in different tissues. Differential distribution insights at normal physiology would also portent better basis for further clarification of its interactions and proteolytic modifications under pathological conditions like numerous cancer, ischemic stroke and immunomodulation. We therefore raised an antibody against the C terminal of PTMA to use in tandem with available antibody against the N terminal in a murine model to explicate the differences in its distribution in brain cell types and major peripheral organs through western blotting and immunohistochemical approaches. The newly generated antibody was applied against the N-terminal antibody to distinguish truncated versions of PTMA or deduce possible masking of the protein by other interacting molecules. Western blot analysis indicated presence of a truncated form of the protein only in the thymus, while immunohistochemical analysis showed that in brain hippocampus the full-length PTMA was stained prominently in the nucleus whereas in the stomach full-length PTMA staining was not observed in the nucleus but in the cytoplasm. Truncated PTMA could not be detected by western blotting when both antibodies were applied in all tissues examined except the thymus. However, immunohistochemistry revealed differential staining by these antibodies suggesting possible masking of epitopes by interacting

  16. Confocal laser scanning microscope, Raman microscopy and Western blotting to evaluate inflammatory response after myocardial infarction.

    Science.gov (United States)

    Riezzo, Irene; Cantatore, Santina; DeCarlo, Dania; Fiore, Carmela; Neri, Margherita; Turillazzi, Emanuela; Fineschi, Vittorio

    2015-01-01

    Cardiac muscle necrosis is associated with inflammatory cascade that clears the infarct from dead cells and matrix debris, and then replaces the damaged tissue with scar, through three overlapping phases: the inflammatory phase, the proliferative phase and the maturation phase. Western blotting, laser confocal microscopy, Raman microscopy are valuable tools for studying the inflammatory response following myocardial infarction both humoral and cellular phase, allowing the identification and semiquantitative analysis of proteins produced during the inflammatory cascade activation and the topographical distribution and expression of proteins and cells involved in myocardial inflammation. Confocal laser scanning microscopy (CLSM) is a relatively new technique for microscopic imaging, that allows greater resolution, optical sectioning of the sample and three-dimensional reconstruction of the same sample. Western blotting used to detect the presence of a specific protein with antibody-antigen interaction in the midst of a complex protein mixture extracted from cells, produced semi-quantitative data quite easy to interpret. Confocal Raman microscopy combines the three-dimensional optical resolution of confocal microscopy and the sensitivity to molecular vibrations, which characterizes Raman spectroscopy. The combined use of western blotting and confocal microscope allows detecting the presence of proteins in the sample and trying to observe the exact location within the tissue, or the topographical distribution of the same. Once demonstrated the presence of proteins (cytokines, chemokines, etc.) is important to know the topographical distribution, obtaining in this way additional information regarding the extension of the inflammatory process in function of the time stayed from the time of myocardial infarction. These methods may be useful to study and define the expression of a wide range of inflammatory mediators at several different timepoints providing a more

  17. Western Blotting Is an Efficient Tool for Differential Diagnosis of Paracoccidioidomycosis and Pulmonary Tuberculosis

    Science.gov (United States)

    Bertoni, Thâmara Aline; Perenha-Viana, Maysa Cláudia Zolin; Patussi, Eliana Valéria; Cardoso, Rosilene Fressatti

    2012-01-01

    Sputum and sera from 134 patients screened for tuberculosis (TB) were analyzed to investigate TB and paracoccidioidomycosis (PCM). Of these patients, 11 (8.2%) were confirmed to have TB, but six (4.5%) were positive only for PCM. All patients with PCM presented anti-43-kDa-component antibodies in Western blotting (WB) assays, while in the TB-positive patients these antibodies did not appear. This preliminary study suggests WB as a potential tool for differential laboratory diagnosis between TB and PCM. PMID:22971781

  18. Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

    Science.gov (United States)

    Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

    2013-06-01

    Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

  19. Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

    Directory of Open Access Journals (Sweden)

    Elizabeth Anaya

    1997-01-01

    Full Text Available Se separaron extractos de proteínas totales de Brucella melitensis en gel 15% SDS-PAGE. Su seroreactividad fue analizada por Western Blot con resultados satisfactorios. Para éste propósito sueros controles negativos (n=03, sueros de pacientes con brucelosis (n=34, cólera (n=12, tifoidea (n=02 y tuberculosis (n=02 fueron usados. Esta prueba inmunodiagnóstica detectó bandas seroreactivas altamente específicas (100% correspondientes a 8,14,18, un complejo de 25-48 y 58kDa. La sensibilidad del test fue del 90% usando los sueros antes mencionados.

