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Sample records for bacterial antigen detection

  1. [Clinical analysis of patients with bacterial meningitis in childhood and reevaluation of rapid antigen detection methods].

    Science.gov (United States)

    Nakamura, A; Kuroki, H; Ohshima, H; Sugioka, T; Ishiwada, N; Takeda, N; Aizawa, J; Ohkusu, K

    1999-09-01

    Twenty-eight cases of bacterial meningitis during the recent ten years were analyzed retrospectively, and the following results were obtained. 1. Pathogens were as follows; H. influenzae 13 (46.4%), S. pneumoniae 8 (28.6%), S. agalactiae 4 (14.3%), E. coli 2 (7.1%), and L. monocytogenes 1 case (3.6%). 2. Twelve out of the thirteen H. influenzae cases were caused by serotype b (Hib), and 2 strains were beta-lactamase producer. Fifty percent of the S. pneumoniae cases were caused by penicillin-resistant strains. And all these resistant strains belonged to serotype 19 or 23. 3. Underlying diseases related to the onset of meningitis were found in 46% of the cases, and these consisted of CNS shunt operated 5, asplenia or polysplenia 2, Mondini's anomaly 1, sacral dermal sinus 1, and neonate 4 cases. 4. Prognosis of these cases were three deaths, four with neurologic sequelae, and twenty-one complete recoveries. 5. On admission, 85% (17/20) of the cases were diagnosed correctly by the rapid antigen detection. Sensitivity and specificity of the rapid antigen detection by using latex particle agglutination is 90% and 100% in the Hib cases, and 83% and 100% in the S. pneumoniae cases respectively. Moreover, the bacteriologically unknown 2 cases caused by parenteral partial treatment were also diagnosed by the detection of antigen in concentrated urine.

  2. Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

    Science.gov (United States)

    Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

    2012-01-01

    This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

  3. Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

    Science.gov (United States)

    Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

    2016-10-01

    Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    -polysaccharide AbSC of the IgG isotype, the increase was as high as 7.4-11.8 times. Evidence is presented that the pronounced improvement in the detection of the latter is due to the presence of aggregating anti-IgG antibody from the beginning of the assay. It is proposed that in the case of low affinity of anti...

  5. Evaluating the use of dedicated swab for rapid antigen detection ...

    African Journals Online (AJOL)

    Background: Group A streptococcus (GAS) is the most common and fearful bacterial cause in pediatric acute pharyngitis due to its serious complications. Several generations of rapid antigen detection tests (RADTs) have been developed to facilitate rapid detection of GAS pharyngitis. We assessed the value of using a ...

  6. [Acute bacterial meningitis with soluble antigen detected by latex particle agglutination tests at the Sourô-Sanou University Hospital of Bobo-Dioulasso (Burkina Faso)].

    Science.gov (United States)

    Ouédraogo, S M; Yaméogo, T M; Kyelem, C G; Poda, G E A; Ouédraogo, N F; Millogo, A; Ouédraogo, A; Ouédraogo-Traoré, A; Drabo, Y J

    2012-01-01

    Acute bacterial meningitis constitutes a major public health problem in Burkina Faso, in part because of its high lethality rate, estimated in 2004 at 17.5%. Failure to confirm suspected cases of meningitis results in overestimating reported cases and incorrectly treating false positives. The latex particle agglutination test is a diagnostic alternative that overcomes these limitations. Determine the bacteriological and therapeutic profile as well as the course of cases of acute meningitis confirmed by the latex agglutination test at Sourô-Sanou University Hospital. This prospective longitudinal study took place over a one-year period (2008 to 2009). Data were collected from clinical and laboratory records. The diagnosis of meningitis was confirmed by testing for specific soluble antigens in the spinal fluid. We used the Pastorex(™) Meningitis Kit for that purpose. The threshold of significance selected for our study was 0.05. In all, 457 samples of spinal fluid from patients with suspected acute bacterial meningitis were analyzed and the latex test was performed in 438 of these samples: 154 (35.2%) were positive. The average age of our cases confirmed by the latex test was 13.2 ± 4.2 years old. This test confirmed more cases than any other method of identification. The therapeutic strategy used from one to four treatment agents. Streptococcus pneumoniae was the most virulent and the most lethal pathogen, with a 64.7% lethality rate. The earliness of the consultation and the treatment of the bacterial meningitis seem to have a positive effect on the course of disease.

  7. Radiometric detection of bacterial metabolism

    International Nuclear Information System (INIS)

    Camargo, E.E.; Wagner Junior, H.N.

    1979-01-01

    The measurement of 14 CO 2 produced by the bacterial oxidation of labelled compounds is discussed as a means of evaluating the bacterial metabolism. The following items are discussed:automated radiometric detection, types of graphs, clinical applications of the radiometric system and influential factors. Complementary studies on bacterial assimilation of substances are presented. (M.A.) [pt

  8. House dust mites as potential carriers for IgE sensitization to bacterial antigens.

    Science.gov (United States)

    Dzoro, S; Mittermann, I; Resch-Marat, Y; Vrtala, S; Nehr, M; Hirschl, A M; Wikberg, G; Lundeberg, L; Johansson, C; Scheynius, A; Valenta, R

    2018-01-01

    IgE reactivity to antigens from Gram-positive and Gram-negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis. Plasma samples from patients with AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified HDM bodies, and the IgE-reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments. IgE reactivity to bacterial antigens was significantly more frequent in patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune-blotted HDM extract, and the presence of IgE-reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments. House dust mites (HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens. © 2017 The Authors. Allergy Published by John Wiley & Sons Ltd.

  9. Purification of antibodies to bacterial antigens by an immunoadsorbent and a method to quantify their reaction with insoluble bacterial targets

    International Nuclear Information System (INIS)

    Mathews, H.L.; Minden, P.

    1979-01-01

    A combination of procedures was employed to develop a radioimmunoassay which quantified the binding of antibodies to antigens of either intact Propionibacterium acnes or to antigens of insoluble extracts derived from the bacteria. Reactive antibody populations were purified by use of bacterial immunoadsorbents which were prepared by coupling P. acnes to diethylaminoethyl cellulose. Binding of antibodies was detected with [ 125 I]staphylococcal protein A ([ 125 I]SpA) and optimal conditions for the assay defined by varying the amounts of antibodies, bacterial antigenic targets and [ 125 I]SpA. In antibody excess, 100% of available [ 125 I]SpA was bound by the target-antibody complexes. However, when antibody concentration was limiting, a linear relationship was demonstrated between per cent specific binding of[ 125 I]SpA and antibodies bound to bacterial targets. These results were achieved only with immunoadsorbent-purified antibody populations and not with hyperimmune sera or IgG. The radioimmunoassay detected subtle antigenic differences and similarities between P. acnes, P. acnes extracts and a variety of unrelated microorganisms. (Auth.)

  10. Identification of bacterial antigens and super antigens in synovial fluid of patients with arthritis: a cross sectional study

    Directory of Open Access Journals (Sweden)

    Samileh Noorbakhsh

    2013-02-01

    negative SF culture. Rapid identification of bacterial antigens (LPA or S.aureus superantigens (toxins are valuable for diagnosis in cases with negative cultures. We recommend usage of complementary methods (e.g. antigen detection tests in children. Those tests are cheaper and easier in comparison with PCR as a complex and time-taking method. Identification of S.aureus superantigens in SF of all cases w

  11. Molecular detection of human bacterial pathogens

    National Research Council Canada - National Science Library

    Liu, Dongyou

    2011-01-01

    .... Molecular Detection of Human Bacterial Pathogens addresses this issue, with international scientists in respective bacterial pathogen research and diagnosis providing expert summaries on current...

  12. Lectin binding patterns and immunohistochemical antigen detection ...

    African Journals Online (AJOL)

    Ibrahim Eldaghayes

    2018-02-09

    Feb 9, 2018 ... examined by histological, lectin-histochemical, immunohistochemical and cultural techniques. B. abortus antigens were immunohistochemically detected in fetal lungs and placenta. An increase in the labeling with UEA-1, DBA,. PNA, RCA-1 and SBA was found in the lungs and an increase in the labeling ...

  13. Microconductometric Detection of Bacterial Contamination

    Directory of Open Access Journals (Sweden)

    Sarra EL ICHI

    2014-05-01

    Full Text Available Several approaches can be used for the electrochemical detection of bacterial contamination. Their performance can be assessed by the ability to detect bacteria at very low concentrations within a short-time response. We have already demonstrated that a conductometric biosensor based on interdigitated thin-film electrodes is adapted to detect bacteria in clinical samples like serum and compatible with microfluidic fabrication. The type of interdigitated microelectrodes influences the performance of the biosensor. This was shown by the results obtained in this work. A magnetic-nanoparticles based immunosensor was designed using gold screen-printed electrodes. The immunosensor was able to specifically detect E. coli in the range of 1-103 CFU mL-1. The new transducer offered a larger active sensing surface with a lower cost and a robust material. Accuracy of the conductance value was enhanced by differential measurements. The immunosensor is compatible with a microfluidic system.

  14. Mucosal delivery of antigens using adsorption to bacterial spores.

    Science.gov (United States)

    Huang, Jen-Min; Hong, Huynh A; Van Tong, Hoang; Hoang, Tran H; Brisson, Alain; Cutting, Simon M

    2010-01-22

    The development of new-generation vaccines has followed a number of strategic avenues including the use of live recombinant bacteria. Of these, the use of genetically engineered bacterial spores has been shown to offer promise as both a mucosal as well as a heat-stable vaccine delivery system. Spores of the genus Bacillus are currently in widespread use as probiotics enabling a case to be made for their safety. In this work we have discovered that the negatively charged and hydrophobic surface layer of spores provides a suitable platform for adsorption of protein antigens. Binding can be promoted under conditions of low pH and requires a potent combination of electrostatic and hydrophobic interactions between spore and immunogen. Using appropriately adsorbed spores we have shown that mice immunised mucosally can be protected against challenge with tetanus toxin, Clostridium perfringens alpha toxin and could survive challenge with anthrax toxin. In some cases protection is actually greater than using a recombinant vaccine. Remarkably, killed or inactivated spores appear equally effective as live spores. The spore appears to present a bound antigen in its native conformation promoting a cellular (T(h)1-biased) response coupled with a strong antibody response. Spores then, should be considered as mucosal adjuvants, most similar to particulate adjuvants, by enhancing responses against soluble antigens. The broad spectrum of immune responses elicited coupled with the attendant benefits of safety suggest that spore adsorption could be appropriate for improving the immunogenicity of some vaccines as well as the delivery of biotherapeutic molecules.

  15. Detection of poliovirus antigen by enzyme immunoassay.

    OpenAIRE

    Ukkonen, P; Huovilainen, A; Hovi, T

    1986-01-01

    A solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Rabbit and guinea pig antisera for the assay were raised against purified poliovirus type 3/Fin (strain 3/Fin/K) isolated from a fecal specimen from a meningitis patient during an outbreak of poliomyelitis in Finland in 1984. The EIA was highly specific for poliovirus type 3, and it was about 30 times more sensitive for strain 3/Fin/K than for strain 3/Saukett used in the inactivated poliovirus vacci...

  16. A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

    Science.gov (United States)

    Cortina, María E; Balzano, Rodrigo E; Rey Serantes, Diego A; Caillava, Ana J; Elena, Sebastián; Ferreira, A C; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

    2016-06-01

    Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei.

    Science.gov (United States)

    Caboni, Mariaelena; Pédron, Thierry; Rossi, Omar; Goulding, David; Pickard, Derek; Citiulo, Francesco; MacLennan, Calman A; Dougan, Gordon; Thomson, Nicholas R; Saul, Allan; Sansonetti, Philippe J; Gerke, Christiane

    2015-03-01

    Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

  18. An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei.

    Directory of Open Access Journals (Sweden)

    Mariaelena Caboni

    2015-03-01

    Full Text Available Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg of the lipopolysaccharide (LPS plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

  19. Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

    Directory of Open Access Journals (Sweden)

    Edith S Málaga-Machaca

    2017-11-01

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

  20. A Safe Bacterial Microsyringe for In Vivo Antigen Delivery and Immunotherapy

    Science.gov (United States)

    Le Gouëllec, Audrey; Chauchet, Xavier; Laurin, David; Aspord, Caroline; Verove, Julien; Wang, Yan; Genestet, Charlotte; Trocme, Candice; Ahmadi, Mitra; Martin, Sandrine; Broisat, Alexis; Cretin, François; Ghezzi, Catherine; Polack, Benoit; Plumas, Joël; Toussaint, Bertrand

    2013-01-01

    The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the “killed but metabolically active” (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery. PMID:23531551

  1. Detection of antibody activity in human sera against meningococcal cell wall antigens using a gel-immuno-radio-assay (GIRA)

    International Nuclear Information System (INIS)

    Poolman, J.T.; Zanen, H.C.

    1980-01-01

    The authors recently described the application of the SDS-polyacrylamide-gel-electrophoresis-immuno-peroxidase (SGIP) technique to the analysis of meningococcal cell walls. However, it appeared that SGIP was not sensitive enough to detect low levels of human antibodies against meningococcal cell wall antigens. They therefore replaced the peroxidase labeled anti-IgG by 125 I-labeled protein A in order to detect antibody binding by bacterial antigens separated in gels, resulting in gel-immuno-radio-assay (GIRA). (Auth.)

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

    African Journals Online (AJOL)

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

  3. Biosensors for Whole-Cell Bacterial Detection

    Science.gov (United States)

    Rushworth, Jo V.; Hirst, Natalie A.; Millner, Paul A.

    2014-01-01

    SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost. PMID:24982325

  4. Detection of dentin antigenic fractions by salivary immunoglobulin G ...

    African Journals Online (AJOL)

    Detection of dentin antigenic fractions by salivary immunoglobulin G in patients undergoing orthodontic treatment. TMP Soares da Costa, S de Paula Ramos, MM Hidalgo, A Consolaro, SA Khan, EN Itano ...

  5. Participation of CD1 molecules in the presentation of bacterial protein antigens in humans.

    Science.gov (United States)

    Ulanova, M; Tarkowski, A; Hahn-Zoric, M; Hanson, L A

    1999-10-01

    Human CD1 molecules, expressed on the surface of professional antigen-presenting cells (including dendritic cells, Langerhans' cells, B cells and activated monocytes) are structurally homologous to major histocompatibility complex (MHC) class I and class II molecules. CD1b and CD1c have been shown to present nonpeptide bacterial antigens to T cells. We hypothesized that CD1 molecules may also be involved in the presentation of bacterial protein antigens. Human peripheral blood mononuclear cells (PBMC) were exposed to two medically important proteins, tetanus toxoid (TT) and purified protein derivative (PPD), with and without murine monoclonal antibodies (MoAbs) specific for CD1a, CD1b and CD1c. All the MoAbs substantially inhibited the proliferative responses of PBMC to TT and PPD. Simultaneous interaction of CD1 and MHC class II molecules was even more inhibitory to these antigen-specific proliferative responses. In contrast, neither mixed lymphocyte reaction nor superantigen and mitogenic responses were affected by CD1-specific antibodies, indicating a certain restriction pattern in antigen presentation. Our findings suggest that, besides MHC class I and II molecules, there is a family of nonpolymorphic cell surface molecules that is able to present certain bacterial protein antigens to T cells.

  6. Mini-review: Strategies for Variation and Evolution of Bacterial Antigens

    Directory of Open Access Journals (Sweden)

    Janet Foley

    2015-01-01

    Full Text Available Across the eubacteria, antigenic variation has emerged as a strategy to evade host immunity. However, phenotypic variation in some of these antigens also allows the bacteria to exploit variable host niches as well. The specific mechanisms are not shared-derived characters although there is considerable convergent evolution and numerous commonalities reflecting considerations of natural selection and biochemical restraints. Unlike in viruses, mechanisms of antigenic variation in most bacteria involve larger DNA movement such as gene conversion or DNA rearrangement, although some antigens vary due to point mutations or modified transcriptional regulation. The convergent evolution that promotes antigenic variation integrates various evolutionary forces: these include mutations underlying variant production; drift which could remove alleles especially early in infection or during life history phases in arthropod vectors (when the bacterial population size goes through a bottleneck; selection not only for any particular variant but also for the mechanism for the production of variants (i.e., selection for mutability; and overcoming negative selection against variant production. This review highlights the complexities of drivers of antigenic variation, in particular extending evaluation beyond the commonly cited theory of immune evasion. A deeper understanding of the diversity of purpose and mechanisms of antigenic variation in bacteria will contribute to greater insight into bacterial pathogenesis, ecology and coevolution with hosts.

  7. Bacterial ghosts (BGs)--advanced antigen and drug delivery system.

    Science.gov (United States)

    Kudela, Pavol; Koller, Verena Juliana; Lubitz, Werner

    2010-08-16

    Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2-3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.

  8. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    Science.gov (United States)

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  9. Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis

    Directory of Open Access Journals (Sweden)

    Malkit Singh

    2013-01-01

    Full Text Available Background: Reactive arthritis (ReA/Reiter′s syndrome (RS may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. Aim: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. Materials and Methods: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. Results and Conclusions: A common 30 kDa antigen was found to be specifically present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients′ sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with post-dysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the first time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

  10. 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 ...

  11. Detection of canine parvovirus antigen in dogs in Kumasi, Ghana ...

    African Journals Online (AJOL)

    Background: Canine Parvovirus (CPV) in dogs has been documented in many countries. However, evidence of the infection is scanty in Ghana. This study was conducted to detect canine parvovirus antigen in dogs presented with diarrhoea to the Government Veterinary Clinic in Kumasi, Ghana. Materials and Methods: ...

  12. Detection of Rabies Antigen in the Brain Tissues of Apparetly ...

    African Journals Online (AJOL)

    Rabies is a serious public health hazard and recently outbreaks of the disease have been reported in three local government areas in Cross River State. Detection of rabies antigen in the brain tissues of apparently healthy dogs indicates the presence of rabies virus and this is a significant factor in the transmission and ...

  13. Antigen detection of entamoeba histolytica intestinal infection: cost ...

    African Journals Online (AJOL)

    Purpose: Laboratory diagnosis of Entamoeba histolytica infection is still being made through compound light microscopy in resource limited countries despite the associated flaws. This study is aimed at applying and determining the usefulness of ELISA antigen detection technique for E. histolytica intestinal infection ...

  14. Bacteriophages for detection of bacterial pathogens

    International Nuclear Information System (INIS)

    Kutateladze, M.