  20. Construction of multiple-epitope tag sequence by PCR for sensitive Western blot analysis.

    OpenAIRE

    Nakajima, K; Yaoita, Y

    1997-01-01

    Epitope tagging is a powerful technique to characterize a recombinantly expressed protein encoded by cDNA without the purification of the protein and the immunization of animals. In some cases, however, the expression of a tagged protein is too low to analyze by Western blot. We have developed a simple method to generate tandem repetitive nucleotide sequence by PCR, which allows us to label a protein of interest with a multiple-epitope tag. When five myc epitopes were attached to vaccinia vir...

  1. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.

    Science.gov (United States)

    Tian, Geng; Tang, Fangrong; Yang, Chunhua; Zhang, Wenfeng; Bergquist, Jonas; Wang, Bin; Mi, Jia; Zhang, Jiandi

    2017-08-29

    Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this "Big Data" era.

  2. Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation.

    Science.gov (United States)

    Rey, María-Dolores; Prieto, Pilar

    2017-01-01

    Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare , Secale cereale and Agropyron cristatum in the wheat background was also reported .

  3. Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca

    2009-01-01

    A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.

  4. An overview of technical considerations for Western blotting applications to physiological research.

    Science.gov (United States)

    Bass, J J; Wilkinson, D J; Rankin, D; Phillips, B E; Szewczyk, N J; Smith, K; Atherton, P J

    2017-01-01

    The applications of Western/immunoblotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e., resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection, to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate, and troubleshoot the WB process, to produce reproducible and reliable blots. © 2016 The Authors. Scandinavian Journal of Medicine & Science in Sports published by John Wiley & Sons Ltd.

  5. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  6. Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1.

    Science.gov (United States)

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-02-01

    A common way to measure the induction of autophagy in yeast and mammalian cells is to compare the amount of Atg8/LC3-I with that of Atg8-PE/LC3-II by using western blot analysis. This is because changes in the amount of LC3-II correlate closely with changes in the number of autophagosomes present in cells. Atg8/LC3 is initially synthesized as an unprocessed form, which is proteolytically processed to form Atg8/LC3-I, and then this is modified into the phosphatidylethanolamine (PE)-conjugated Atg8-PE/LC3-II form. Atg8/LC3-II is membrane bound, whereas Atg8-PE/LC3-I is cytosolic. By associating with both the inner and outer membranes of the autophagosome, Atg8-PE/LC3-II is the only autophagy reporter that is reliably associated with completed autophagosomes. In the nematode Caenorhabditis elegans, the ortholog of Atg8/LC3 is LGG-1. Here, we discuss how changes in the levels of LGG-1-II (and the paralog LGG-2) protein can, with appropriate controls, be used to monitor autophagy activity in nematodes and present a protocol for monitoring changes in the protein levels of different forms of LGG-1 by western blotting. © 2016 Cold Spring Harbor Laboratory Press.

  7. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    Science.gov (United States)

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

  8. Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

    Science.gov (United States)

    Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

    2017-11-01

    The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    Energy Technology Data Exchange (ETDEWEB)

    Lee, C.Y.G. (Univ. of British Columbia, Vancouver, Canada); Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-06-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

  10. The limits for detection of activated caspases of spermatozoa by western blot in human semen.

    Science.gov (United States)

    Brugnon, F; Pons-Rejraji, H; Artonne, C; Janny, L; Grizard, G

    2012-08-01

    Detection of activated caspases of spermatozoa could be helpful to evaluate male infertility. Although western blot is validated as a highly specific method to detect the proteins extracted from cells, the ability of this technique to detect activated sperm caspases in human semen may be limited. Indeed, round cells, which potentially contain some activated caspases, may be present in semen and interfere with the detection of activated sperm caspases. Moreover, it is necessary to evaluate the minimum amount of spermatozoa necessary to optimise the detection of activated caspases in semen samples. Our results showed that interference due to round cells contained in semen with activated caspase-3 requires separation of spermatozoa by density migration. This sperm preparation selects a mature sperm population that does not reflect the whole sperm population, and in infertile men with oligoasthenoteratozoospermia, the amount of spermatozoa thus selected is usually low. Moreover, the western blot technique's low detection sensitivity and the low level of caspase enzyme activity in human spermatozoa for activated caspase-3, -8 and -9 mean that large quantities of spermatozoa are needed to detect the expression of the activated caspases. These limitations prevent this method being used for routine analysis in clinical practice. © 2012 Blackwell Verlag GmbH.