    2009-01-01

    The G. Eliava Institute of Bacteriophages, Microbiology and Virology (Tbilisi, Georgia) is one of the most famous institutions focused on bacteriophage research for the elaboration of appropriate phage methodologies for human and animal protection. The main direction of the institute is the study and production of bacteriophages against intestinal disorders (dysentery, typhoid, intesti) and purulent-septic infections (staphylococcus, streptococcus, pyophage, etc.). These preparations were successfully introduced during the Soviet era, and for decades were used throughout the former Soviet Union and in other Socialist countries for the treatment, prophylaxis, and diagnosis of various infectious diseases, including those caused by antibiotic-resistant bacterial strains. Bacteriophages were widely used for identifying and detecting infections caused by the most dangerous pathogens and causative agents of epidemiological outbreaks. The specific topic of this presentation is the phage typing of bacterial species, which can be an important method for epidemiological diagnostics. Together with different genetic methodologies - such as PCR-based methods, PFGE, plasmid fingerprinting, and ribosomal typing - phage typing is one method for identifying bacterial pathogens. The method has a high percentage of determination of phage types, high specificity of reaction, and is easy for interpretation and use by health workers. Phage typing was applied for inter-species differentiation of different species of Salmonella, S. typhi, Brucella spp, Staphylococcus aureus, E. col,i Clostridium deficile, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, Lysteria monocytogenes, Clostridium perfringens, Clostridium tetani, plant pathogens, and other bacterial pathogens. In addition to addressing the utility and efficacy of phage typing, the paper will discuss the isolation and selection of diagnostic typing phages for interspecies differentiation of pathogens that is necessary

  15. O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.

    Science.gov (United States)

    Rapicavoli, Jeannette N; Kinsinger, Nichola; Perring, Thomas M; Backus, Elaine A; Shugart, Holly J; Walker, Sharon; Roper, M Caroline

    2015-12-01

    Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Conventional and molecular methods to detect bacterial pathogens in mussels.

    Science.gov (United States)

    Gugliandolo, C; Lentini, V; Spanò, A; Maugeri, T L

    2011-01-01

    To detect Aeromonas spp., Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in mussels and water samples from a farming area, conventional and molecular methods were applied to enrichment cultures. The aerolysin gene (aero) of Aeromonas spp., the invasion plasmid antigen B (ipaB) gene of Salmonella spp., the enterotoxin secretion protein (epsM) gene of V. cholerae, the species-specific region of 16S rRNA gene of V. vulnificus, the 16S-23S rDNA (IGS) gene of V. parahaemolyticus and the pR72H fragment of V. parahaemolyticus were amplified by multiplex polymerase chain reaction (PCR) assays on DNA extracted from enrichment cultures. The haemolysin gene (tdh) of pathogenic V. parahaemolyticus was also amplified. Conventional culture method allowed the isolation of V. parahaemolyticus and V. vulnificus from water and mussels. The genes aero, epsM and 16S rRNA of V. vulnificus were occasionally detected in the enrichment cultures. In mussels, the ipaB and IGS genes were detected from June to September and from April to November, respectively. All genes, except aero, were amplified from mussels collected in September, when pathogenic V. parahaemolyticus (tdh+) strains were also isolated. Multiplex-PCR assays were more sensitive and faster than conventional procedures. The results emphasize the need of an accurate and rapid detection of bacterial pathogens in mussels to protect human health. © 2010 The Authors. Letters in Applied Microbiology © 2010 The Society for Applied Microbiology.

  17. Bacterial antigen induced release of soluble vascular endothelial growth factor (VEGF) and VEGFR1 before and after surgery

    DEFF Research Database (Denmark)

    Svendsen, Mads N; Lykke, J; Werther, Kim

    2005-01-01

    OBJECTIVE: The influence of surgery on release of soluble vascular endothelial growth factor (sVEGF) and the soluble inhibitory receptor (sVEGFR1) is unknown. The effect of major and minor surgery on variations in sVEGF and sVEGFR1 concentrations in vivo was studied, and on bacterial antigen...... concentrations in plasma changed during surgery. In vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in sVEGF (p Bacterial antigen-induced release of sVEGF correlated...... significantly with neutrophil cell counts (0.53 Bacterial antigen-induced sVEGFR1 release did not correlate with cell counts. CONCLUSIONS: Plasma sVEGF and sVEGFR1 concentrations did not change during surgery. In vitro bacterial stimulation led to increased release of sVEGF, which...

  18. Benchtop Antigen Detection Technique using Nanofiltration and Fluorescent Dyes

    Science.gov (United States)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2009-01-01

    The designed benchtop technique is primed to detect bacteria and viruses from antigenic surface marker proteins in solutions, initially water. This inclusive bio-immunoassay uniquely combines nanofiltration and near infrared (NIR) dyes conjugated to antibodies to isolate and distinguish microbial antigens, using laser excitation and spectrometric analysis. The project goals include detecting microorganisms aboard the International Space Station, space shuttle, Crew Exploration Vehicle (CEV), and human habitats on future Moon and Mars missions, ensuring astronaut safety. The technique is intended to improve and advance water contamination testing both commercially and environmentally as well. Lastly, this streamlined technique poses to greatly simplify and expedite testing of pathogens in complex matrices, such as blood, in hospital and laboratory clinics.

  19. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

    Science.gov (United States)

    Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

    2006-03-01

    We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

  20. Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling.

    Directory of Open Access Journals (Sweden)

    Amit A Lugade

    Full Text Available The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2(-/- DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively, our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.

  1. Immunoglobulin (Ig) D in Labeo rohita is widely expressed and differentially modulated in viral, bacterial and parasitic antigenic challenges.

    Science.gov (United States)

    Basu, Madhubanti; Lenka, Saswati S; Paichha, Mahismita; Swain, Banikalyan; Patel, Bhakti; Banerjee, Rajanya; Jayasankar, Pallipuram; Das, Surajit; Samanta, Mrinal

    2016-10-15

    Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. 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.

  3. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    Directory of Open Access Journals (Sweden)

    He Junkun

    2012-06-01

    Full Text Available Abstract Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to

  4. Understanding the bacterial polysaccharide antigenicity of Streptococcus agalactiae versus Streptococcus pneumoniae.

    Science.gov (United States)

    Kadirvelraj, Renuka; Gonzalez-Outeiriño, Jorge; Foley, B Lachele; Beckham, Meredith L; Jennings, Harold J; Foote, Simon; Ford, Michael G; Woods, Robert J

    2006-05-23

    Bacterial surface capsular polysaccharides (CPS) that are similar in carbohydrate sequence may differ markedly in immunogenicity and antigenicity. The structural origin of these phenomena is poorly understood. Such a case is presented by the Gram-positive bacteria Streptococcus agalactiae (Group B Streptococcus; GBS) type III (GBSIII) and Streptococcus pneumoniae (Pn) type 14 (Pn14), which share closely related CPS sequences. Nevertheless, antibodies (Abs) against GBSIII rarely cross-react with the CPS from Pn14. To establish the origin for the variation in CPS antigenicity, models for the immune complexes of CPS fragments from GBSIII and Pn14, with the variable fragment (Fv) of a GBS-specific mAb (mAb 1B1), are presented. The complexes are generated through a combination of comparative Ab modeling and automated ligand docking, followed by explicitly solvated 10-ns molecular dynamics simulations. The relationship between carbohydrate sequence and antigenicity is further quantified through the computation of interaction energies using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method, augmented by conformational entropy estimates. Despite the electrostatic differences between Pn14 and GBSIII CPS, analysis indicates that entropic penalties are primarily responsible for the loss of affinity of the highly flexible Pn14 CPS for mAb 1B1. The similarity of the solution conformation of the relatively rigid GBSIII CPS with that in the immune complex characterizes the previously undescribed 3D structure of the conformational epitope. The analysis provides a comprehensive interpretation for a large body of biochemical and immunological data related to Ab recognition of bacterial polysaccharides and should be applicable to other Ab-carbohydrate interactions.

  5. Application of bead array technology to simultaneous detection of human leucocyte antigen and human platelet antigen antibodies.

    Science.gov (United States)

    Fujiwara, K; Shimano, K; Tanaka, H; Sekine, M; Kashiwase, K; Uchikawa, M; Satake, M; Nakajima, K

    2009-04-01

    Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described. Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.

  6. Detection of Carcinoembryonic Antigens Using a Surface Plasmon Resonance Biosensor

    Science.gov (United States)

    Su, Fengyu; Xu, Chunye; Taya, Minoru; Murayama, Kimie; Shinohara, Yasuro; Nishimura, Shin-Ichiro

    2008-01-01

    Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection with high sensitivity, though its ability to detect CEA in human serum was highly dependent on the analytical conditions employed. We investigated the influences of various analytical conditions including immobilization methods for anti-CEA antibody and composition of sensor surface on the selective and sensitive detection of CEA. The results show that anti-CEA antibody immobilized via Protein A or Protein G caused a large increase in the resonance signal upon injection of human serum due to the interactions with IgGs in serum, while direct covalent immobilization of anti-CEA antibody could substantially reduce it. An optimized protocol based on further kinetic analysis and the use of 2nd and 3rd antibodies for the sandwich assay allowed detecting spiked CEA in human serum as low as 25 ng/mL. Furthermore, a self-assembled monolayer of mixed ethylene-glycol terminated alkanethiols on gold was found to have a comparable ability in detecting CEA as CM5 with thick dextran matrix and C1 with short flat layer on gold. PMID:27879935

  7. Bacterial antigens alone can influence intestinal barrier integrity, but live bacteria are required for initiation of intestinal inflammation and injury.

    Science.gov (United States)

    Sydora, Beate C; Martin, Sarah M; Lupicki, Maryla; Dieleman, Levinus A; Doyle, Jason; Walker, John W; Fedorak, Richard N

    2006-06-01

    Intestinal flora plays a critical role in the initiation and perpetuation of inflammatory bowel disease. This study examined whether live fecal bacteria were necessary for the initiation of this inflammatory response or whether sterile fecal material would provoke a similar response. Three preparations of fecal material were prepared: (1) a slurry of live fecal bacteria, (2) a sterile lysate of bacterial antigens, and (3) a sterile filtrate of fecal water. Each preparation was introduced via gastric gavage into the intestines of axenic interleukin-10 gene-deficient mice genetically predisposed to develop inflammatory bowel disease. Intestinal barrier integrity and degrees of mucosal and systemic inflammations were determined for each preparation group. Intestinal barrier integrity, as determined by mannitol transmural flux, was altered by both live fecal bacterial and sterile lysates of bacterial antigens, although it was not altered by sterile filtrates of fecal water. However, only live fecal bacteria initiated mucosal inflammation and injury and a systemic immune response. Fecal bacterial antigens in the presence of live bacteria and sterile fecal bacterial antigens have different effects on the initiation and perpetuation of intestinal inflammation.

  8. Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Ertürk, Gizem [Department of Biotechnology, Lund University, Lund (Sweden); Department of Biology, Hacettepe University, Ankara (Turkey); Hedström, Martin [Department of Biotechnology, Lund University, Lund (Sweden); CapSenze HB, Medicon Village, SE-223 63 Lund (Sweden); Tümer, M. Aşkın [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil [Department of Chemistry, Hacettepe University, Ankara (Turkey); Mattiasson, Bo, E-mail: Bo.Mattiasson@biotek.lu.se [Department of Biotechnology, Lund University, Lund (Sweden); CapSenze HB, Medicon Village, SE-223 63 Lund (Sweden)

    2015-09-03

    Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL{sup −1}–100 ng mL{sup −1}. The detection limits were found as 8.0 × 10{sup −5} ng mL{sup −1} (16 × 10{sup −17} M) and 6.0 × 10{sup −4} ng mL{sup −1} (12 × 10{sup −16} M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations. - Highlights: • Microcontact imprinting method was used for preparing the sensor chip for capacitive biosensing. • High sensitivity was obtained. • Good selectivity was

  9. Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors

    International Nuclear Information System (INIS)

    Ertürk, Gizem; Hedström, Martin; Tümer, M. Aşkın; Denizli, Adil; Mattiasson, Bo

    2015-01-01

    Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL −1 –100 ng mL −1 . The detection limits were found as 8.0 × 10 −5  ng mL −1 (16 × 10 −17  M) and 6.0 × 10 −4  ng mL −1 (12 × 10 −16  M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations. - Highlights: • Microcontact imprinting method was used for preparing the sensor chip for capacitive biosensing. • High sensitivity was obtained. • Good selectivity was demonstrated. • Stability of the

  10. DETECTION OF CANINE PARVOVIRUS ANTIGEN IN DOGS IN KUMASI, GHANA.

    Science.gov (United States)

    Folitse, R D; Kodie, D O; Amemor, E; Dei, D; Tasiame, W; Burimuah, V; Emikpe, B O

    2018-01-01

    Canine Parvovirus (CPV) in dogs has been documented in many countries. However, evidence of the infection is scanty in Ghana. This study was conducted to detect canine parvovirus antigen in dogs presented with diarrhoea to the Government Veterinary Clinic in Kumasi, Ghana. Faecal samples from 72 dogs presented with diarrhoea were tested for the presence of canine parvovirus antigen using commercially available rapid test kit (BIT ® Rapid Colour Canine Parvovirus Ag Test Kit, BIOINDIST Co. Ltd, Korea) based on the principle of immunochromatography. Influence of breed, sex, age, vaccination history and the nature of diarrhoea were assessed. Data obtained was analysed with SPSS and subjected to the chi-square test. Significance was at α 0.05 . We found 61.11% tested positive (44/72) for CPV. Based on sex, 61.54% of males (20/33) and 60.61% of females tested positive (24/39). A total of 65.67% of samples from puppies below 6 months were positive. 56.25% of CPV vaccinated dogs and 70.83% of unvaccinated dogs were positive respectively. 69.05% of samples from haemorrhagic diarrhoeic dogs and 50.00% from non-haemorrhagic diarrhoeic dogs were positive of CPV. The study is the first documented evidence of the existence of CPV in Ghana. It also revealed that absence of bloody diarrhoea does not necessarily rule out CPV infection.

  11. A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis.

    Science.gov (United States)

    Ciocchini, Andrés E; Serantes, Diego A Rey; Melli, Luciano J; Guidolin, Leticia S; Iwashkiw, Jeremy A; Elena, Sebastián; Franco, Cristina; Nicola, Ana M; Feldman, Mario F; Comerci, Diego J; Ugalde, Juan E

    2014-08-27

    Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Laboratory diagnosis of bovine brucellosis mainly relies on serological diagnosis using serum and/or milk samples. Although there are several serological tests with different diagnostic performance and capacity to differentiate vaccinated from infected animals, there is still no standardized reference antigen for the disease. Here we validate the first recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of bovine brucellosis. This antigen can be produced in homogeneous batches without the need of culturing pathogenic brucellae; all characteristics that make it appropriate for standardization. An indirect immunoassay based on the detection of anti O-polysaccharide IgG antibodies in bovine samples was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept and to validate the antigen, we analyzed serum, whole blood and milk samples obtained from non-infected, experimentally infected and vaccinated animals included in a vaccination/infection trial performed in our laboratory as well as more than 1000 serum and milk samples obtained from naturally infected and S19-vaccinated animals from Argentina. Our results demonstrate that OAg-AcrA-based assays are highly accurate for diagnosis of bovine brucellosis, even in vaccinated herds, using different types of samples and in different platforms. We propose this novel recombinant glycoprotein as an antigen suitable for the development of new standard immunological tests for screening and confirmatory diagnosis of bovine brucellosis in regions or countries with brucellosis-control programs. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Rapid detection of cryptococcal antigen by a flow assay

    Directory of Open Access Journals (Sweden)

    Graziano Bargiggia

    2017-10-01

    Full Text Available Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii. Tests for quick detection of the cryptococcal antigen are needed. This study compares the performance of a lateral flow assay (LFA to the latex agglutination method. Thirty-five cryopreserved positive samples (sera and cerebrospinal fluids plus three negative sera for control have been examined. LFA does not need high-temperature incubation or enzyme pre-treatment. All the results, except for one serum, agree with previous obtained with latex agglutination method. LFA has an important clinical utility for its rapidity and sensitivity, and it also can be used as a point-of-care test.

  13. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

    Directory of Open Access Journals (Sweden)

    Christoph B Geier

    Full Text Available Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  14. Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay

    Directory of Open Access Journals (Sweden)

    O. Opota

    2015-03-01

    Full Text Available Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity.

  15. Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay.

    Science.gov (United States)

    Opota, O; Desgraz, B; Kenfak, A; Jaton, K; Cavassini, M; Greub, G; Prod'hom, G; Giulieri, S

    2015-03-01

    Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity.

  16. Bacterial contamination of platelet concentrates: pathogen detection and inactivation methods

    Directory of Open Access Journals (Sweden)

    Dana Védy

    2009-04-01

    Full Text Available Whereas the reduction of transfusion related viral transmission has been a priority during the last decade, bacterial infection transmitted by transfusion still remains associated to a high morbidity and mortality, and constitutes the most frequent infectious risk of transfusion. This problem especially concerns platelet concentrates because of their favorable bacterial growth conditions. This review gives an overview of platelet transfusion-related bacterial contamination as well as on the different strategies to reduce this problem by using either bacterial detection or inactivation methods.

  17. AgdbNet – antigen sequence database software for bacterial typing

    Directory of Open Access Journals (Sweden)

    Maiden Martin CJ

    2006-06-01

    Full Text Available Abstract Background Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. Results Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. Conclusion The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated.

  18. Sensitive Detection of Deliquescent Bacterial Capsules through Nanomechanical Analysis.

    Science.gov (United States)

    Nguyen, Song Ha; Webb, Hayden K

    2015-10-20

    Encapsulated bacteria usually exhibit strong resistance to a wide range of sterilization methods, and are often virulent. Early detection of encapsulation can be crucial in microbial pathology. This work demonstrates a fast and sensitive method for the detection of encapsulated bacterial cells. Nanoindentation force measurements were used to confirm the presence of deliquescent bacterial capsules surrounding bacterial cells. Force/distance approach curves contained characteristic linear-nonlinear-linear domains, indicating cocompression of the capsular layer and cell, indentation of the capsule, and compression of the cell alone. This is a sensitive method for the detection and verification of the encapsulation status of bacterial cells. Given that this method was successful in detecting the nanomechanical properties of two different layers of cell material, i.e. distinguishing between the capsule and the remainder of the cell, further development may potentially lead to the ability to analyze even thinner cellular layers, e.g. lipid bilayers.

  19. Detecting Nosocomial Intrinsic Infections through Relating Bacterial ...

    African Journals Online (AJOL)

    Sierra Leone Journal of Biomedical Research ... Surgical procedures often lead to both intrinsic and extrinsic infections. ... This study demonstrated surgical procedures as precursory to intrinsic infections and that bacterial pathogens found on wounds and endogenous indicators of surgery are links to intrinsic infection.

  20. Investigation of contactless detection using a giant magnetoresistance sensor for detecting prostate specific antigen.