  11. HOPE technique enables Western blot analysis from paraffin-embedded tissues.

    Science.gov (United States)

    Uhlig, U; Uhlig, S; Branscheid, D; Zabel, P; Vollmer, E; Goldmann, T

    2004-01-01

    In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

  12. [Western blot, ELISA and indirect immunofluorescence test evaluation of Leishmania (Leishmania) infantum-infected dogs].

    Science.gov (United States)

    Vargas-Duarte, Jimmy J; López-Páez, Myriam C; Escovar-Castro, Jesús E; Fernández-Manrique, José

    2009-08-01

    Evaluating canine visceral leishmaniasis diagnostic test performance in Colombia and adapting the Western blot test in naturally and experimentally infected dogs. Sera were obtained from 10 experimentally L. Infantum-infected dogs, 5 naturally infected dogs, 16 healthy dogs, 26 Babesia canis, Erhlichia canis, Dirofilaria immitis, Trypanosoma cruzi and Leishmania (Viannia) spp infected dogs, 40 dogs from non-endemic areas and 150 from endemic areas. Sera were tested for L. infantum infection using immunofluorescent antibody (IFAT), ELISA and Western blot (WB) tests. Positives results were obtained for 73 % of known infected dogs by the IFAT test and false positives were obtained for 2.5 % of non-infected dogs using WB. ELISA was not efficient for diagnosis. 24 antigenic fractions were recognised in tested sera using WB; however, 29, 34, 50, 69, 75, 86, 99 and 123 kDa bands were recognised in sera from dogs from non-endemic areas, healthy dogs and Trypanosoma cruzi, Erhlichia canis, Dirofilaria immitis and Babesia canis infected dogs. The 13 kDa fraction proved potentially useful for diagnosing canine visceral leishmaniasis. The separate use of parasitological and serological test could lead to misdiagnosis of Leishmania infection; using both kinds of technique simultaneously is thus highly recommended.

  13. A Western blot and molecular genetic investigation of the estrogen receptor beta in giant cell arteritis.

    Science.gov (United States)

    Larsson, K; Nordborg, C; Moslemi, A-R; Nordborg, E

    2006-01-01

    The epidemiology of giant cell arteritis (GCA) may indicate a pathogenetic relationship between GCA and female sex hormone metabolism; GCA is two to four times more common in women compared with men. Our previous analyses gave no support for the hypothesis that the pathogenesis of GCA should be related to somatic mutations in the estrogen receptor alpha (ERalpha) gene. The object of the present study was to investigate the size of the estrogen receptor beta (ERBeta), and the size and nucleotide sequence of the ERBeta gene in temporal arteries in GCA. The ERBeta protein was analyzed by Western blot technique and the ERBeta gene by RT-PCR and direct sequencing of the PCR product. Western blot analysis revealed an ERBeta of normal size. There were no aberrations in size or nucleotide sequence in the ERBeta gene in the GCA patients. The present observations gave no support for the hypothesis that somatic mutations in the ERBeta gene should be involved in the pathogenesis of GCA.

  14. Western blot evaluation of siRNA delivery by pH-responsive peptides.

    Science.gov (United States)

    Liang, Wanling; Mason, A James; Lam, Jenny K W

    2013-01-01

    Gene silencing, via RNA interference (RNAi) technologies using small interfering RNA (siRNA), has been developed as an important tool for target identification and validation in drug discovery and has huge therapeutic potential. However, effective delivery into cells presents a major challenge to the use of siRNA. pH-responsive cell-penetrating peptides have attracted considerable attention in recent years as delivery vectors due to their ability to transport their cargos across the biological membrane and/or to promote endosomal escape and prevent lysosomal degradation. To evaluate the in vitro transfection efficiency of the pH-responsive peptide-based siRNA delivery system, the western blotting technique is commonly employed. This method offers a simple, efficient and economical way to study the gene silencing effect of the siRNA by analysing the protein of interest in a sample with minimum equipment requirement. This chapter provides a description of siRNA delivery and analysis by western blotting protocols for qualitatively and quantitatively assessing gene silencing efficiency and selectivity.

  15. Quantitative detection for plant virus's RNA-loading by dot-blot

    International Nuclear Information System (INIS)

    Chai Lihong; Xu Bujin; Chen Jishuang

    2003-01-01

    A new method, RNA dot blot combined with direct determination of the radioactivity by BIO-Imaging Analyzer (dRH-dBIA) was used for detecting RNA of plant virus in infected plant tissue. This method was used for the influence of RNA-loading level of tobacco mosaic virus (TMV) in tobacco leave tissues after treatment of a plant hormone relatives (n-Propyl dihydro-jasmonate, PDJ) in the concentration range of 0.001-10 ppm. The results indicate that after PDJ application onto tobacco leaves for 3 days all PDJ treatments cause increase of TMV RNA-loading level except 0.001 ppm treatment, and the higher the concentration, the more obvious increase was observed. This phenomenon was confirmed with semi-leaf lesion spot on Nicotiana glutinosa as a local lesion host. The dRH-dBIA method is applicable in quantitative determination of RNA without obvious artificial influence

  16. Profiling protein expression in circulating tumour cells using microfluidic western blotting.

    Science.gov (United States)

    Sinkala, Elly; Sollier-Christen, Elodie; Renier, Corinne; Rosàs-Canyelles, Elisabet; Che, James; Heirich, Kyra; Duncombe, Todd A; Vlassakis, Julea; Yamauchi, Kevin A; Huang, Haiyan; Jeffrey, Stefanie S; Herr, Amy E

    2017-03-23

    Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.

  17. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

    DEFF Research Database (Denmark)

    Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

    2017-01-01

    genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render...... many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers...... that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting...

  18. A novel method that improves sensitivity of protein detection in PAGE and western blot

    Science.gov (United States)

    Vallejo-Illarramendi, Ainara; Marciano, Denise K.; Reichardt, Louis F.

    2013-01-01

    We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a 5-fold increase in the sensitivity of antigen detection in a western blot. As a result less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available. PMID:23400834

  19. Post-stained Western blotting, a useful approach in immunoproteomic studies.

    Science.gov (United States)

    Reguera-Brito, Mercedes; Fernández-Garayzábal, José F; Blanco, M Mar; Aguado-Urda, Mónica; Gibello, Alicia

    2014-12-15

    The precise localisation of immunogenic proteins on stained two-dimensional electrophoresis (2DE) gels is occasionally difficult, contributing to the erroneous identification of unrelated non-immunogenic proteins, which is expensive and time consuming. This inconvenience can be solved by performing immunoblotting using previously stained polyacrylamide gels. This approach was proposed nearly 20 years ago but is now almost forgotten. We have evaluated the suitability of this approach to identify immunogenic proteins from Lactococcus garvieae. Some of the immunogenic proteins identified in L. garvieae, such as Gls24, have been considered important as immunotarget in different bacterial species. Post-staining western blotting facilitated the correct selection of immunogenic proteins of interest in 2D gels before their identification. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Isolation and Western Blotting of Latex-Bead Phagosomes to Track Phagosome Maturation.

    Science.gov (United States)

    Härtlova, Anetta; Peltier, Julien; Bilkei-Gorzo, Orsolya; Trost, Matthias

    2017-01-01

    Phagocytosis plays an essential role in the immune system for the defense against invading microorganisms and the clearing of apoptotic cells. After internalization, the newly formed phagosome is constantly remodeled by fusion with early endosomes, late endosomes, and lysosomes. These changes ultimately deliver the engulfed material into the terminal degradative compartments known as phagolysosomes. However, defective phagosome maturation can result in inflammatory or autoimmune disease depending on the type of phagosome cargo. Therefore, characterization of the components involved in phagosome formation and maturation is important for a better understanding of macrophage physiological and pathological functions. In this chapter we describe a step-by-step protocol for the isolation of large-scale latex/polystyrene bead phagosome preparations with high degrees of purity for Western blotting analysis of phagosome maturation.

  1. Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.

    Science.gov (United States)

    Horinouchi, Takahiro; Terada, Koji; Higashi, Tsunehito; Miwa, Soichi

    2016-01-01

    Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

  2. Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

    Directory of Open Access Journals (Sweden)

    Bowlin Gary L

    2007-10-01

    Full Text Available Abstract Background Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools. Methods The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized. Results Nitrocellulose electrospun from a starting concentration of Conclusion The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use.

  3. A novel method that improves sensitivity of protein detection in PAGE and Western blot.

    Science.gov (United States)

    Vallejo-Illarramendi, Ainara; Marciano, Denise K; Reichardt, Louis F

    2013-04-01

    We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Western blot analysis of cells encapsulated in self-assembling peptide hydrogels.