    Science.gov (United States)

    Sun, Xuecheng; Zhi, Shaotao; Lei, Chong; Zhou, Yong

    2016-08-01

    This paper presents a contactless detection method for detecting prostate specific antigen with a giant magnetoresistance sensor. In contactless detection case, the prostate specific antigen sample preparation was separated from the sensor that prevented the sensor from being immersed in chemical solvents, and made the sensor implementing in immediately reuse without wash. Experimental results showed that applied an external magnetic field in a range of 50 Oe to 90 Oe, Dynabeads with a concentration as low as 0.1 μg/mL can be detected by this system and could give an approximate quantitation to the logarithmic of Dynabeads concentration. Sandwich immunoassay was employed for preparing PSA samples. The PSA capture was implemented on a gold film modified with a self-assembled monolayer and using biotinylated secondary antibody against PSA and streptavidinylated Dynabeads. With DC magnetic field in the range of 50 to 90 Oe, PSA can be detected with a detection limit as low as 0.1 ng/mL. Samples spiked with different concentrations of PSA can be distinguished clearly. Due to the contactless detection method, the detection system exhibited advantages such as convenient manipulation, reusable, inexpensive, small weight. So, this detection method was a promising candidate in biomarker detection, especially in point of care detection.

  1. Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

    DEFF Research Database (Denmark)

    Shilova, N V; Navakouski, M J; Huflejt, M

    2011-01-01

    The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted...... pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization....

  2. Bacterial antigen detection in cerebrospinal fluid by the latex agglutination test Detecção de antígenos bacterianos no líquido cefalorraquidiano através do teste de aglutinação de latex

    Directory of Open Access Journals (Sweden)

    Ilka Maria Landgraf

    1995-06-01

    Full Text Available Eighty purulent cerebrospinal fluid (CSF samples from patients with clinical evidence of meningitis were studied using the Directigen latex agglutination (LA kit to determine the presence of bacterial antigen in CSF. The results showed a better diagnostic performance of the LA test than bacterioscopy by Gram stain, culture and counterimmunoelectrophoresis (CIE, as far as Neisseria meningitidis groups B and C, and Haemophilus influenzae type b are concerned, and a better performance than bacterioscopy and culture considering Streptococcus pneumoniae. Comparison of the results with those of culture showed that the LA test had the highest sensitivity for the Neisseria meningitidis group C. Comparing the results with those of CIE, the highest levels of sensitivity were detected for N. meningitidis groups B and C. Regarding specificity, fair values were obtained for all organisms tested. The degree of K agreement when the LA test was compared with CIE exhibited better K indices of agreement for N. meningitidis groups B and C.Oitenta amostras purulentas de líquido cefalorraquidiano (LCR de pacientes com evidência clínica de meningite foram estudadas empregando-se Kit Directigen de aglutinação de latex (AL para demonstrar antígeno bacteriano no LCR. Os resultados mostraram que o teste de AL apresentou melhor desempenho diagnóstico do que bacterioscopia através da coloração de Gram, cultura e imunoeletroforese cruzada (IEC em relação à Neisseria meningitidis grupos B e C, e ao Haemophilus influenzae tipo b, e melhor do que coloração de Gram e cultura quando Streptococcus pneumoniae foi avaliado. A comparação dos resultados com os de cultura mostrou o maior nível de sensibilidade considerando-se N. meningitidis grupo C. Quanto à especificidade, os valores foram satisfatórios para todos os microrganismos testados. O grau de concordância K em relação à IEC exibiu melhores índices K de concordância para N. meningitidis grupos B e C.

  3. Impact of heat treatment on Dirofilaria immitis antigen detection in shelter dogs.

    Science.gov (United States)

    DiGangi, Brian A; Dworkin, Carly; Stull, Jason W; O'Quin, Jeanette; Elser, Morgan; Marsh, Antoinette E; Groshong, Lesli; Wolfson, Wendy; Duhon, Brandy; Broaddus, Katie; Gingrich, Elise N; Swiniarski, Emily; Berliner, Elizabeth A

    2017-11-09

    The diagnosis and management of canine heartworm disease is a growing concern for shelter veterinarians. Although the accuracy of commercial antigen test kits has been widely studied, recent reports have renewed interest in antigen blocking as a causative factor for false "no antigen detected" results. The objectives of this study were to determine the prevalence of false "no antigen detected" results in adult dogs entering shelters in northern, southern, and western regions of the country and to identify historical and clinical risk factors for such results. Serum samples were evaluated for Dirofilaria immitis antigen using a commercially available point-of-care ELISA; samples in which no antigen was detected underwent a heat treatment protocol and repeat antigen testing. Whole blood samples underwent Knott testing to identify the presence of microfilariae. Historical and clinical findings were analyzed using exact logistic regression. A total of 616 samples were analyzed. Overall prevalence of positive antigen test results (prior to heat treatment) was 7.3% and frequency of false "no antigen detected" results due to antigen blocking (ie, samples with no antigen detected prior to heat treatment and positive after heat treatment) was 5.2%. Among dogs that had no detectable antigen on the initial tests, dogs that had microfilariae detected via modified Knott testing (OR = 32.30, p-value = 0.013) and dogs that previously received a heartworm preventive (OR = 3.81, p-value = 0.016) had greater odds of antigen blocking than dogs without these factors. Among dogs that were heartworm positive, those without microfilariae detected had greater odds of antigen blocking than dogs with this factor (OR = 11.84, p-value = 0.0005). Geographic region of origin was significantly associated with occurrence of antigen blocking (p = 0.0036); however, blocking occurred in all regions sizably contributing to heartworm diagnoses. Of the 74 dogs found to be infected with

  4. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    Directory of Open Access Journals (Sweden)

    Yuan-Cheng Cao

    2015-01-01

    Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

  5. Simultaneous detection of Hepatitis B surface antigen and its antibody by radioimmunoassay

    International Nuclear Information System (INIS)

    Crouzat-Reynes, Gerard; Perigois, Francois; Lecureuil, Michel; Lejeune, Bernard

    1981-01-01

    The authors describe an original radioimmunoassay which allows the simultaneous detection of hepatitis B surface antigen and its antibody in a biological sample. Antigen and antibody are indiscriminately detected in a first step and then distinguished in a second step using the same reagents [fr

  6. Clinicopathological features and immunohistochemical detection of antigens in acute experimental Streptococcus agalactiae infection in red tilapia (Oreochromis spp.).

    Science.gov (United States)

    Abdullah, Syuhaidah; Omar, Noraini; Yusoff, Sabri Mohd; Obukwho, Emikpe Benjamin; Nwunuji, Tanko Polycarp; Hanan, Latifah; Samad, Jamil

    2013-01-01

    This study investigates the clinicopathological features of acute experimental streptococcosis in red tilapia using various routes of infection; intraperitoneal (IP), immersion (IM) and immersion cut (IC). Twenty four red tilapia in duplicates were inoculated intraperitoneally with 10(9) CFU/ml of S. agalactiae while another sets: intact, one with sharp cut at the tail end were exposed to bacterial inoculums 10(9) CFU/ml diluted in water while two groups of control fish were similarly manipulated. Clinical signs were recorded; samples from the gills, brain, eyes and kidneys were also taken for bacterial isolation and histopathology. Immunohistochemistry (IHC) and polymerase chain reaction (PCR) were employed to detect the antigen. The diseased fish showed skin, fin haemorrhages and exophthalmia with obvious signs in IP at 2 hpc followed by IC and IM at 4 hpc. The lesions were noticed earlier in the kidney and most severe in IP. IHC detected antigen as early as PCR and isolation with intense staining in blood vessel lumen and wall, macrophages in choroid, focal haemorrhage in the renal interstitium and meninges especially in IP followed by IC and IM. The immunolocalisation of the antigen described for the first time further explain the pathogenesis of streptococcosis in red tilapia.

  7. Pre-treatment with heat facilitates detection of antigen of Dirofilaria immitis in canine samples.

    Science.gov (United States)

    Little, Susan E; Munzing, Candace; Heise, Steph R; Allen, Kelly E; Starkey, Lindsay A; Johnson, Eileen M; Meinkoth, James; Reichard, Mason V

    2014-06-16

    Diagnosis of Dirofilaria immitis infection in dogs is largely dependent on detection of antigen in canine serum, plasma, or whole blood, but antigen may be bound in immune complexes and thus not detected. To develop a model for antigen blocking, we mixed serum from a microfilaremic, antigen-positive dog with that of a hypergammaglobulinemic dog not currently infected with D. immitis and converted the positive sample to antigen-negative; detection of antigen was restored when the mixed sample was heat-treated, presumably due to disruption of antigen/antibody complexes. A blood sample was also evaluated from a dog that was microfilaremic and for which microfilariae were identified as D. immitis by morphologic examination. Antigen of D. immitis was not detected in this sample prior to heating but the sample was strongly positive after heat treatment of whole blood. Taken together, our results indicate that blood samples from some dogs may contain factors that inhibit detection of antigen of D. immitis, and that heat treatment of these samples prior to testing could improve the sensitivity of these assays in some patients. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Illuminating the detection chain of bacterial bioreporters

    NARCIS (Netherlands)

    Meer, J.R. van der; Tropel, D.; Jaspers, M.

    2004-01-01

    Engineering bacteria for measuring chemicals of environmental or toxicological concern (bioreporter bacteria) has grown slowly into a mature research area. Despite many potential advantages, current bioreporters do not perform well enough to comply with environmental detection standards. Basically,

  9. Impact of heat treatment on Dirofilaria immitis antigen detection in shelter dogs

    Directory of Open Access Journals (Sweden)

    Brian A. DiGangi

    2017-11-01

    Full Text Available Abstract Background The diagnosis and management of canine heartworm disease is a growing concern for shelter veterinarians. Although the accuracy of commercial antigen test kits has been widely studied, recent reports have renewed interest in antigen blocking as a causative factor for false “no antigen detected” results. The objectives of this study were to determine the prevalence of false “no antigen detected” results in adult dogs entering shelters in northern, southern, and western regions of the country and to identify historical and clinical risk factors for such results. Methods Serum samples were evaluated for Dirofilaria immitis antigen using a commercially available point-of-care ELISA; samples in which no antigen was detected underwent a heat treatment protocol and repeat antigen testing. Whole blood samples underwent Knott testing to identify the presence of microfilariae. Historical and clinical findings were analyzed using exact logistic regression. Results A total of 616 samples were analyzed. Overall prevalence of positive antigen test results (prior to heat treatment was 7.3% and frequency of false “no antigen detected” results due to antigen blocking (ie, samples with no antigen detected prior to heat treatment and positive after heat treatment was 5.2%. Among dogs that had no detectable antigen on the initial tests, dogs that had microfilariae detected via modified Knott testing (OR = 32.30, p-value = 0.013 and dogs that previously received a heartworm preventive (OR = 3.81, p-value = 0.016 had greater odds of antigen blocking than dogs without these factors. Among dogs that were heartworm positive, those without microfilariae detected had greater odds of antigen blocking than dogs with this factor (OR = 11.84, p-value = 0.0005. Geographic region of origin was significantly associated with occurrence of antigen blocking (p = 0.0036; however, blocking occurred in all regions sizably contributing to

  10. Non-labeled QCM Biosensor for Bacterial Detection using Carbohydrate and Lectin Recognitions

    Science.gov (United States)

    Shen, Zhihong; Huang, Mingchuan; Xiao, Caide; Zhang, Yun; Zeng, Xiangqun; Wang, Peng G.

    2008-01-01

    High percentages of harmful microbes or their secreting toxins bind to specific carbohydrate sequences on human cells at the recognition and attachment sites. A number of studies also show that lectins react with specific structures of bacteria and fungi. In this report, we take advantage of the fact that a high percentage of microorganisms have both carbohydrate and lectin binding pockets at their surface. We demonstrate here for the first time that a carbohydrate non-labeled mass sensor in combination with lectin-bacterial O-antigen recognition can be used for detection of high molecular weight bacterial targets with remarkably high sensitivity and specificity. A functional mannose self-assembled monolayer (SAM) in combination with lectin Con A was used as molecular recognition elements for the detection of E. coli W1485 using Quartz Crytsal Microbalance (QCM) as a transducer. The multivalent binding of Concanavalin A (Con A) to the Escherichia coli (E. coli) surface O-antigen favors the strong adhesion of E. coli to mannose modified QCM surface by forming bridges between these two. As a result, the contact area between cell and QCM surface increases that leads to rigid and strong attachment. Therefore it enhances the binding between E. coli and the mannose. Our results show a significant improvement of the sensitivity and specificity of carbohydrate QCM biosensor with a experimental detection limit of a few hundred bacterial cells. The linear range is from 7.5 × 102 to 7.5 × 107 cells/mL that is four decade wider than the mannose alone QCM sensor. The change of damping resistances for E. coli adhesion experiments was no more than 1.4% suggesting that the bacterial attachment was rigid, rather than a viscoelastic behavior. Little non-specific binding was observed for Staphylococcus aureus and other proteins (Fetal Bovine serum, Erythrina cristagalli lectin). Our approach not only overcomes the challenges of applying QCM technology for bacterial detection but

  11. Impact of heat treatment on antigen detection in sera of Angiostrongylus vasorum infected dogs

    OpenAIRE

    Gillis-Germitsch, Nina; Schnyder, Manuela

    2017-01-01

    BACKGROUND: In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vas...

  12. Direct testing of blood cultures for detection of streptococcal antigens.

    Science.gov (United States)

    Wetkowski, M A; Peterson, E M; de la Maza, L M

    1982-07-01

    A direct, rapid, and simple method for the detection of streptococcal antigens of Lancefield groups A, B, C, D, and G from blood cultures was developed by using a coagglutination test. Fifty-five clinical specimens and 117 simulated blood cultures containing gram-positive cocci were tested. Out of 6,261 clinical blood cultures screened, 55 cultures from 53 patients were positive, with organisms resembling streptococci, by Gram stain. Of these cultures, 78% (43 of 55) were pure cultures of streptococci, and 22% (12 of 55) were mixed with at least one other organism. Of the 43 pure cultures only, correct reactions were obtained (grouping correctly or giving no cross-reactions, or both) with 86% (37 of 43) of the isolates, 12% (5 of 43) exhibited cross-reactions, and 2% (1 of 43) gave false-negative reactions. All of the cross-reacting isolates were Streptococcus pneumoniae, which reacted with the group C reagent, and the false-negative reaction occurred with a Streptococcus bovis isolate. However, by using a direct modified bile solubility test, the correct identification of the S. pneumoniae isolates was obtained. Therefore, by using the modified bile solubility test in conjunction with the direct grouping method, 98% (42 of 43) of the isolates in pure culture could be identified accurately and rapidly after the detection of a positive Gram stain. Correct grouping reactions were obtained with 83% (10 of 12) of the mixed blood cultures, and false-negative results occurred with 17% (2 of 12) of them. Both cultures contained an enterococcus and a gram-negative rod. Of the 117 simulated blood cultures, there was only one incorrect grouping reaction; this occurred with an S. bovis isolate that cross-reacted with the group C reagent. The direct grouping reaction was positive when blood cultures contained a minimum of 1 x 10(8) to 8 x 10(8) colony-forming units per ml. In general, this procedure provided information on the identification of the organism 24 h earlier than

  13. DETECTION OF PNEUMOCOCCAL CAPSULAR ANTIGEN IN THE PRESENCE OF PENICILLIN IN-VITRO

    NARCIS (Netherlands)

    HOLLOWAY, Y; BOERSMA, WG; KUTTSCHRUTTER, H; SNIJDER, JAM

    1993-01-01

    Eight strains of Streptococcus pneumoniae were tested in vitro for their ability to produce capsular antigen in the presence of penicillin. It was found that, provided 10(6) to 10(7) pneumococci/ml were present, capsular antigen could be detected during the 72 h in which the experiment was

  14. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Dileep Tiwari

    2017-04-01

    Conclusion: Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application.

  15. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  16. A Printed Multicomponent Paper Sensor for Bacterial Detection.

    Science.gov (United States)

    Ali, M Monsur; Brown, Christine L; Jahanshahi-Anbuhi, Sana; Kannan, Balamurali; Li, Yingfu; Filipe, Carlos D M; Brennan, John D

    2017-09-26

    We present a simple all-in-one paper-based sensor for E. coli detection using a composite ink made of a fluorogenic DNAzyme probe for bacterial recognition and signal generation, lysozyme that lyses whole bacterial cells, and pullulan/trehalose sugars that stabilize printed bioactive molecules. The paper sensor is capable of producing a fluorescence signal as a readout within 5 minutes upon contacting E. coli, can achieve a limit of detection of 100 cells/mL, in a variety of sample matrixes, without sample enrichment, and remains stable for at least 6 months when stored at ambient temperature. Therefore, this simple paper sensor provides rapid bacterial testing on site, and can be shipped and stored under ambient conditions to benefit users living in resource-limited regions.

  17. Increased detection of Dirofilaria immitis antigen in cats after heat pretreatment of samples.

    Science.gov (United States)

    Gruntmeir, Jeff M; Adolph, Chris B; Thomas, Jennifer E; Reichard, Mason V; Blagburn, Byron L; Little, Susan E

    2017-10-01

    Objectives To determine whether pretreating diagnostic samples with heat increases the detection of Dirofilaria immitis antigen in adult cats, we evaluated feline serum and plasma samples collected in heartworm-endemic areas of the southern United States. Methods Commercial microtiter well assays for detection of D immitis antigen were used to evaluate serum or plasma samples from 385 shelter and free-roaming cats from the southcentral and southeastern United States before and after heat treatment; commercial antibody tests were performed on a subset of samples. Results Prior to sample heat treatment, 1/220 (0.5%) shelter cats and 4/165 (2.4%) free-roaming cats had detectable D immitis antigen. After heat pretreatment, the detection rate increased to 13/220 (5.9%) and 13/165 (7.9%), respectively. Antibody reactive to D immitis was significantly more common ( P cats that were antigen positive after heat treatment (10/13; 76.9%) than serum from cats that remained antigen negative after heat treatment (22/163; 13.5%). Conclusions and relevance Heat pretreatment of feline samples increased antigen detection by commercial assays for D immitis and improved overall concordance of antigen and antibody test results in antigen-positive samples in this population. Although further work to investigate the specificity of D immitis antigen assays when using pre-treated samples is warranted, this approach may be useful in the diagnosis of heartworm infection in individual cats and may increase the accuracy of surveys based on antigen detection.

  18. Detection of chlamydial antigenic material in ovarian, prostatic, ectopic pregnancy and semen samples of culture-negative subjects.