    Science.gov (United States)

    Burgess, Kyle A; Miller, Aline F; Oceandy, Delvac; Saiani, Alberto

    2017-12-01

    Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell-matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies.

  5. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    Science.gov (United States)

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10 5 cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  6. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  7. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis,

    Directory of Open Access Journals (Sweden)

    Jaqueline Dario Capobiango

    Full Text Available Abstract Objective: To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii (IgG-WB in the serum of children with suspected congenital toxoplasmosis. Methods: We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. Results: 15 children (15.1% met the criteria for congenital toxoplasmosis and 32 (32.3% had the diagnosis excluded. The symptoms were observed in 12 (80.0% children and the most frequent were cerebral calcification in 9 (60.0%, chorioretinitis in 8 (53.3%, and hydrocephalus in 4 (26.6%. IgM antibodies anti-T. gondii detected by chemiluminescence (CL were found in 6 (40.0% children and the polymerase chain reaction (PCR for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%. The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%. The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. Conclusions: The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers.

  8. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Science.gov (United States)

    Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

    2013-01-01

    Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation

  9. Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method.

    Science.gov (United States)

    Hemmatzadeh, F; Kazemimanesh, M

    2017-01-01

    Newcastle disease virus (NDV) is an economically important poultry pathogen with a worldwide distribution that may infect a wide range of domestic and wild avian species. The identification of different pathotypes of NDVs plays an important role in the diagnosis and development of vaccines to control and eradicate NDV infections. In our previous study, we showed that mono-specific antibodies can differentiate velogenic and lentogenic strains of NDV in Agar Gel Immuno-Diffusion tests. To evaluate the ability of the specific antibodies to detect NDV specific antigens, this study was conducted with a range of NDV isolates. The samples included 9 NDV neuropathogenic/velogenic isolates from diseased chickens collected from poultry farms in central and northern parts of Iran plus La-Sota and B1 vaccine strains. All samples were propagated in embryonated chicken eggs and concentrated and purified by ultra-centrifugation. All samples were subjected to 12.5% SDS-PAGE and Western blotting using the specific antibodies mentioned previously. In SDS-PAGE all velogenic and vaccine strains showed the same electrophoretic pattern. The detected bands included 15, 38, 46, 48, 53, 55, 68, 74 and 220 kDa proteins. In Western blotting analysis, the mono-specific antibodies reacted specifically to the viral proteins with 15, 38, 48, 55, 74 and 220 kDa and non-specifically to the viral protein with 53 kDa. The results suggest that specific anti-NDV antibodies can react specifically to glycoproteins (haemagglutin-neuraminidase and fusion proteins) but not to internal proteins (nucleoprotein or matrix protein) of NDV strains.

  10. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis.

    Science.gov (United States)

    Capobiango, Jaqueline Dario; Monica, Thaís Cabral; Ferreira, Fernanda Pinto; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico; Garcia, João Luis; Reiche, Edna Maria Vissoci

    To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  11. Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

    Science.gov (United States)

    Kang, Chi-Chih; Lin, Jung-Ming G; Xu, Zhuchen; Kumar, Sanjay; Herr, Amy E

    2014-10-21

    Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

  12. Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR.

    Science.gov (United States)

    Fontanot, Marco; Iacumin, Lucilla; Cecchini, Francesca; Comi, Giuseppe; Manzano, Marisa

    2014-10-01

    The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth. The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 10(2)C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Far Western blotting as a rapid and efficient method for detecting interactions between DNA replication and DNA repair proteins.

    Science.gov (United States)

    Walsh, Brian W; Lenhart, Justin S; Schroeder, Jeremy W; Simmons, Lyle A

    2012-01-01

    Protein-protein interactions are required for the proper function of many biological pathways. Numerous biochemical and protein blotting methods are available for probing direct and indirect interactions between two protein-binding partners. Here, we describe the methodology of far Western blotting, or immunodot blotting, as a technique for probing direct interactions between two proteins. We describe the utility of this approach as a rapid, qualitative screen for identifying novel protein-binding partners. We also describe the importance of this technique for measuring differences in interaction between wild-type and mutant forms of a known binding partner. Far Western blotting is a rapid and highly reproducible experimental approach for identifying and understanding the interaction between protein-binding partners leading to new discoveries in the function and regulation of biological pathways.

  14. One-day detection of PCR amplified Chlamydia trachomatis DNA in clinical samples: ELISA versus Southern blot hybridisation.

    OpenAIRE

    Roymans, R T; Onland, G; Postma, B H

    1996-01-01

    AIMS: To compare ELISA and Southern blot hybridisation for the detection of PCR amplified Chlamydia trachomatis DNA extracted from clinical samples; to assess the value of the ELISA method in a clinical setting. METHODS: DNA was extracted from urogenital samples of 508 patients, purified and amplified using C trachomatis specific primers, one of which was endlabelled with biotin. Amplification products were detected by Streptavidin biotin based ELISA and non-radioactive Southern blotting. RES...

  15. Application of Western Blotting for the Immunodiagnosis of Fasciola hepatica in Cattle Using Excretory/Secretory Antigens

    OpenAIRE

    SARIMEHMETOĞLU, H. Oğuz

    2002-01-01

    In this study, protein bands of excretory/secretory antigens of Fasciola hepatica were determined using SDS-PAGE and Western blotting. Blood and faecal samples were obtained from cattle brought to Kazan slaughterhouse. After examining the organs and faecal samples of these cattle for Fasciola hepatica and other helminths, serum samples were divided into two groups as positive (10 cattle) and negative (5 cattle) for F. hepatica. The sera of these two groups were tested using Western blotting. ...

  16. V3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting.

    Science.gov (United States)

    Posch, Anton; Kohn, Jonathan; Oh, Kenneth; Hammond, Matt; Liu, Ning

    2013-12-30

    The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.

  17. A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method.

    Science.gov (United States)

    Tomisawa, Satoshi; Abe, Chiharu; Kamiya, Masakatsu; Kikukawa, Takashi; Demura, Makoto; Kawano, Keiichi; Aizawa, Tomoyasu

    2013-01-01

    Western blotting is a widely used technique for the detection and quantification of proteins and peptides. However, it is challenging to detect small peptides efficiently by the conventional Western blotting method with shaking, in part because the peptides readily detach from the blotted membrane. Although some modified Western blotting protocols have been developed to overcome this problem, it remains difficult to prevent peptide detachment from the membrane. In this study, we show that the previously developed vacuum-assisted detection method greatly improves the detection of small peptides without additional protocol modification. The vacuum-assisted method was developed to shorten the time required for all immunodetection steps, and all the Western blotting solutions penetrated the membrane quickly and efficiently by this method. By using this vacuum method, we succeeded in detecting small peptides that were completely undetectable by the conventional Western blotting method. We also confirmed that peptide detachment was induced even by gentle shaking in the case of the conventional method, and the detachment was accelerated when detergent was present in the buffer. Unlike in the conventional method, there is no need to shake the membrane in solution in the vacuum method. Therefore, it is thought that the small peptides could be detected sensitively only by the vacuum method.

  18. Canine pyometra: a study of the urinary proteins by SDS-PAGE and Western blot.

    Science.gov (United States)

    Zaragoza, C; Barrera, R; Centeno, F; Tapia, J A; Mañe, M C

    2004-05-01

    Canine pyometra often causes glomerulonephritis by immune complex deposition in the glomeruli. Proteinuria, ranging from moderate to severe, may be present secondary to renal damage. To determine urinary protein excretion due to pyometra, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted on urine from 15 bitches with pyometra and 10 healthy bitches. To characterize urinary immunoglobin excretion, Western blot analysis of the urine samples using antibodies to canine IgG and IgA was also performed. Nine bands were detected by electrophoresis in bitches with pyometra, while only four were detected in the healthy animals. The urinary proteins from bitches with pyometra were primarily of glomerular origin; 58% were of medium-high molecular weight (MW), and the remainder were low MW. None of the healthy dogs had IgG or IgA in their urine, whereas three bitches with pyometra had IgG in their urine and another bitch with pyometra had both IgG and IgA. The low proportion of bitches with urinary immunoglobins was probably be due to early diagnosis of the disease. Although only a limited number of dogs was used, this study is apparently the first to characterize the electrophoretic pattern of urinary proteins and to quantify urinary excretion of IgG and IgA in bitches with pyometra.

  19. Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

    Science.gov (United States)

    Fischer, Peter U; Curtis, Kurt C; Folk, Scott M; Wilkins, Patricia P; Marcos, Luis A; Weil, Gary J

    2013-06-01

    Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

  20. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.