    Science.gov (United States)

    Toth, M; Patton, D L; Campbell, L A; Carretta, E I; Mouradian, J; Toth, A; Shevchuk, M; Baergen, R; Ledger, W

    2000-04-01

    The pathogenesis of long-term sequelae in Chlamydia trachomatis infection is poorly understood. While serology indicates previous chlamydial infection, culture studies are frequently negative. We wanted to know whether in chronic cases the bacterium is absent or persists in a dormant state where it evades detection. Using immunoperoxidase (IP) staining and in situ hybridization (ISH), we examined tissues of culture-negative subjects. Ovarian biopsy specimens from 19 culture-negative women with pelvic adhesions and/or tubal infertility were analyzed by both methods. Samples of prostates from 10 culture-negative men undergoing prostatectomy for benign hypertrophy, two sets of semen samples from culture-negative sexual partners of 28 women with PID and/or bacterial vaginosis (BV), and ten endometrium-tube sample-pairs from ectopic pregnancies (EPs) were examined by IP only. Seven of the nineteen ovarian specimens tested positive for Chlamydia antigen or deoxyribonucleic acid (DNA) (36%). Of the 10 hypertrophic prostates examined, 4 (40%) were positive. Of the 28 semen samples examined, 10 (35%) tested positive. Tissue samples of 3 cases of EP were positive by IP. 1. C. trachomatis antigen and nucleic acid can be frequently demonstrated in asymptomatic, culture-negative men and women with chronic infection. 2. Chlamydia antigens may have an etiologic role in benign prostate hypertrophy and EP. 3. Antigenic material may be sexually transmissible. 4. IP and ISH identify temporarily inactive bacteria that may continue to act as immunostimulants and potentially reactivate as Chlamydia infection.

  19. Molecular analysis of cross-bacterial contamination detected in ...

    African Journals Online (AJOL)

    ... the isolate Delftia acidovorans BP(R2) and it is also coupled to protein with molecular weight 25-26 KDa. As well as, this bacterial contamination was the reason for the false positive results observed during the detection of HCV infections. Journal of Applied Sciences and Environmental Management Vol. 9(1) 2005: 5-10.

  20. Automated alarm to detect antigen excess in serum free immunoglobulin light chain kappa and lambda assays.

    Science.gov (United States)

    Urdal, Petter; Amundsen, Erik K; Toska, Karin; Klingenberg, Olav

    2014-10-01

    Antigen excess causing a falsely low concentration result may occur when measuring serum free immunoglobulin light chains (SFLC). Automated antigen excess detection methods are available only with some analyzers. We have now developed and verified such a method. Residuals of sera with known SFLC-κ and -λ concentrations were analyzed using Binding Site reagents and methods adapted to the Roche Cobas® c.501 analyzer. We analyzed 117 sera for SFLC-κ and -λ and examined how the absorbance increased with time during the 7 minutes of reaction (absorbance reading points 12-70). From this an antigen excess alarm factor (ratio of absorbance increases between reading points 68-60 and 20-12, multiplied by 100) was defined. Upon our request, Roche added to our two SFLC assays a program which calculated this antigen excess alarm factor and triggered an alarm when the factor was below a defined value. We verified this antigen excess alarm function by analyzing serum from 325 persons of whom 143 were multiple myeloma patients. All samples with a known concentration above 30 mg/L triggered either an antigen excess alarm, an 'above test' alarm or both. Also, all samples above 200 mg/L (SFLC-λ) and 300 mg/L (SFLC-κ) triggered the antigen excess alarm and all but one triggered the above test alarm. The antigen excess alarm function presented here detected all known antigen excess samples at no increased time of analysis, a reduced workload and reduced reagent cost.

  1. Carcinoembryonic Antigen Level in Primary Sclerosing Cholangitis Is Not Influenced by Dominant Strictures or Bacterial Cholangitis.

    Science.gov (United States)

    Wannhoff, Andreas; Rupp, Christian; Friedrich, Kilian; Knierim, Johannes; Flechtenmacher, Christa; Weiss, Karl Heinz; Stremmel, Wolfgang; Gotthardt, Daniel N

    2017-02-01

    Carcinoembryonic antigen (CEA) can be used to screen for biliary tract cancer in patients with primary sclerosing cholangitis (PSC). To study the influence of benign dominant strictures (DS), superimposed bacterial cholangitis (SBC), smoking status, and inflammatory bowel disease on CEA serum levels. A retrospective analysis of CEA values in cancer-free PSC patients was performed. We included the maximal CEA value obtained during follow-up and information on the presence of DS and SBC at that time, and we analyzed the CEA values in the presence and absence of DS and SBC. Results are reported as medians with the interquartile range (IQR). The median maximal CEA level, which was 1.8 ng/mL (IQR 1.2-2.9) in the final 270 PSC patients included in the study, was not influenced by the presence of either DS or SBC (P = 0.320). Moreover, in 49 patients, the first CEA value available at the time of DS (1.5 ng/mL; IQR 1.2-2.1) and that at a time without DS (1.6 ng/mL; IQR 1.1-2.3) did not differ significantly (P = 0.397). Lastly, in 24 patients, the median CEA values at a time without SBC (1.8 ng/mL; IQR 1.2-2.5) and at the time of SBC (1.8 ng/mL; IQR 1.0-3.0) were comparable (P = 0.305). Smoking did not influence CEA-based cancer screening. Serum CEA level is not influenced by the presence of DS or SBC and might therefore serve as a favorable parameter for improving cancer screening in PSC patients.

  2. HLA-DR antigen detection in giant cell lesions.

    Science.gov (United States)

    Regezi, J A; Zarbo, R J; Lloyd, R V

    1986-09-01

    Sixty-six giant cell lesions ranging from inflammatory to neoplastic were evaluated for HLA-DR antigens using formalin/paraffin tissue and a monoclonal antibody labelled by the avidin-biotin peroxidase. HLA-DR antigens were expressed in nearly all lesions, predominantly on round, macrophage-like cells. Granulomatous inflammatory lesions were generally more immunoreactive than non-inflammatory lesions. Multinucleate giant cells were relatively unreactive in non-inflammatory lesions as compared to inflammatory lesions. Determination of HLA-DR expression does not appear to be helpful in discriminating between the various giant cell lesions.

  3. Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species

    Directory of Open Access Journals (Sweden)

    Stone Joshua K

    2012-11-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. Results PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. Conclusions This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.

  4. PNEUMOCOCCAL CAPSULAR ANTIGEN-DETECTION AND PNEUMOCOCCAL SEROLOGY IN PATIENTS WITH COMMUNITY ACQUIRED PNEUMONIA

    NARCIS (Netherlands)

    BOERSMA, WG; LOWENBERG, A; HOLLOWAY, Y; KUTTSCHRUTTER, H; SNIJDER, JAM; KOETER, GH

    1991-01-01

    Background Methods to determine the microbial cause of community acquired pneumonia include detection of pneumococcal antigen and measurement of pneumococcal capsular antibody response. Their usefulness compared with conventional microbiological techniques was investigated in patients with

  5. Detection of serum prostate specific antigen in lactating, pregnant ...

    African Journals Online (AJOL)

    Introduction: Although prostate-specific antigen (PSA) is the most valuable tumor marker for the diagnosis and management of prostate carcinoma, it is widely accepted that PSA is not prostate specific. Objectives: The aim of this study is to address the possibility of using the PSA as marker for the sex assignment in different ...

  6. Detection of non-jejuni and -coli Campylobacter species from stool specimens with an immunochromatographic antigen detection assay.

    Science.gov (United States)

    Couturier, Brianne A; Couturier, Marc Roger; Kalp, Kim J; Fisher, Mark A

    2013-06-01

    The STAT! Campy immunochromatographic assay for Campylobacter antigen was compared to culture for 500 clinical stool specimens. Antigen was detected in six culture-negative, PCR-positive specimens. C. upsaliensis, a pathogenic species that is traditionally difficult to recover in routine stool cultures, was detected in two of these culture-negative specimens. This study provides evidence that antigen testing may cross-react with at least one additional non-jejuni and -coli Campylobacter species that may be missed by routine culture for campylobacteriosis.

  7. Detection of filarial antigen and antibody in serum and hydrocele fluid of 100 patients of hydrocele.

    Science.gov (United States)

    Goel, Ravishankar S; Verma, Nimesh S; Mullan, Sumaiya A; Ashdown, Andrew C

    2006-05-01

    The present study was carried out to detect an association between isolated non-communicable hydrocoele and filariasis and to provide awareness to positive patients regarding sequel and advising methods for the reduction of morbidity. Blood samples and hydrocoele fluids were used to detect filarial antigen and antibody by ICT Kit, Trop-bio kit and Sevafilachek Kit. These were followed by statistical evaluation by chi2 test. 14% of cases were positive for filarial antigen and antibody in hydrocoele patient serum, while 15% of cases were positive for filarial antigen and antibody in the serum of non-hydrocoele patients. Probability is less than 0.05, which is statistically significant.

  8. Improved Diagnosis of Acute Pulmonary Histoplasmosis by Combining Antigen and Antibody Detection.

    Science.gov (United States)

    Richer, Sarah M; Smedema, Melinda L; Durkin, Michelle M; Herman, Katie M; Hage, Chadi A; Fuller, Deanna; Wheat, L Joseph

    2016-04-01

    Acute pulmonary histoplasmosis can be severe, especially following heavy inoculum exposure. Rapid diagnosis is critical and often possible by detection of antigen, but this test may be falsely negative in 17% of such cases. Antibody detection by enzyme immunoassay (EIA) may increase sensitivity and permit the measurement of immunoglobulin M (IgM) and immunoglobulin G (IgG) classes of antibodies separately. Microplates coated with Histoplasma antigen were used for testing of serum from patients with acute pulmonary histoplasmosis and controls in the MVista Histoplasma antibody EIA. Results for IgG and IgM were reported independently. IgG antibodies were detected in 87.5%, IgM antibodies in 67.5%, and IgG and/or IgM antibodies in 88.8% of patients with acute pulmonary histoplasmosis in this assay, while immunodiffusion, complement fixation, and antigen testing showed sensitivities of 55.0%, 73.1%, and 67.5%, respectively (n = 80). Combining antigen and antibody detection increased the sensitivity to 96.3%. The MVista Histoplasma antibody EIA offers increased sensitivity over current antibody tests while also allowing separate detection of IgG and IgM antibodies and complementing antigen detection. Combining antigen and EIA antibody testing provides an optimal method for diagnosis of acute pulmonary histoplasmosis. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

  9. Detection of Bacterial Endospores in Soil by Terbium Fluorescence

    Directory of Open Access Journals (Sweden)

    Andrea Brandes Ammann

    2011-01-01

    Full Text Available Spore formation is a survival mechanism of microorganisms when facing unfavorable environmental conditions resulting in “dormant” states. We investigated the occurrence of bacterial endospores in soils from various locations including grasslands (pasture, meadow, allotment gardens, and forests, as well as fluvial sediments. Bacterial spores are characterized by their high content of dipicolinic acid (DPA. In the presence of terbium, DPA forms a complex showing a distinctive photoluminescence spectrum. DPA was released from soil by microwaving or autoclaving. The addition of aluminium chloride reduced signal quenching by interfering compounds such as phosphate. The highest spore content (up to 109 spores per gram of dry soil was found in grassland soils. Spore content is related to soil type, to soil depth, and to soil carbon-to-nitrogen ratio. Our study might provide a basis for the detection of “hot spots” of bacterial spores in soil.

  10. Diagnosis of bacterial infection

    African Journals Online (AJOL)

    rapid and easy-to-use test for bacterial infections. Clearly, this is a very ... detect antigens or specific antibodies, e.g. group A streptococcal antigen testing can be employed to reduce antibiotic use. Culture-based tests are often ... White blood cell count 12 000 cells/mm³; or the presence of >10% ...

  11. Detection of HBs antigens and antibodies by immunoenzymology and radioimmunology. Comparison of the results

    International Nuclear Information System (INIS)

    Fauchet, R.; Genetet, B.; Phengsavath

    1981-01-01

    The respective sensitivity threshold of immunoenzymology and radioimmunology in HBs antigen and antibody detection were compared and the optical density (o.d.) and counts per minute (cpm) values were correlated with the concentration in ng/ml of a given sample. The sensitivity comparison between RIA and EIA appears in favour of the former technique for both HBs antigen and HBs antibody detection. However the procedure tried here to detect HBs antibodies by immunoenzymology is simple, requires only the choice of neutralising antigenic pool and sensitivity threshold and could usefully be generalized for teams not authorized to handle radioelements. Quantification of the HBs antigen content in ng/ml is a difficult operation, needing great care in the dilution of the sera tested. The reproducibility is not perfect. The semi-quantitative RIA determination expressed in R.U. (radioimmunological units) seems adequate as a mean to follow the progress of an HBs antibody in both liver disease and vaccination [fr

  12. [Detection of Chlamydia trachomatis antigen in cases of cervicitis and patients with vaginal discharge].

    Science.gov (United States)

    Aksoy, A M

    1993-10-01

    This study was carried on 129 women of 16-62 age group, with complaints of vaginal discharge, genital itching and soreness, dysuria and pollakiuria. Endocervical specimens were investigated for Chlamydia trachomatis (C. trachomatis) antigen by ELISA. Risk factors for several gynecologic and obstetric pathologies and the role of C. trachomatis in mucopurulent cervicitis were emphasized. C. trachomatis antigen was found to be positive in 9 (7%) specimens. We concluded that, in cases of cervicitis, especially to prevent complications and social problems, the presence of C. trachomatis should also be investigated in addition to several viral, bacterial and fungal agents.

  13. An improved gold nanoparticle probe-based assay for HCV core antigen ultrasensitive detection.

    Science.gov (United States)

    Yin, Hui-Qiong; Ji, Chang-Fu; Yang, Xi-Qin; Wang, Rui; Yang, Shu; Zhang, He-Qiu; Zhang, Jin-Gang

    2017-05-01

    A gold nanoparticle probe-based assay (GNPA) was developed for ultrasensitive detection of Hepatitis C virus (HCV) core antigen. In the GNPA, after anti-HCV core antigen polyclonal antibodies and single-stranded barcode signal DNA were labeled on gold nanoparticle probe (NP), DNA enzyme was used to degrade the unbound barcode DNAs. The anti-HCV core antigen monoclonal antibodies were coated on magnetic microparticles probe (MMP). Then the NP-HCV core antigen-MMP sandwich immuno-complex was formed when the target antigen protein was added and captured. Magnetically separated, the immuno-complex containing the single-stranded barcode signal DNA was characterized by TaqMan probe based real-time fluorescence PCR. A detection limit of 1 fg/ml was determined for the HCV core antigen which is magnitude greater than that of ELISA (2ng/ml). The coefficients of variation (CV) of intra-assay and inter-assay respectively ranged from 0.22-2.62% and 1.92-3.01%. The improved GNPA decreased the interference of unbound barcode DNAs and may be an new way for HCV core antigen detection. Copyright © 2017. Published by Elsevier B.V.

  14. Detection of antibiotic resistance in clinical bacterial strains from pets

    OpenAIRE

    Poeta, P.; Rodrigues, J.

    2008-01-01

    The identification of different bacterial strains and the occurrence of antibiotic resistance were investigated in several infection processes of pets as skin abscess with purulent discharge, bronco alveolar fluid, earwax, urine, mammary, and eye fluid. Streptococcus spp. and Staphylococcus spp. were the most detected in the different samples. A high frequency of antimicrobial resistance has been observed and this could reflect the wide use of antimicrobials in pets, making the effectiveness ...

  15. Development and Evaluation of a Rapid Antigen Detection and Serotyping Lateral Flow Antigen Detection System for Foot-and-Mouth Disease Virus.

    Directory of Open Access Journals (Sweden)

    Kazuki Morioka

    Full Text Available We developed a lateral flow strip using monoclonal antibodies (MAbs which allows for rapid antigen detection and serotyping of foot-and-mouth disease virus (FMDV. This FMDV serotyping strip was able to detect all 7 serotypes and distinguish serotypes O, A, C and Asia1. Its sensitivities ranged from 10(3 to 10(4 of a 50% tissue culture infectious dose of each FMDV stain; this is equal to those of the commercial product Svanodip (Boehringer Ingelheim Svanova, Uppsala, Sweden, which can detect all seven serotypes of FMDV, but does not distinguish them. Our evaluation of the FMDV serotyping strip using a total of 118 clinical samples (vesicular fluids, vesicular epithelial emulsions and oral and/or nasal swabs showed highly sensitive antigen detection and accuracy in serotyping in accordance with ELISA or RT-PCR. To the best of our knowledge, this is the first report on any FMDV serotyping strip that provides both rapid antigen detection and serotyping of FMDV at the same time on one strip without extra devices. This method will be useful in both FMD-free countries and FMD-infected countries, especially where laboratory diagnosis cannot be carried out.

  16. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    Science.gov (United States)

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Detection of toxoplasma gondii antigens in sera from experimentally infected mice

    International Nuclear Information System (INIS)

    Shojaee, S.; Keshavarz, H.; Rezaian, M.; Mohebali, M.

    2007-01-01

    Detection of Toxoplasma antigen in serum of mice by Immunoblotting. strain. IgG isolated from rabbits that were immunized with T. gondii Immunoblotting was performed to detect T. gondii antigens in sera of mice. Serum samples from mice experimentally infected with T. gondii RH strain. The value of Immunoblotting in diagnosis of toxoplasmosis in acute stage of infection. The antigen bands detected in serum sample of mice were experimentally infected with T. gondii tachyzoite in immunoblotting. Six bands demonstrated on seventh post infection day six bands were identified. Similarly on sixth day four bands, on day five three bands and on fourth post infection day two bands were identified. No band was detected in control group sera. Immunoblotting is a sensitive method for diagnosis of acute stage of toxoplasmosis. (author)

  18. Diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infections using antigen detection methods.

    Science.gov (United States)

    Stamm, W E

    1986-03-01

    Rapid antigen detection methods have great potential value in managing sexually transmitted gonococcal and chlamydial infections. Ideally, such tests should be rapid, technically simple, inexpensive, accurate, and applicable to all sites of infection commonly sampled (cervix, urethra, pharynx). For gonorrhea, the Gram stain fulfills these criteria in men with symptomatic urethritis, but lacks sensitivity when used at other sites or in asymptomatic patients. Antigen detection for gonorrhea would thus be of greatest value in 1) the diagnosis of gonococcal cervical infections in women with mucopurulent cervicitis or pelvic inflammatory disease, 2) the diagnosis of gonococcal proctitis in homosexual men, and 3) in situations requiring lengthy specimen transport. Because culture confirmation of Chlamydia trachomatis infections is not widely available, antigen detection tests could be of great value in management of these infections. Major uses include 1) confirming infection in women with cervicitis, endometritis, and pelvic inflammatory disease; 2) screening for asymptomatic infections in high risk groups of women; and 3) confirmation of Chlamydia trachomatis infections in infants and in adult males. The currently available methods for diagnosis of gonococcal and chlamydial infection by antigen detection are reviewed herein. Continued experience with antigen detection tests in well defined populations having high and low risk of gonococcal and chlamydial infection is needed to more fully determine how best to utilize these assays.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-05-01

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

  20. Radioimmunoassay for detecting antibodies against murine malarial parasite antigens: monoclonal antibodies recognizing Plasmodium yoelii antigens

    International Nuclear Information System (INIS)

    Kim, K.J.; Taylor, D.W.; Evans, C.B.; Asofsky, R.

    1980-01-01

    A solid-phase radioimmunoassay (SPRIA) in microtiter wells was established for detecting antibodies against Plasmodium yoelii Ag. The SPRIA was found (1) to require as little as 5 μg of crude parasite Ag per well, (2) to be able to detect 0.5 ng of monoclonal Ab, and (3) to be 10 4 times more sensitive than the indirect fluorescent Ab staining technique. In a modification of the above assay using intact RBC as an Ag, hyperimmune serum showed significant binding to the surface of erythrocytes of mice infected with P. yoelii parasites but not to RBC of normal mice. Hybridomas were prepared by fusing infected mouse spleen cells with myeloma cells. Using the SPRIA, hybrids secreting Ab against P. yoelii 17XL Ag were detected

  1. Nucleic acid detection technologies and marker molecules in bacterial diagnostics.

    Science.gov (United States)

    Scheler, Ott; Glynn, Barry; Kurg, Ants

    2014-05-01

    There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

  2. Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

    Science.gov (United States)

    Singh, Gulshan; Manohar, Murli; Adegoke, Anthony Ayodeji; Stenström, Thor Axel; Shanker, Rishi

    2017-01-01

    The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1-100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

  3. Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Gulshan, E-mail: gsingh.gulshan@gmail.com [Durban University of Technology, Institute for Water and Wastewater Technology (IWWT) (South Africa); Manohar, Murli [Jamia Hamdard (Hamdard University), Department of Biochemistry (India); Adegoke, Anthony Ayodeji; Stenström, Thor Axel [Durban University of Technology, Institute for Water and Wastewater Technology (IWWT) (South Africa); Shanker, Rishi [Ahmedabad University, Division of Biological & Life Sciences, School of Arts & Sciences (India)

    2017-01-15

    The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1–100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

  4. Comparative Evaluation of Native Antigens for the Development of Brucellosis Antibody Detection System

    Directory of Open Access Journals (Sweden)

    Yasmin Bano

    2015-09-01

    Full Text Available Brucellosis is a highly infectious zoonotic disease and an economically important infection of humans and livestock with a worldwide distribution. The main mode of transmission of this disease to humans is through the consumption of infected milk, milk products, and uncooked or raw meat. The present study was designed to prepare few native antigens, that is, sonicated antigen (SA, cell envelope (CE antigen, and freeze and thaw (FT antigen from Brucella abortus S99 culture and to test them in a highly sensitive and specific indirect enzyme-linked immunosorbent assay (I-ELISA in both a microtiter plate and a dot-blot format for the development of field-based diagnosis. All 50 suspected bovine samples were tested by plate as well as in dot ELISA formats for all the three antigens prepared. The CE antigen was found to be more suitable as it had the maximum agreement with the Rose Bengal plate agglutination test results followed by the SA and the least agreement was found with that of the FT antigen. This detection system in microtiter plates and a dot-blot format will be useful for the rapid screening of samples for the disease surveillance and routine diagnosis.

  5. Impact of heat treatment on antigen detection in sera of Angiostrongylus vasorum infected dogs.

    Science.gov (United States)

    Gillis-Germitsch, Nina; Schnyder, Manuela

    2017-09-16

    In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vasorum antigen in sera of experimentally (n = 21, 119 follow-up sera) and naturally (n = 18) infected animals. In addition, sera of dogs showing clinical signs consistent with angiostrongylosis (n = 10), of randomly selected dogs (n = 58) and of dogs with other parasitic infections (n = 15) were evaluated. Sera were subjected to heat treatment at 100 °C after addition of 0.5 M EDTA (dilution 1:5) and tested with ELISAs for detection of circulating A. vasorum antigen before and after treatment. Between 5 and 11 weeks post-inoculation (wpi) the percentage of positive untreated samples (experimentally infected dogs) increased over time from 33.3 to 90%. Single samples were still negative between 12 and 15 wpi. Overall, between 5 and 15 wpi, 50.6% (45/89) of the available samples were seropositive. From 3 to 6 wpi EDTA/heat treatment caused a change in 8/34 (23.5%) of the samples, with most (n = 6, 17.6%) converting from positive to negative. In contrast, from 7 to 10 wpi, treatment induced a change in 19/52 (36.5%) samples, with all but one converting from negative to positive. Thirteen of 18 naturally infected dogs were antigen positive before and 15 after EDTA/heat treatment, respectively. Untreated samples of 3 dogs with suspected angiostrongylosis were antigen positive, of which only one remained positive after EDTA/heat treatment. One of 58 untreated random samples was antigen positive; this sample became negative after treatment, while another turned positive. One of 15

  6. Dengue NS1 Antigen - for Early Detection of Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Amol Hartalkar

    2015-08-01

    Full Text Available Objectives: To evaluate the efficacy of NS1 antigen assay for early diagnosis of dengue virus infection in a tertiary care hospital. Methods: This cross sectional study was carried out in department of Medicine from August to December 2013. Total 100 patients with dengue fever were included. Complete blood count, alanine aminotransferase (ALT, aspartate aminotransferase (AST, Dengue NS1 antigen and IgM and IgG antibodies of dengue virus were done in all cases. Results: Of the 100 sera tested, 75% were positive for dengue virus infection based on dengue NS1 antigen, IgM antibody and IgG antibody. Dengue NS1 antigen and IgM, IgG antibody were able to detect dengue virus infection between day 1 to day 8 in 92% of samples, 86.7% of samples and 82.6% of samples respectively. Sixty nine percent (69% were found positive for dengue NS1 antigen, 65% were IgM positive and 62% were IgG positive. Based on the dengue NS1 antigen and IgM antibody combination, 74% were positive for dengue virus infections. Sensitivity of Dengue NS1 antigen was 92.3% and specificity of 74.28% in comparison to IgM antibody. Detection rate increased to 75%, based on the antigen and IgG antibody combination. Sensitivity of dengue NS1 antigen was 90.3% and specificity of 65.8% in comparison to IgG antibody. Conclusion: Dengue NS1 antigen is a useful, sensitive and specific test for early diagnosis of dengue virus infection and it improves diagnostic efficiency in combination with antibody test. Key words: Dengue fever, NS1 antigen. Introduction: Dengue fever (DF is the most common arboviral illness in humans. Each year, an estimated 50-100 million cases of dengue fever and 500,000 cases of dengue hemorrhagic fever occur worldwide, with 30000 deaths (mainly in children. Globally 2.5-3 billion people in approximately 112 tropical and subtropical countries are at risk of dengue.of samples respectively. Sixty nine percent (69% were found positive for dengue NS1 antigen, 65% were Ig

  7. Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Han, Xiangan; Ding, Chan; Wang, Shaohui; Peng, Daxin

    2014-01-01

    We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants. PMID:24752516

  8. Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

    Science.gov (United States)

    Maurer, John J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, Susan; Bolton, Lance; Lee, Margie D.

    1999-01-01

    The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157. PMID:10388689

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

    Directory of Open Access Journals (Sweden)

    Anak Agung Ayu Mirah Adi

    2013-07-01

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

  10. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  11. Usefulness of pneumococcal antigen detection in pleural fluid samples by immunochromatographic assay for diagnosis of pneumococcal pneumonia.

    Science.gov (United States)

    Andreo, F; Domínguez, J; Ruiz-Manzano, J; Prat, C; Blanco, S; Lores, L; Sánchez, M D; Latorre, I; Giménez, M; Ausina, V

    2006-07-01

    This study investigated the utility of an immunochromatographic test (ICT) for the detection of Streptococcus pneumoniae antigens in pleural fluid. Antigen was detected in 15 of 19 (79%) patients with pneumococcal pneumonia. The ICT was always negative in patients with non-pneumococcal pneumonia, but was positive in three cases with a non-infectious aetiology. In patients with pneumonia for which no pathogen was identified, antigen was detected in one of 24 pleural fluids tested. The ICT can be a valuable tool for the management of pneumonia because it can detect pneumococcal antigen in pleural effusion samples.

  12. Smart phone based bacterial detection using bio functionalized fluorescent nanoparticles

    International Nuclear Information System (INIS)

    Rajendran, Vinoth Kumar; Bakthavathsalam, Padmavathy; Ali, Baquir Mohammed Jaffar

    2014-01-01

    We are describing immunochromatographic test strips with smart phone-based fluorescence readout. They are intended for use in the detection of the foodborne bacterial pathogens Salmonella spp. and Escherichia coli O157. Silica nanoparticles (SiNPs) were doped with FITC and Ru(bpy), conjugated to the respective antibodies, and then used in a conventional lateral flow immunoassay (LFIA). Fluorescence was recorded by inserting the nitrocellulose strip into a smart phone-based fluorimeter consisting of a light weight (40 g) optical module containing an LED light source, a fluorescence filter set and a lens attached to the integrated camera of the cell phone in order to acquire high-resolution fluorescence images. The images were analysed by exploiting the quick image processing application of the cell phone and enable the detection of pathogens within few minutes. This LFIA is capable of detecting pathogens in concentrations as low as 10 5 cfu mL −1 directly from test samples without pre-enrichment. The detection is one order of magnitude better compared to gold nanoparticle-based LFIAs under similar condition. The successful combination of fluorescent nanoparticle-based pathogen detection by LFIAs with a smart phone-based detection platform has resulted in a portable device with improved diagnosis features and having potential application in diagnostics and environmental monitoring. (author)

  13. A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals.

    Science.gov (United States)

    Ulrich, Sophia Arlena; Lehnert, Kristina; Siebert, Ursula; Strube, Christina

    2015-09-02

    Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well. For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed. Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100% (95% CI: 87.23-100%) and a sensitivity of 97.83% (95% CI: 88.47-99.94%). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies

  14. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Kvistborg, Pia; Frøsig, Thomas Mørch

    2012-01-01

    Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specif......(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.......Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen......-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two...

  15. ELISA for the detection of venom antigens in experimental and clinical envenoming by Loxosceles intermedia spiders.

    Science.gov (United States)

    Chávez-Olórtegui, C; Zanetti, V C; Ferreira, A P; Minozzo, J C; Mangili, O C; Gubert, I C

    1998-04-01

    Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Loxosceles intermedia spider venom. Hyperimmune horse anti-Loxosceles intermedia IgGs were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate the circulating antigens in mice that were experimentally inoculated with L. intermedia venom from those inoculated with L. gaucho, L. laeta, and Phoneutria nigriventer spider venoms, Tityus serrulatus scorpion venom and Bothrops jararaca, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.8 ng of venom per assay. The ELISA also detected antigens in the sera of patients envenomed by L. intermedia. Therefore, after standardization for clinical use this ELISA may be a valuable tool for clinicians and epidemiologists.

  16. Detection of avian influenza antibodies and antigens in poultry and ...

    African Journals Online (AJOL)

    Using HI test, the wild birds were negative for AI (H5) antibodies but ELISA detected AI (NP) antibodies in Black Stork (Ciconia nigra) with an overall seroprevalence of 4.5% and mean titre of 24.50±2.400 EU. Cloacal swabs from the same species of wild birds that were tested for antibodies and 710 oropharyngeal swabs ...

  17. Detection of antigens of allergic diseases in children by radioallergosorbent test (RAST), 1

    International Nuclear Information System (INIS)

    Hirooka, Junko

    1977-01-01

    Detection of antigens mainly by RAST, measurement of immunoglobulin, and investigation of clinical history were performed on children with intractable bronchial asthma. The results were compared with those in cases of mild or moderate degree, and they were discussed. The obtained results were as follows: 1) Cases, of which the occurrence age of disease was under 2 years old, hold a majority in intractable cases, and the ratio was twice that of the control group. 2) A result of skin test was generally lower positive rate in the intractable group as compared to the control group. However, a result of prick test for buckwheat antigen in the intractable group showed higher positive rate than that in the control group. The intractable group tended to be separated into two extreme groups, one which showed positive to most of inhaled antigens in skin test, and another which showed negative to all antigens. 3) As a result of RAST, 13% of the intractable group showed positive to egg white out of food antigens, and it was three times the ratio of positive in the control group. One case showed strong positive to rice. 4) Two thirds of cases which showed positive in RAST for food antigens showed negative in prick test. 5) Total IgE in the serum of the intractable group was clearly lower in values than that of the control group. (Tsunoda, M.)

  18. Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization

    International Nuclear Information System (INIS)

    Horenstein, A.L.; Feinstein, R.E.

    1985-01-01

    Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody. (orig.)

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    plates and avoids cross-reactivity due to conserved domains in the lipid A. Furthermore, the covalent binding of the polysaccharide antigens are compatible with harsh assay conditions, such as extensive washing procedures and buffers with high salt concentrations with no risk of antigen leakage. Here we......In this study we demonstrate a new UV irradiation technique for covalent coupling of bacterial polysaccharides derived from lipopolysaccharides to microtiter plates and the use of such plates in an enzyme linked immunosorbent assay (ELISA). Lipopolysaccharides were cleaved by mild acid hydrolysis...... into the lipid A part and the polysaccharide part. The polysaccharide was conjugated regiospecifically to a photochemically active compound, anthraquinone, resulting in a polysaccharide-anthraquinone conjugate. Anthraquinones forms active radicals when exposed to soft UV irradiation (350 nm) permitting...

  20. Detection of antibodies to Helicobacter pylori cell surface antigens.

    Science.gov (United States)

    Guruge, J L; Schalén, C; Nilsson, I; Ljungh, A; Tyszkiewicz, T; Wikander, M; Wadström, T

    1990-01-01

    Serum IgG antibodies of Helicobacter pylori were detected in single-dilution ELISA using glycine extracted material. Among 148 endoscopy patients 59% displayed antibodies; as expected, a higher occurrence (90%) was found in patients with positive gastric culture for H. pylori than in culture negative patients (37%). Among 68 blood donors the frequency of H. pylori antibodies was 28%. In 73 children less than 15 years of age examined for unrelated disorders the occurrence was 4%. By immunoblotting using the same extract, 3 prominent bands, 29K, 54K and 60K and several weak bands were identified. These were formed by 57%, 92%, and 65%, respectively, of the ELISA positive patient sera. Comparing culture positive and negative patients, the 3 bands occurred more often among the culture positive subjects though between 18 and 61% of the sera from culture negative patients gave either of the bands. When comparing the glycine extracts of 4 different H. pylori strains with separate haemagglutinating patterns no differences in the position of the major bands emerged. By absorption experiments no immunological cross-reactivity with components of Escherichia coli, Klebsiella pneumoniae, Campylobacter jejuni or C. fetus was found. Thus, the glycine extract seemed specific for the detection of antibodies to H. pylori.

  1. Picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface-enhanced Raman spectroscopy.

    Science.gov (United States)

    Zou, Kun; Gao, Zhigang; Deng, Quanfeng; Luo, Yong; Zou, Lijuan; Lu, Yao; Zhao, Weijie; Lin, Bingcheng

    2016-03-01

    Carcinoembryonic antigen (CEA) is a wide-spectrum biomarker. Clinically, we generally use serum sample to detect CEA, which needs to be centrifuged to pretreat the raw blood sample. In this study, we realized direct CEA detection in raw blood samples exploiting microfluidics. The LOD was as low as 10(-12) M. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Biochemical principle of Limulus test for detecting bacterial endotoxins.

    Science.gov (United States)

    Iwanaga, Sadaaki

    2007-05-01

    A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1,3)-β-D-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, and factor G, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. The molecular structures of these proteins have also been elucidated. Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel. Here, I will focus on the biochemical principle of Limulus test for detecting bacterial endotoxins, and its activation and regulation mechanism on the LPS-mediated coagulation cascade.

  3. Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells.

    Science.gov (United States)

    Kudela, Pavol; Koller, Verena Juliana; Mayr, Ulrike Beate; Nepp, Johannes; Lubitz, Werner; Barisani-Asenbauer, Talin

    2011-05-20

    The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Vesicles for Signal Amplification in a Biosensor for the Detection of Low Antigen Concentrations

    Directory of Open Access Journals (Sweden)

    Dorothee Grieshaber

    2008-12-01

    Full Text Available The sensitivity of biosensors is often not sufficient to detect diagnostically relevant biomarker concentrations. In this paper we have utilized a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D to detect dissipative losses induced by the attachment of intact vesicles. We modified a sandwich assay by coupling the secondary antibodies to vesicles. This resulted in an increase of detection sensitivity, achieving a diagnostically relevant detection limit of 5 ng/ml or 30 pM antigens. In addition, we could combine the individual assay steps to decrease the total time to result in about 30 minutes.

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

    Directory of Open Access Journals (Sweden)

    Nguekam

    2003-03-01

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

  6. Detecting rare gene transfer events in bacterial populations

    Directory of Open Access Journals (Sweden)

    Kaare Magne Nielsen

    2014-01-01

    Full Text Available Horizontal gene transfer (HGT enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research.

  7. Specific and common antigens of Trichomonas vaginalis detected by monoclonal antibodies.

    Science.gov (United States)

    Torian, B E; Connelly, R J; Stephens, R S; Stibbs, H H

    1984-01-01

    Monoclonal antibodies to Trichomonas vaginalis were prepared by immunizing mice with a cloned isolate of T. vaginalis. Eight antibodies reacted with the same four isolates or strains but did not react with the other T. vaginalis strains or isolates tested. All eight antibodies reacted uniformly with both the body and flagella of T. vaginalis in the immunofluorescence assay but were unreactive by immunoblotting. The antigen(s) recognized by these antibodies was determined to be present on the surface membrane by indirect immunofluorescence assay of live organisms. The antigen(s) was found to be sensitive to periodate oxidation but resistant to pronase digestion. In addition, one monoclonal antibody was derived which reacted with all T. vaginalis isolates or strains tested, as well as with Trichomonas gallinae, Tritrichomonas foetus, and Giardia lamblia. This antibody reacted with the body but not the flagella of Formalin-fixed protozoa in the immunofluorescence assay but failed to react with live organisms. The antigen was found to be periodate resistant but pronase labile. In the immunoblot assay, this antibody detected a single T. vaginalis polypeptide with a molecular weight of 62,000. Images PMID:6360900

  8. The detection of hepatitis c virus core antigen using afm chips with immobolized aptamers.

    Science.gov (United States)

    Pleshakova, T O; Kaysheva, A L; Bayzyanova, J М; Anashkina, А S; Uchaikin, V F; Ziborov, V S; Konev, V A; Archakov, A I; Ivanov, Y D

    2018-01-01

    In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10 -10 М to 10 -13 М. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Detection of targeted carcinoembryonic antigens using a micro-fluxgate-based biosensor

    Science.gov (United States)

    Lei, Jian; Lei, Chong; Wang, Tao; Yang, Zhen; Zhou, Yong

    2013-11-01

    In this work, a micro-fluxgate-based biosensor was designed for the detection of carcinoembryonic antigen (CEA) labeled by Dynabeads. The sensor with Fe-based amorphous core and three dimension solenoid coils was fabricated by Micro Electro-Mechanical system technology. Sandwich assays are performed using antibody-antigen pair combination of biotin-streptavidin assay on a separated Au film substrate surface with a self-assembled layer. With dc magnetic fields in the range of 560 μT to 875 μT, detection of CEAs with different concentrations was performed and a minimum detectable concentration of 1 pg/ml was achieved. Furthermore, CEA samples with different concentrations can be distinguished.

  10. Detection of circulating Fasciola gigantica antigen in experimental and natural infections of sheep with fasciolosis.

    Science.gov (United States)

    Guobadia, E E; Fagbemi, B O

    1996-10-15

    Detection of circulating Fasciola gigantica antigen was performed in sera of sheep with experimental and natural F. gigantica infections using the direct enzyme-linked immunosorbent assay method. Sera from sheep with monoinfections of Schistosoma bovis, Dicrocoelium hospes and Paramphistomum microbothrium were included in the assay to ascertain specificity. Circulating F. gigantica antigen (CFA) was detected as early as 1 week after infection in the experimentally infected sheep. No detectable CFA was observed 2 weeks after chemotherapy. Positivity rates of 82.5%, 12.5%, 10% and 10% were found in sera with monospecific infections of F. gigantica, P. microbrothrium, D. hospes and S. bovis, respectively. Acid treatment of the sera did not enhance the sensitivity of the assay.

  11. Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection

    DEFF Research Database (Denmark)

    Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H

    2017-01-01

    Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial...... of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method....

  12. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system.

    Science.gov (United States)

    Dunbar, Sherry A; Vander Zee, Coe A; Oliver, Kerry G; Karem, Kevin L; Jacobson, James W

    2003-05-01

    Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification

  13. Immunostimulation using bacterial antigens – mechanism ofaction and clinical practice inviral respiratory tract infections

    Directory of Open Access Journals (Sweden)

    Wojciech Feleszko

    2015-12-01

    Full Text Available Recurrent respiratory tract infections constitute a significant problem in the practice of a general practitioner and paediatrician. Antibiotic resistance of bacterial strains, which has been growing for years, prompts the search for alternative ways of combating pathogens. One of them is the usage of preparations based on cell lysis of various bacterial strains. Bacterial lysates have been available in Europe for many years. In preclinical trials, they are characterised by the capability of reducing infections caused by bacteria and viruses that are not the components of the preparations. A range of clinical trials have demonstrated their usefulness in reducing the frequency of seasonal respiratory tract infections and antibiotic use. Moreover, patients with chronic obstructive pulmonary disease gain an additional advantage in the form of the reduction of the risk of hospitalization due to disease exacerbations and a positive influence on the survival curve. The action of bacterial lysates is based on oral immunostimulation of gut-associated lymphoid tissue, which results in increased antibody production. Moreover, they activate a range of mucosal mechanisms of non-specific immunity, mainly by enhancing the activity of TLR-dependent mechanisms. The efficacy of this group of drugs has been confirmed in a range of clinical trials, systematic reviews and meta-analyses. Recent studies also indicate their immunoregulatory potential, suggesting that they might be used in the future in preventing allergies, asthma and autoimmune diseases. To conclude, physicians (paediatricians, laryngologists, pulmonologists should consider reducing the use of antibiotics in their daily practice. Instead, they should offer preparations that promote the immune system, thus controlling infections in a better way.

  14. Novel antigens used to detect cell-mediated immune responses over time in Mycobacterium avium subsp. paratuberculosis infected cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

    consisting of undefined antigens with possible cross reactions toward other environmental bacteria. The objective of the study was to optimize the IFN-γ test using different types of novel antigens for stimulation. Fourteen novel antigen candidates were selected for testing, including 4 peptides of the ESAT...... on the same 30 heifers from a known MAP infected herd. Determination of cut-off for each antigen was based on samples from a non-infected herd, including 60 heifers. Based on PPDj stimulations, more than 50% of the heifers tested MAP positive at the first two samplings, whereas only 20% tested positive...... at third sampling. The resulted showed that PPDj detect a high percentage as MAP positive animals, as this crude antigen mixture is expected to induce non-specific IFN-γ production. However, the tested latency antigens, some secreted proteins and some peptides of the ESAT-6 family detected a comparable...

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

    Science.gov (United States)

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

    1998-01-01

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

  16. Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

    Science.gov (United States)

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

    2015-09-01

    New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

  17. Evaluation of Hepatitis C Virus Core Antigen Assays in Detecting Recombinant Viral Antigens of Various Genotypes ▿

    Science.gov (United States)

    Saeed, Mohsan; Suzuki, Ryosuke; Kondo, Madoka; Aizaki, Hideki; Kato, Takanobu; Mizuochi, Toshiaki; Wakita, Takaji; Watanabe, Haruo; Suzuki, Tetsuro

    2009-01-01

    A single substitution within the hepatitis C virus core antigen sequence, A48T, which is observed in ∼30% of individuals infected with genotype 2a virus, reduces the sensitivity of a commonly used chemiluminescence enzyme immunoassay. Quantitation of the antigen is improved by using a distinct anticore antibody with a different epitope. PMID:19812276

  18. Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin.

    Science.gov (United States)

    Ticehurst, John R; Aird, Deborah Z; Dam, Lisa M; Borek, Anita P; Hargrove, John T; Carroll, Karen C

    2006-03-01

    We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were > or = 99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by approximately 75 to 80% and two-step testing was complete in CCNA alone had been performed on all 5,887 specimens.

  19. Mechanisms of recurrent otitis media: importance of the immune response to bacterial surface antigens.

    Science.gov (United States)

    Murphy, T F; Yi, K

    1997-12-29

    Otitis-prone children experience recurrent episodes of otitis media due to nontypeable H. influenzae (NTHI). A protective immune response occurs following infection, but this immune response is specific for the infecting strain, leaving the child susceptible to infection by other strains of NTHI. Little is known about the mechanism by which a strain-specific antibody response occurs to nonencapsulated bacteria. To explore the mechanism by which this strain-specific response occurs, animals were inoculated with whole bacterial cells and the antibody response was studied. The antibody response was predominantly directed to a highly strain-specific, immunodominant surface loop on the major outer membrane protein. This exquisitely restricted immune response leaves the host susceptible to recurrent infections by many strains of NTHI. The ability of the bacterium to direct the host to make a strain-specific antibody response has important implications in understanding the immune response to otitis media due to NTHI and in designing strategies for vaccine development.

  20. MHC-based detection of antigen-specific CD8(+) T cell responses

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker; Schumacher, Nana Maria Pii

    2010-01-01

    epitopes in limited biological material. These technologies are based on the joint binding of differentially labelled MHC multimers on the T cell surface, thereby providing each antigen-specific T cell population with a unique multicolour code. This strategy of 'combinatorial encoding' enables detection...... of many (at least 25) different T cell populations per sample and should be of broad value for both T cell epitope identification and immunomonitoring...

  1. Evaluation of a Combination Rapid Immunoassay for Detection of Giardia and Cryptosporidium Antigens

    OpenAIRE

    Chan, Raymond; Chen, Jing; York, Mary K.; Setijono, Norman; Kaplan, Raymond L.; Graham, Fitzroy; Tanowitz, Herbert B.

    2000-01-01

    A combination cassette format nonenzymatic rapid immunoassay for detection of Giardia and Cryptosporidium antigens was evaluated by using 556 patient stool specimens from three clinical laboratories. This assay (Genzyme Diagnostics Contrast Giardia/Cryptosporidium), which can be used with fresh or formalin-fixed specimens, had unadjusted sensitivities and specificities of 96.1 and 98.5% for Giardia and 100 and 98.7% for Cryptosporidium, respectively, in this study.

  2. Antigen Detection in the Diagnosis of Histoplasmosis: A Meta-analysis of Diagnostic Performance.

    Science.gov (United States)

    Fandiño-Devia, Estefanía; Rodríguez-Echeverri, Carolina; Cardona-Arias, Jaiberth; Gonzalez, Angel

    2016-04-01

    We performed a meta-analysis of diagnostic data to evaluate the performance of Histoplasma antigen detection tests for diagnosing histoplasmosis. We included all studies involving human subjects that assessed the performance of any antigen detection test for histoplasmosis in urine or serum by carrying out an exhaustive and reproducible search of the literature between 1980 and 2014 from four databases. Quality of the articles was assessed, and meta-analysis was performed under the random effects model, calculating sensitivity, specificity, likelihood and odds ratios, and ROC curve using Meta-DiSc(es). Nine out of a total of 23 studies met strict quality criteria and were therefore included. The overall sensitivity for antigen detection in serum and urine was 81% (95% CI 78-83%), while specificity was 99% (95% CI 98-99%). Sensitivity for antigenuria and antigenemia was 79% (95% CI 76-82%) and 82% (95% CI 79-85%), respectively; specificity values were 99% (95% CI 98-100%) in urine and 97% (95% CI 96-98%) in serum. The positive and negative likelihood ratios were 49.5 (95% CI 20.7-118.7) and 0.19 (95% CI 0.14-0.26), respectively, while the diagnostic OR was 362 (95% CI 121.2-1080.3) and area under the curve was 0.99. In conclusion, the performance of Histoplasma antigen detection assay of urine was not significantly different from that of blood, indicating that antigenuria and antigenemia have equal diagnostic value in histoplasmosis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-05-01

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

  4. Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation.

    Science.gov (United States)

    Caro-Oleas, José Luis; González-Escribano, María Francisca; González-Roncero, Francisco Manuel; Acevedo-Calado, María José; Cabello-Chaves, Virginia; Gentil-Govantes, Miguel Ángel; Núñez-Roldán, Antonio

    2012-03-01

    Clinical relevance of donor-specific antibodies (DSAs) detected by a single antigen Luminex virtual crossmatch in pre-transplant serum samples from patients with a negative cytotoxicity-dependent complement crossmatch is controversial. The aim of this study was to analyse the influence of a pre-transplant positive virtual crossmatch in the outcome of kidney transplantation. A total of 892 patients who received a graft from deceased donors after a negative cytotoxicity crossmatch were included. Presence of anti-human leucocyte antigen (HLA) antibodies was investigated using a Luminex screening assay and anti-HLA specificities were assigned performing a Luminex single antigen assay. Graft survival was significantly worse among patients with anti-HLA DSA compared to both patients with anti-HLA with no DSA (P = 0.001) and patients without HLA antibodies (P HLA with no DSA and no HLA antibodies patient groups were observed (P = 0.595). Influence of both anti-Class I and anti-Class II DSA was detected (P 1500 (global P > 0.05). The presence of preformed HLA DSA in transplanted patients with a negative cytotoxicity crossmatch is associated with a lower allograft survival. The detection of anti-HLA with no DSA has no influence in the graft outcome. Finally, there were no demonstrable effects of mean fluorescence intensity (MFI) values >1500 on graft survival.

  5. In Vitro Use of Autologous Dendritic Cells Improves Detection of T Cell Responses to Hepatitis B Virus (HBV) Antigens

    NARCIS (Netherlands)

    Carotenuto, Patrizia; Artsen, Andre; Niesters, Hubert G.; Osterhaus, Albert D.; Pontesilli, Oscar

    T lymphocyte responses to hepatitis B virus (HBV) core antigen (HBcAg) are vigorous and easily detectable in vitro during recovery from acute hepatitis B but significantly weaker in patients with chronic HBV infection. In contrast, T cell responses to hepatitis B surface antigen (HBsAg) are almost

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

    Science.gov (United States)

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

    2018-01-01

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

  7. Impedance-Based Miniaturized Biosensor for Ultrasensitive and Fast Prostate-Specific Antigen Detection

    Directory of Open Access Journals (Sweden)

    Ganna Chornokur

    2011-01-01

    Full Text Available This paper reports the successful fabrication of an impedance-based miniaturized biosensor and its application for ultrasensitive Prostate-Specific Antigen (PSA detection in standard and real human plasma solution, spiked with different PSA concentrations. The sensor was fabricated using photolithographic techniques, while monoclonal antibodies specific to human PSA were used as primary capture antibodies. Electrochemical impedance spectroscopy (EIS was employed as a detection technique. The sensor exhibited a detection limit of 1 pg/ml for PSA with minimal nonspecific binding (NSB. This detection limit is an order of magnitude lower than commercial PSA ELISA assays available on the market. The sensor can be easily modified into an array for the detection of other biomolecules of interest, enabling accurate, ultrasensitive, and inexpensive point-of-care sensing technologies.

  8. Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain formation

    DEFF Research Database (Denmark)

    Tian, Bo; Wetterskog, Erik; Qiu, Zhen

    2017-01-01

    anisotropy), and directly increasing the optomagnetic signal (via optical shape anisotropy). We achieve a limit of detection (LOD) of 5.5 pM (0.82 ng/mL) for the detection of a model multivalent molecule, biotinylated anti-streptavidin, in PBS. For the measurements of prostate-specific antigen (PSA) in 50......% serum using the proposed method, we achieve an LOD of 21.6 pM (0.65 ng/mL) and a dynamic detection range up to 66.7 nM (2 µg/mL) with a sample-to-result time of approximately 20 min. The performance for PSA detection therefore well meets the clinical requirements in terms of LOD (the threshold PSA level...... in blood is 4 ng/mL) and detection range (PSA levels span from PSA diagnostics and for other in-situ applications....

  9. Prostate cancer detection is also relevant in low prostate specific antigen ranges.

    Science.gov (United States)

    Lujan, Marcos; Paez, Alvaro; Miravalles, Elena; Fernandez, Inmaculada; Llanes, Luis; Berenguer, Antonio

    2004-02-01

    To address detection rates and clinical features of the cancers detected with low prostate specific antigen (PSA) levels. In the context of a prostate cancer (PCa) screening program 1097 men attended to a new rescreen round. Sextant prostate biopsy was recommended when PSA > or =3 ng/ml. We also recommended to undergo biopsy in the range 1.0-2.99 ng/ml when free to total (f/t) PSA ratio or =3.0 ng/ml. In the group with PSA between 1.0 and 2.99 ng/ml and f/t PSA ratio aggresive cancers is still of concern.

  10. Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection.

    Science.gov (United States)

    Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

    2016-06-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

  11. Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture

    Directory of Open Access Journals (Sweden)

    Jyoti Kumar

    2015-09-01

    Full Text Available Aim: This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. Introduction: M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA, Rpt2 and 12S ribosomal RNA (rRNA genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. Materials and Methods: A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. Results: All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631. Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S r

  12. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

    DEFF Research Database (Denmark)

    Staalsoe, T; Giha, H A; Dodoo, D

    1999-01-01

    BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously...

  13. Comparison of direct immunofluorescence and direct immunoperoxidase procedures for detection of herpes simplex virus antigen in lesion specimens.

    OpenAIRE

    Schmidt, N J; Dennis, J; Devlin, V; Gallo, D; Mills, J

    1983-01-01

    Direct immunofluorescence and direct immunoperoxidase staining were equally sensitive and specific for detection of herpes simplex virus antigen in lesion specimens, and each method showed 82% agreement with virus isolation results.

  14. A novel method to detect bacterial resistance to disinfectants

    Directory of Open Access Journals (Sweden)

    Xiao-Feng He

    2017-09-01

    Full Text Available In clinical practice, the important hygienic prevention of bacterial pathogen spread is disinfection of potentially contaminated area. Benzalkonium bromide and chlorhexidine acetate are commonly used disinfectants with a broad spectrum of anti-microbial effect. It is vital to inhibit the spread of pathogen in hospital. However, a large number of pathogens with the decreased antiseptic susceptibility have been isolated from clinical samples which showed an increased minimal inhibitory concentration (MIC against those antiseptics. These resistant pathogens are the major causes for nosocomial cross-infections in hospital. The present study demonstrated the utility of Oxford plate assay system in determining the potential disinfectant resistance of bacteria. The microbiological assay is based on the inhibitory effect of tested disinfectants upon the strains of Staphylococcus aureus and Escherichia coli. Statistical analysis of the bioassay results indicated the linear correlation (r = 0.87–0.99, P < 0.01 between the diameter of growth inhibition zone and the log dosage of the tested disinfectants. Moreover, comparison of inhibitory efficacy of benzalkonium bromide upon 29 S. aureus strains isolated from clinical samples by both Oxford plate method and broth dilution method showed that the diameter of growth inhibition zone has significantly negative correlation with the minimal inhibitory concentration (MIC (r = −0.574, P < 0.001. These results suggest that the Oxford plate is a simple and time-saving method in detecting potential clinical disinfectant resistance and its usefulness for routine surveillance of pathogenic resistance to disinfectants warrants further investigation.

  15. Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Helena Lúcia Carneiro Santos

    Full Text Available Amebiasis is an infection caused by Entamoeba histolytica. However, differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, precaution of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex -PCR for detection and differentiation of E. histolytica from E. dispar from fresh stool samples in comparison with the coproantigen commercial ELISA. Microscopic examination of stools using the Coprotest method, detection of stool antigen by enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were used for the diagnosis of amoebiasis infection. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21% samples were positive for E. histolytica/E. dispar complex. Among these stool samples, 11 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp and two presenting diagnostic fragment of E. histolytica (132 bp. Among negative samples detected by microscopic examination, three positive samples for E. dispar and one positive for E. histolytica by Multiplex-PCR was observed. This denotes a low sensibility of microscopic examination when a single stool sample is analyzed. Assay for detection of E. histolytica antigen was concordant with multiplex-PCR in relation to E. histolytica. Statistical analysis comparing the sensibility tests was not done because of the low number of E. histolytica cases. The results demonstrate the importance of the specific techniques use for the differentiation between E. histolytica and E. dispar.

  16. Identification of Common Bacterial Antigenic Markers From Bovine Digital Dermatitis Lesions Using Meta-Transcriptomics in Combination With High-Density Peptide-Microarrays

    DEFF Research Database (Denmark)

    Nielsen, Martin W.; Marcatili, Paoli; Sicheritz-Ponten, Thomas

    of collagenous and connective tissues. Multiple Treponema species, many of which are not-yet-cultivable, are strongly implicated in disease progression. Despite the economic and welfare importance of this disease, no effective vaccine is available; and there is presently very little knowledge concerning...... efficacious immunoprophylactic antigens against DD. It is highly likely that DD-associated treponemes possess considerable antigenic variation, as cows exhibit a variable humoral response against different isolates of Treponema. Hence, combinations of antigens from multiple Treponema species should be used...... response directed at the site of infection. By metatranscriptomics we measured the in situ genome-wide transcriptome of the bacterial population in DD-affected skin lesions from 21 dairy cows. From the transcriptome data, we identified a panel of Treponema genes that were highly expressed in multiple...

  17. Rapid detection of bacterial pathogens using flourescence spectroscopy and chemometrics

    Science.gov (United States)

    This work presents the development of a method for rapid bacterial identification based on the fluorescence spectroscopy combined with multivariate analysis. Fluorescence spectra of pure three different genera of bacteria (Escherichia coli, Salmonella, and Campylobacter) were collected from 200...

  18. Case of rhesus antigen weak D type 4.2. (DAR category detection

    Directory of Open Access Journals (Sweden)

    L. L. Golovkina

    2015-01-01

    Full Text Available Serological methods of Rhesus antigens identification in humans cannot identify D-antigen variants. In this article the serological characteristics of Rhesus antigen D weak type 4.2. (Category DAR are described.

  19. Lymphocyte proliferation to mycobacterial antigens is detectable across a spectrum of HIV-associated tuberculosis

    Directory of Open Access Journals (Sweden)

    Bakari Muhammad

    2009-02-01

    Full Text Available Abstract Background Identifying novel TB diagnostics is a major public health priority. We explored the diagnostic characteristics of antimycobacterial lymphocyte proliferation assays (LPA in HIV-infected subjects with latent or active TB. Methods HIV-infected subjects with bacille Calmette Guérin (BCG scars and CD4 counts ≥ 200 cells/mm3 entering a TB booster vaccine trial in Tanzania had baseline in vivo and in vitro immune tests performed: tuberculin skin tests (TST, LPA and five day assays of interferon gamma (IFN-γ release. Assay antigens were early secreted antigenic target 6 (ESAT-6, antigen 85 (Ag85, and Mycobacterium tuberculosis whole cell lysate (WCL. Subjects were screened for active TB at enrollment by history, exam, sputum smear and culture. We compared antimycobacterial immune responses between subjects with and without latent or active TB at enrollment. Results Among 1885 subjects screened, 635 had latent TB and 13 had active TB. Subjects with latent TB were more likely than subjects without TB to have LPA responses to ESAT-6 (13.2% vs. 5.5%, P Conclusion Lymphoproliferative responses to mycobacteria are detectable during HIV-associated active TB, and are less sensitive but more specific than TST. Trial registration ClinicalTrials.gov Identifier NCT00052195.

  20. Development and validation of a high throughput system for discovery of antigens for autoantibody detection.

    Directory of Open Access Journals (Sweden)

    Isabel K Macdonald

    Full Text Available An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor-associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.

  1. Development of a PMMA Electrochemical Microfluidic Device for Carcinoembryonic Antigen Detection

    Science.gov (United States)

    Van Anh, Nguyen; Van Trung, Hoang; Tien, Bui Quang; Binh, Nguyen Hai; Ha, Cao Hong; Le Huy, Nguyen; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

    2016-05-01

    In this study, a poly(methyl methacrylate) (PMMA) microfluidic device fabricated by an inexpensive CO2 laser etching system was developed for detection of carcino-embryonic antigens (CEA). The device was capable of working in continuous mode and was designed with the aid of numerical simulation. The detection of target CEA was based on immuno-assay via magnetic particles and electrochemical sensing. The as-prepared microfluidic can be used to detect CEA at the relatively low concentration of 150 pg mL-1. The device could be reused many times, since the capture and removal of magnetic particles in the assay could be manipulated by an external magnetic field. The proposed approach appears to be suitable for high-throughput and automated analysis of large biomolecules such as tumor markers and pathogens.

  2. Novel means of viral antigen identification: improved detection of avian influenza viruses by proximity ligation.

    Science.gov (United States)

    Schlingemann, Joerg; Leijon, Mikael; Yacoub, Alia; Schlingemann, Heidi; Zohari, Siamak; Matyi-Tóth, Anna; Kiss, István; Holmquist, Göran; Nordengrahn, Ann; Landegren, Ulf; Ekström, Björn; Belák, Sándor

    2010-01-01

    Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safely can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.

  3. Procedures involving lipid media for detection of bacterial contamination in breweries.

    Science.gov (United States)

    Van Vuuren, H J; Louw, H A; Loos, M A; Meisel, R

    1977-01-01

    The liquid equivalent of universal beer agar, designated universal beer liquid medium, and its beer-free equivalent, universal liquid medium (UL), were equally effective in demonstrating bacterial contamination in 120 of 200 samples from different stages of commercial brewing process. Growth of the contaminants after 3 days was consistently more luxuriant in the UL medium. A yeast-water substrate medium failed to reveal many contaminants detected with UL in 392 samples from three breweries and revealed only a few not detected with UL. The use of UL and a lactose-peptone medium, with microscope examination of the media for bacterial growth, permitted detection of 93% of the known contaminants compared to 87%, detected with UL alone; this combination or universal beer liquid medium plus lactose-peptone medium can therefore be recommended for the detection of bacterial contaminants in brewery samples. Bacterial contamination of pitching yeasts appeared to be a particular problem in the breweries investigated. PMID:848948

  4. Comparison of enterovirus detection in cerebrospinal fluid with Bacterial Meningitis Score in children

    Science.gov (United States)

    Pires, Frederico Ribeiro; Franco, Andréia Christine Bonotto Farias; Gilio, Alfredo Elias; Troster, Eduardo Juan

    2017-01-01

    ABSTRACT Objective To measure the role of enterovirus detection in cerebrospinal fluid compared with the Bacterial Meningitis Score in children with meningitis. Methods A retrospective cohort based on analysis of medical records of pediatric patients diagnosed as meningitis, seen at a private and tertiary hospital in São Paulo, Brazil, between 2011 and 2014. Excluded were patients with critical illness, purpura, ventricular shunt or recent neurosurgery, immunosuppression, concomitant bacterial infection requiring parenteral antibiotic therapy, and those who received antibiotics 72 hours before lumbar puncture. Results The study included 503 patients. Sixty-four patients were excluded and 94 were not submitted to all tests for analysis. Of the remaining 345 patients, 7 were in the Bacterial Meningitis Group and 338 in the Aseptic Meningitis Group. There was no statistical difference between the groups. In the Bacterial Meningitis Score analysis, of the 338 patients with possible aseptic meningitis (negative cultures), 121 of them had one or more points in the Bacterial Meningitis Score, with sensitivity of 100%, specificity of 64.2%, and negative predictive value of 100%. Of the 121 patients with positive Bacterial Meningitis Score, 71% (86 patients) had a positive enterovirus detection in cerebrospinal fluid. Conclusion Enterovirus detection in cerebrospinal fluid was effective to differentiate bacterial from viral meningitis. When the test was analyzed together with the Bacterial Meningitis Score, specificity was higher when compared to Bacterial Meningitis Score alone. PMID:28767914

  5. Detection of bacterial blight resistant gene xa5 using linked marker ...

    African Journals Online (AJOL)

    Detection of bacterial blight resistant gene xa5 using linked marker approaches. SA Naveed, M Babar, A Arif, Y Zafar, M Sabar, I Ali, M Chragh, M Arif. Abstract. Rice is the primary source of food for 57% of the world's population. Genetic resistance is important to control many kinds of pathogenic diseases. Bacterial blight ...

  6. Field Effect Transistor Biosensor Using Antigen Binding Fragment for Detecting Tumor Marker in Human Serum

    Science.gov (United States)

    Cheng, Shanshan; Hotani, Kaori; Hideshima, Sho; Kuroiwa, Shigeki; Nakanishi, Takuya; Hashimoto, Masahiro; Mori, Yasuro; Osaka, Tetsuya

    2014-01-01

    Detection of tumor markers is important for cancer diagnosis. Field-effect transistors (FETs) are a promising method for the label-free detection of trace amounts of biomolecules. However, detection of electrically charged proteins using antibody-immobilized FETs is limited by ionic screening by the large probe molecules adsorbed to the transistor gate surface, reducing sensor responsiveness. Here, we investigated the effect of probe molecule size on the detection of a tumor marker, α-fetoprotein (AFP) using a FET biosensor. We demonstrated that the small receptor antigen binding fragment (Fab), immobilized on a sensing surface as small as 2–3 nm, offers a higher degree of sensitivity and a wider concentration range (100 pg/mL–1 μg/mL) for the FET detection of AFP in buffer solution, compared to the whole antibody. Therefore, the use of a small Fab probe molecule instead of a whole antibody is shown to be effective for improving the sensitivity of AFP detection in FET biosensors. Furthermore, we also demonstrated that a Fab-immobilized FET subjected to a blocking treatment, to avoid non-specific interactions, could sensitively and selectively detect AFP in human serum. PMID:28788579

  7. Evolution of MHC-based technologies used for detection of antigen-responsive T cells.

    Science.gov (United States)

    Bentzen, Amalie Kai; Hadrup, Sine Reker

    2017-05-01

    T cell-mediated recognition of peptide-major histocompatibility complex (pMHC) class I and II molecules is crucial for the control of intracellular pathogens and cancer, as well as for stimulation and maintenance of efficient cytotoxic responses. Such interactions may also play a role in the development of autoimmune diseases. Novel insights into this mechanism are crucial to understanding disease development and establishing new treatment strategies. MHC multimers have been used for detection of antigen-responsive T cells since the first report by Altman et al. showed that tetramerization of pMHC class I molecules provided sufficient stability to T cell receptor (TCR)-pMHC interactions, allowing detection of MHC multimer-binding T cells using flow cytometry. Since this breakthrough the scientific community has aimed for expanding the capacity of MHC multimer-based detection technologies to facilitate large-scale epitope discovery and immune monitoring in limited biological material. Screening of T cell specificity using large libraries of pMHC molecules is suitable for analyses of T cell recognition potentially at genome-wide levels rather than analyses restricted to a selection of model antigens. Such strategies provide novel insights into the immune specificities involved in disease development and response to immunotherapy, and extend fundamental knowledge related to T cell recognition patterns and cross-recognition by TCRs. MHC multimer-based technologies have now evolved from detection of 1-2 different T cell specificities per cell sample, to include more than 1000 evaluable pMHC molecules using novel technologies. Here, we provide an overview of MHC multimer-based detection technologies developed over two decades, focusing primarily on MHC class I interactions.

  8. Detection of bacterial blight resistance genes in basmati rice landraces.

    Science.gov (United States)

    Ullah, I; Jamil, S; Iqbal, M Z; Shaheen, H L; Hasni, S M; Jabeen, S; Mehmood, A; Akhter, M

    2012-07-20

    Aromatic basmati rice is vulnerable to bacterial blight disease. Genes conferring resistance to bacterial blight have been identified in coarse rice; however, their incorporation into basmati varieties compromises the prized basmati aroma. We identified bacterial blight resistance genes Xa4, xa5, Xa7, and xa13 in 52 basmati landraces and five basmati cultivars using PCR markers. The Xa7 gene was found to be the most prevalent among the cultivars and landraces. The cultivars Basmati-385 and Basmati-2000 also contained the Xa4 gene; however, xa5 and xa13 were confined to landraces only. Ten landraces were found to have multiple resistance genes. Landraces Basmati-106, Basmati-189 and Basmati-208 contained Xa4 and Xa7 genes. Whereas, landraces Basmati-122, Basmati-427, Basmati-433 were observed to have xa5 and Xa7 genes. Landraces Basmati-48, Basmati-51A, Basmati-334, and Basmati-370A possessed Xa7 and xa13 genes. The use of landraces containing recessive genes xa5 and xa13 as donor parents in hybridization with cultivars Basmati-385 and Basmati-2000, which contain the genes Xa4 and Xa7, will expedite efforts to develop bacterial blight-resistant basmati rice cultivars through marker assisted selection, based on a pyramiding approach, without compromising aroma and grain quality.

  9. Detecting bacterial endophytes in tropical grasses of the Brachiaria ...

    African Journals Online (AJOL)

    Plant-growth-promoting (PGP) bacteria include a diverse group of soil bacteria thought to stimulate plant growth by various mechanisms. Brachiaria forage grasses, of African origin, are perennials that often grow under low-input conditions and are likely to harbour unique populations of PGP bacteria. Three bacterial strains ...

  10. Detection of carcinoembryonic antigen using functional magnetic and fluorescent nanoparticles in magnetic separators

    Science.gov (United States)

    Tsai, H. Y.; Chang, C. Y.; Li, Y. C.; Chu, W. C.; Viswanathan, K.; Bor Fuh, C.

    2011-06-01

    We combined a sandwich immunoassay, anti-CEA/CEA/anti-CEA, with functional magnetic ( 80 nm) and fluorescent ( 180 nm) nanoparticles in magnetic separators to demonstrate a detection method for carcinoembryonic antigen (CEA). Determination of CEA in serum can be used in clinical diagnosis and monitoring of tumor-related diseases. The CEA concentrations in samples were deduced and determined based on the reference plot using the measured fluorescent intensity of sandwich nanoparticles from the sample. The linear range of CEA detection was from 18 ng/mL to 1.8 pg/mL. The detection limit of CEA was 1.8 pg/mL. In comparison with most other detection methods, this method had advantages of lower detection limit and wider linear range. The recovery was higher than 94%. The CEA concentrations of two serum samples were determined to be 9.0 and 55 ng/mL, which differed by 6.7% (9.6 ng/mL) and 9.1% (50 ng/mL) from the measurements of enzyme-linked immunosorbent assay (ELISA), respectively. The analysis time can be reduced to one third of ELISA. This method has good potential for other biomarker detections and biochemical applications.

  11. Detection of carcinoembryonic antigen using functional magnetic and fluorescent nanoparticles in magnetic separators

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, H. Y., E-mail: annetsai@csmu.edu.tw [Chung Shan Medical University, Department of Applied Chemistry (China); Chang, C. Y.; Li, Y. C.; Chu, W. C.; Viswanathan, K.; Bor Fuh, C., E-mail: cbfuh@ncnu.edu.tw [National Chi Nan University, Department of Applied Chemistry (China)

    2011-06-15

    We combined a sandwich immunoassay, anti-CEA/CEA/anti-CEA, with functional magnetic ({approx}80 nm) and fluorescent ({approx}180 nm) nanoparticles in magnetic separators to demonstrate a detection method for carcinoembryonic antigen (CEA). Determination of CEA in serum can be used in clinical diagnosis and monitoring of tumor-related diseases. The CEA concentrations in samples were deduced and determined based on the reference plot using the measured fluorescent intensity of sandwich nanoparticles from the sample. The linear range of CEA detection was from 18 ng/mL to 1.8 pg/mL. The detection limit of CEA was 1.8 pg/mL. In comparison with most other detection methods, this method had advantages of lower detection limit and wider linear range. The recovery was higher than 94%. The CEA concentrations of two serum samples were determined to be 9.0 and 55 ng/mL, which differed by 6.7% (9.6 ng/mL) and 9.1% (50 ng/mL) from the measurements of enzyme-linked immunosorbent assay (ELISA), respectively. The analysis time can be reduced to one third of ELISA. This method has good potential for other biomarker detections and biochemical applications.

  12. Detection of anti-dsDNA by IgG ELISA test using two different sources of antigens: calf thymus versus E.coli

    Directory of Open Access Journals (Sweden)

    Mohammadi M

    2009-04-01

    Full Text Available "nBackground: Anti-dsDNA antibodies frequently found in the sera Systemic Lupus Erythematosus patients, particularly in active disease stage. Nowadays exploit different eukaryotic and prokaryotic dsDNA as antigen source and different reagents as binder. The aim of this study to compared two dsDNA different sources and tow different kinds of reagents for binder in ELISA test. "nMethods: In this study bacterial genomic DNA from E.coli (ATCC 25922 and genomic DNA from calf thymus extracted with high purity and were used as antigens for IgG anti-dsDNA detection by ELISA. To coat dsDNA in microtiter wells, tow different kinds of reagents including methylated -BSA and poly-l-lysine (for pre-coating are used. Sera from systemic lupus erythematosus patients and from normal blood donors are used to assess sensitivity and specificity of our ELISA test in compared with IF test and commercial kits. "nResults: Our results displayed pre-coating of microtiter plates with methylated -BSA reduce nonspecific binding reaction and the relative sensitivity and specificity of ELISA increased when calf thymus DNA is employed as antigenic source in compared with IF test and commercial kits 80%, 88% and 100%, 98% respectively, but when E.coli DNA is used 73%, 69% and 85%, 79%, respectively. "nConclusion: The genomic DNA from calf thymus is a potentially useful source of antigen for detection of anti-dsDNA by ELISA. Also the use of methylatted- BSA could have an effective role in reducing of nonspecific binding reactions.

  13. Effective Detection of Toxigenic Clostridium difficile by a Two-Step Algorithm Including Tests for Antigen and Cytotoxin

    OpenAIRE

    Ticehurst, John R.; Aird, Deborah Z.; Dam, Lisa M.; Borek, Anita P.; Hargrove, John T.; Carroll, Karen C.

    2006-01-01

    We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were ≥99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by ≈75 to 80% and two-step testing was complete in ≤3 days, we decided that this algorithm would be effective. Over 6 months...

  14. Graphene oxide supported rhombic dodecahedral Cu2O nanocrystals for the detection of carcinoembryonic antigen.

    Science.gov (United States)

    Feng, Taotao; Chen, Xiaoyu; Qiao, Xiuwen; Sun, Zhao; Wang, Haining; Qi, Yu; Hong, Chenglin

    2016-02-01

    In this work, a simple electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) based on rhombic dodecahedral Cu2O nanocrystals-graphene oxide-gold nanoparticles (rCu2O-GO-AuNPs). GO as the template and surfactant resulting in rCu2O exhibit improved rhombic dodecahedral structure uniformity and excellent electrochemical performance. Moreover, GO was found to be able to effectively improve the long stability of rCu2O on the electrode response. Under optimal conditions, the immunosensor showed a low limit of detection (0.004 ng ml(-1)) and a large linear range (0.01-120 ng ml(-1)). This work presents a potential alternative for the diagnostic applications of GO-supported special morphology materials in biomedicine and biosensors. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Are species-specifi antigen detection tests needed in the diagnosis of Giardia duodenalis infection?

    Directory of Open Access Journals (Sweden)

    Flávia Fernandes de Mendonça Uchôa

    2017-08-01

    Full Text Available Objective: To assess the diagnostic performance in human stool samples of a rapid, qualitative, solid-phase immunochromatographic test (Alere® originally developed to detect Giardia duodenalis antigens in fecal samples of dogs. Methods: Samples from 54 patients with a previous diagnosis of giardiasis were tested by the microscopic examination to assess the performance of an immunochromatographic kit developed to detect Giardia duodenalis coproantigen in dog feces. Results: The agreement between the microscopic and the immunological methods was 83.3%. These findings are consistent with those of other studies using human specific kits. Conclusions: It is suggested that the same immunochromatographic test could be used for Giardia diagnosis in both species.

  16. Role of digital rectal examination and prostate specific antigen in detecting carcinoma prostate

    International Nuclear Information System (INIS)

    Iqbal, N.; Bhatti, A.N.; Husain, S.

    2003-01-01

    Objective: To investigate the ability of digital rectal examination (DRE) and prostrate specific antigen (PSA) in detecting carcinoma prostrate. Results: Digital rectal examination has shown sensitivity of 63% and specificity of 73.22%. The sensitivity of PSA was 87% and specificity was 70.8%. The positive predictive value for DRE and PSA was 57.5% and 78.04% respectively. Negative predictive value was 73.22% and 85.22% respectively. P value was statistically significant <0.037. In the second part of study a baseline serum PSA level in age-matched 200 patients without history of prostatism was estimated. Conclusion: It was concluded that PSA represent an important adjunct to DRE for detection of prostate carcinoma. (author)

  17. Bacterial contamination of platelet components not detected by BacT/ALERT®.

    Science.gov (United States)

    Abela, M A; Fenning, S; Maguire, K A; Morris, K G

    2018-02-01

    To investigate the possible causes for false negative results in BacT/ALERT ® 3D Signature System despite bacterial contamination of platelet units. The Northern Ireland Blood Transfusion Service (NIBTS) routinely extends platelet component shelf life to 7 days. Components are sampled and screened for bacterial contamination using an automated microbial detection system, the BacT/ALERT ® 3D Signature System. We report on three platelet components with confirmed bacterial contamination, which represent false negative BacT/ALERT ® results and near-miss serious adverse events. NIBTS protocols for risk reduction of bacterial contamination of platelet components are described. The methodology for bacterial detection using BacT/ALERT ® is outlined. Laboratory tests, relevant patient details and relevant follow-up information are analysed. In all three cases, Staphylococcus aureus was isolated from the platelet residue and confirmed on terminal sub-culture using BacT/ALERT ® . In two cases, S. aureus with similar genetic makeup was isolated from the donors. Risk reduction measures for bacterial contamination of platelet components are not always effective. Automated bacterial culture detection does not eliminate the risk of bacterial contamination. Visual inspection of platelet components prior to release, issue and administration remains an important last line of defence. © 2017 British Blood Transfusion Society.

  18. Suitability of Optical, Physical and Chemical Measurements for Detection of Changes in Bacterial Drinking Water Quality

    Science.gov (United States)

    Ikonen, Jenni; Pitkänen, Tarja; Miettinen, Ilkka T.

    2013-01-01

    In this study, different optical, physical and chemical measurements were tested for their capacity to detect changes in water quality. The tests included UV-absorbance at 254 nm, absorbance at 420 nm, turbidity, particle counting, temperature, pH, electric conductivity (EC), free chlorine concentration and ATP concentration measurements. Special emphasis was given to investigating the potential for measurement tools to detect changes in bacterial concentrations in drinking water. Bacterial colony counts (CFU) and total bacterial cell counts (TBC) were used as reference methods for assessing the bacterial water quality. The study consists of a series of laboratory scale experiments: monitoring of regrowth of Pseudomonas fluorescens, estimation of the detection limits for optical measurements using Escherichia coli dilutions, verification of the relationships by analysing grab water samples from various distribution systems and utilisation of the measurements in the case of an accidentally contaminated distribution network. We found significant correlations between the tested measurements and the bacterial water quality. As the bacterial contamination of water often co-occurs with the intrusion of matrixes containing mainly non-bacterial components, the tested measurement tools can be considered to have the potential to rapidly detect any major changes in drinking water quality. PMID:24284353

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

    Directory of Open Access Journals (Sweden)

    Om Parkash

    2014-11-01

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

  20. Biochemical principle of Limulus test for detecting bacterial endotoxins

    OpenAIRE

    Iwanaga, Sadaaki

    2007-01-01

    A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that ther...

  1. Carcinoma-associated antigens

    International Nuclear Information System (INIS)

    Bartorelli, A.; Accinni, R.

    1981-01-01

    This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125 I-labeled forms of said antigens and methods of detecting said antigens in serum or plasma. The invention also relates to a diagnostic kit containing standardised antigens or antisera or marked forms thereof for the detection of said antigens in human blood, serum or plasma. (author)

  2. Clinical characteristics and prostate-specific antigen kinetics of prostate cancer detected in repeat annual population screening in Japan.

    Science.gov (United States)

    Kitagawa, Yasuhide; Sawada, Kiyoshi; Mizokami, Atsushi; Nakashima, Kazuyoshi; Koshida, Kiyoshi; Nakashima, Takao; Miyazaki, Kimiomi; Takeda, Yasuo; Namiki, Mikio

    2014-05-01

    To clarify the present status regarding repeat examination in the annual population screening system in Japan, and to analyze the clinical characteristics and prostate-specific antigen kinetics of prostate cancer detected in this setting. We summarized the annual individual data of prostate-specific antigen-based population screening in Kanazawa, Japan, and analyzed the prostate cancer detection rates at first and repeat screening. The clinical characteristics were compared between patients detected at first and repeat screening. The patients were classified according to favorable or unfavorable clinical characteristics of cancer, and prostate-specific antigen kinetics were compared between the two groups. From 2000 to 2011, 19 620 men participated in this screening program, and a total of 59 019 screenings were carried out. The total annual numbers of examinees increased, and the annual rates of first examinees gradually decreased. The annual detection rates of cancer at total screening decreased in the second year. The annual detection rate at first screening was not different from that in the first year. The rate of patients with favorable cancer features was significantly higher among patients detected at repeat screening than at first screening. The rates of patients with high prostate-specific antigen velocity and low prostate-specific antigen doubling time were significantly higher in unfavorable than favorable cancer patients in repeat screening. Repeat population screening could contribute to early detection of prostate cancer, and it seems that prostate-specific antigen kinetics might predict the cancer characteristics in repeat screening. © 2013 The Japanese Urological Association.

  3. [The detection of virus antigen in the lower respiratory tract of the patients with lung cancer].

    Science.gov (United States)

    Ou, M; Wang, H; Chen, H

    1999-12-01

    To find out about the viral infection situation of lower respiratory tract of the patients with lung cancer. The excretion from the surface of bronchiogenic carcinoma was brushed under fibrobronchoscopy. The respiratory virus antigen was detected and analysed by reagent kit produced by the 262nd Hospital of Beijing Military Region. The respiratory virus antigen was positive in eight cases of lung cancer group, the positive rate was 17.4%(8/46), it was significantly higher than that in non-lung cancer group (P < 0.05). Among them, there were one case of influenza virus A, two cases of influenza virus B, two cases of para-influenza 1,3, two cases of adenovirus and one case of respiratory syncytial virus. The carcinoma accompanied with viral infection were 4,3,1, cases in order of squamous carcinoma, small cell lung carcinoma and adenocarcinoma. The results showed that a relationship existed between lung cancer and viral infection of respiratory tract statistically. The viral infection increased in patients with lung cancer, this is worthy to pay attention to.

  4. On the importance of controlling film architecture in detecting prostate specific antigen

    Science.gov (United States)

    Graça, Juliana Santos; Miyazaki, Celina Massumi; Shimizu, Flavio Makoto; Volpati, Diogo; Mejía-Salazar, J. R.; Oliveira, Osvaldo N., Jr.; Ferreira, Marystela

    2018-03-01

    Immunosensors made with nanostructured films are promising for detecting cancer biomarkers, even at early stages of the disease, but this requires control of film architecture to preserve the biological activity of immobilized antibodies. In this study, we used electrochemical impedance spectroscopy (EIS) to detect Prostate Specific Antigen (PSA) with immunosensors produced with layer-by-layer (LbL) films containing anti-PSA antibodies in two distinct film architectures. The antibodies were either adsorbed from solutions in which they were free, or from solutions where they were incorporated into liposomes of dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation into DPPG liposomes was confirmed with surface plasmon resonance experiments, while the importance of electrostatic interactions on the electrical response was highlighted using the Finite Difference Time-Domain Method (FDTD). The sensitivity of both architectures was sufficient to detect the threshold value to diagnose prostate cancer (ca. 4 ng mL-1). In contrast to expectation, the sensor with the antibodies incorporated into DPPG liposomes had lower sensitivity, though the range of concentrations amenable to detection increased, according to the fitting of the EIS data using the Langmuir-Freundlich adsorption model. The performance of the two film architectures was compared qualitatively by plotting the data with a multidimensional projection technique, which constitutes a generic approach for optimizing immunosensors and other types of sensors.

  5. Gold nanoparticle-based low limit of detection Love wave biosensor for carcinoembryonic antigens.

    Science.gov (United States)

    Li, Shuangming; Wan, Ying; Su, Yan; Fan, Chunhai; Bhethanabotla, Venkat R

    2017-09-15

    In this work, a Love wave biosensing platform is described for detecting cancer-related biomarker carcinoembryonic antigen (CEA). An ST 90°-X quartz Love wave device with a layer of SiO 2 waveguide was combined with gold nanoparticles (Au NPs) to amplify the mass loading effect of the acoustic wave sensor to achieve a limit of detection of 37pg/mL. The strategy involves modifying the Au NPs with anti-CEA antibody conjugates to form nanoprobes in a sandwich immunoassay. The unamplified detection limit of the Love wave biosensor is 9.4ng/mL. This 2-3 order of magnitude reduction in the limit of detection brings the SAW platform into the range useful for clinical diagnosis. Measurement electronics and microfluidics are easily constructed for acoustic wave biosensors, such as the Love wave device described here, allowing for robust platforms for point of care applications for cancer biomarkers in general. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Characteristics of prostate cancers detected at prostate specific antigen levels less than 2.5 ng/ml.

    Science.gov (United States)

    Meeks, Joshua J; Loeb, Stacy; Helfand, Brian T; Kan, Donghui; Smith, Norm D; Catalona, William J

    2009-06-01

    The Prostate Cancer Prevention Trial reported that 15% of men with a prostate specific antigen less than 4 ng/ml and a normal digital rectal examination have biopsy detectable prostate cancer. However, limited published data describe the tumor features of prostate cancer detected at low prostate specific antigen levels (less than 2.5 ng/ml). A total of 1,278 men underwent radical retropubic prostatectomy by 1 surgeon between 2003 and 2008. We describe the clinicopathological features of 77 patients with a preoperative prostate specific antigen of less than 2.5 ng/ml. Of the men with a low prostate specific antigen (less than 2.5 ng/ml) tumor 51 (66%) had findings suspicious for prostate cancer on digital rectal examination. Indications for prostate biopsy in the remainder of men included an increased prostate specific antigen velocity, hematospermia and abnormal transrectal ultrasound findings. Prostate cancer was detected at transurethral resection of the prostate in the remaining 8% of men. Despite having a low prostate specific antigen at diagnosis 8 (10.4%) and 20 (26%) men, respectively, had biopsy and radical retropubic prostatectomy Gleason grade 7 disease or greater, while 7 (9%) and 6 (7.8%), respectively, had extracapsular tumor extension or positive surgical margins. Compared to men with a normal digital rectal examination mean tumor volume was significantly higher in those with a suspicious digital rectal examination (3.3 vs 1.7 cc, p = 0.018). Despite having a prostate specific antigen of less than 2.5 ng/ml at diagnosis, a considerable proportion of men had aggressive pathological features at radical retropubic prostatectomy. Digital rectal examination remains an important component of early prostate cancer detection.

  7. Universal Breast Cancer Antigens as Targets Linking Early Detection and Therapeutic Vaccination

    National Research Council Canada - National Science Library

    Domchek, Susan M

    2005-01-01

    This grant supports studies to understand the potential of universal tumor antigens for cancer immunotherapy, with a particular focus on the characterization of the human telomerase reverse transcriptase (hTERT) as tumor antigen...

  8. Gallium-67 myocardial imaging for the detection of bacterial endocarditis

    International Nuclear Information System (INIS)

    Wiseman, J.; Rouleau, J.; Rigo, P.; Strauss, H.W.; Pitt, B.

    1976-01-01

    Eleven patients with a clinical diagnosis of bacterial endocarditis underwent scintillation scanning of the precordial region 2--7 days after the intravenous administration of 3 mCi of gallium-67 citrate. Seven had positive scans, 3 of which were confirmed by postmortem imaging at autopsy. Serial images revealed the scans to be frequently negative at 48 hours and positive from 3 to 8 days following injection. Uptake was not seen in the region of the myocardium 48 hours or longer after the injection of 15 patients without endocarditis used as controls

  9. Gallium-67 myocardial imaging for the detection of bacterial endocarditis

    Energy Technology Data Exchange (ETDEWEB)

    Wiseman, J.; Rouleau, J.; Rigo, P.; Strauss, H.W.; Pitt, B.

    1976-07-01

    Eleven patients with a clinical diagnosis of bacterial endocarditis underwent scintillation scanning of the precordial region 2--7 days after the intravenous administration of 3 mCi of gallium-67 citrate. Seven had positive scans, 3 of which were confirmed by postmortem imaging at autopsy. Serial images revealed the scans to be frequently negative at 48 hours and positive from 3 to 8 days following injection. Uptake was not seen in the region of the myocardium 48 hours or longer after the injection of 15 patients without endocarditis used as controls.

  10. 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

  11. Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

    Directory of Open Access Journals (Sweden)

    Suzanne Kalb

    2011-03-01

    Full Text Available Matrix-assisted laser-desorption time-of-flight (MALDI-TOF mass spectrometry (MS is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA which combines with lethal factor (LF and edema factor (EF, forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

  12. Validation and operational application of a rapid method for rabies antigen detection.

    Science.gov (United States)

    Saturday, Greg A; King, Robin; Fuhrmann, Leslie

    2009-01-01

    The Veterinary Laboratory Europe (VLE) deployed a direct, rapid immunohistochemical test (DRIT), developed by the Centers for Disease Control and Prevention (CDC), to the US military veterinary units in Iraq and Afghanistan. The test detects rabies virus antigen in fresh and frozen brainstem samples from various animal species. The goal was to conduct surveillance in 2 countries without current rabies diagnostic capabilities and little historical data on rabies prevalence. During the deployment the DRIT was evaluated, and Veterinary Corps officers and technicians were trained to operate the test. Civilian veterinarians from both Iraq and Afghanistan were organized for a lecture forum on rabies, a sample collection lab, and familiarization with the DRIT. Samples collected in Iraq and Afghanistan were tested in the field with the DRIT, and compared to the traditional laboratory standard utilizing direct fluorescent antibody testing at VLE and the CDC with 100% agreement.

  13. The diagnostic accuracy of carcinoembryonic antigen to detect colorectal cancer recurrence – A systematic review

    DEFF Research Database (Denmark)

    Sørensen, Caspar G; Karlsson, William K; Pommergaard, Hans-Christian

    2016-01-01

    INTRODUCTION: Carcinoembryonic Antigen (CEA) has been used as a tumor marker in the follow-up of colorectal cancer for more than 40 years. Controversy exists regarding its diagnostic applicability due to a relatively low sensitivity and a questionable effect on mortality. The aim of this review...... was to assess the diagnostic accuracy of CEA in detecting recurrence after intended curative surgery for primary colorectal cancer. METHODS: Systematic literature searches were performed in PubMed, EMBASE and Cochrane databases, and articles were chosen based on predefined inclusion criteria. Reference lists...... from included articles were manually searched for additional publications of relevance. RESULTS: Forty-two original studies with generally representative populations and long follow-up were included. Data were reported on outcomes from 9,834 CEA tests during follow-up. Reporting on the reference...

  14. [THE APPLICATION OF DOT-TECHNIQUE FOR DETECTING ANTIGENS OF ADENOVIRUS IN CLINICAL SAMPLES].

    Science.gov (United States)

    Ivanova, I A; Pisareva, M M; Leontieva, G F; Smirnova, T D; Sorokin, E V; Amosova, I V; Petrova, E R; Shaldjian, A A; Sirosh, A A; Maiorova, V G

    2016-02-01

    The article substantiates possibility of application of point enzyme-linked immunosorbent assay (dot-technique) for detecting viral antigens in samples from patients. To diagnose adenovirus infection conjugate of virus-specific monoclonal antibodies and peroxidase of horse-radish were used The chromatographic rectification of conjugate from free peroxidase permits diminishing background coloring of nitrocellulose membrane and therefore to increase sensitivity. The application of direct conjugates on the basis of virus-specific monoclonal antibodies increases specifcity of dot-technique and significantly shortens time period of analysis. As in case of application of direct conjugates on the basis of polyclonal serum, samples from patients require preliminary processing with detergent for preventing non-specific reactions. The dot-technique demonstrates good coincidence with data of polymerase chain reaction and after clinical trials it can be used in diagnostic of human viral infections.

  15. Impact of sensitivity of human leucocyte antigen antibody detection by Luminex technology on graft loss at 1 year

    OpenAIRE

    Szatmary, Peter; Jones, James; Hammad, Abdul; Middleton, Derek

    2013-01-01

    Background The clinical relevance of the detection of human leucocyte antigen (HLA) antibodies in sera of renal transplant recipients by highly sensitive methods such as Luminex alone is uncertain and a matter of debate. The choice of output thresholds affects antibody detection and thus organ allocation, yet there are no internationally agreed threshold levels. This study aims at evaluating our current practice of using an MFI threshold of 1000 in antibody detection. Methods We carried out a...

  16. DETECTION OF BACTERIAL SMALL TRANSCRIPTS FROM RNA-SEQ DATA: A COMPARATIVE ASSESSMENT.

    Science.gov (United States)

    Peña-Castillo, Lourdes; Grüell, Marc; Mulligan, Martin E; Lang, Andrew S

    2016-01-01

    Small non-coding RNAs (sRNAs) are regulatory RNA molecules that have been identified in a multitude of bacterial species and shown to control numerous cellular processes through various regulatory mechanisms. In the last decade, next generation RNA sequencing (RNA-seq) has been used for the genome-wide detection of bacterial sRNAs. Here we describe sRNA-Detect, a novel approach to identify expressed small transcripts from prokaryotic RNA-seq data. Using RNA-seq data from three bacterial species and two sequencing platforms, we performed a comparative assessment of five computational approaches for the detection of small transcripts. We demonstrate that sRNA-Detect improves upon current standalone computational approaches for identifying novel small transcripts in bacteria.

  17. Detection of Hepatitis B Virus Antigens in Paraffin-embedded Liver Specimens from the Amazon Region, Brazil

    Directory of Open Access Journals (Sweden)

    Simonetti SRR

    2002-01-01

    Full Text Available Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg and hepatitis B core antigen (HBcAg by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4% histological samples were HBsAg reactive and 5 (6.3% were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

  18. Development of a double-antibody radioimmunoassay for detecting ovarian tumor-associated antigen fraction OCA in plasma

    International Nuclear Information System (INIS)

    Knauf, S.; Urbach, G.I.

    1978-01-01

    Ovarian tumor-associated antigen isolated from human tumor tissue was shown to have a different mobility from that of carcinoembryonic antigen (CEA) in both acrylamide gel electrophoresis and immunoelectrophoresis in agarose. The ovarian tumor antigen is composed of six species with different electrophoretic mobility in acrylamide gel electrophoresis. Three of these species were detected in Sephadex G-100 ovarian fraction OCA (from the void volume peak) and the other three species of lower apparent molecular weight were detected in fraction OCD (from the second peak). Fractions OCA and OCD did not share common antigenic determinants as determined by immunodiffusion. CEA was shown to share antigenic determinants with both OCA and OCD. A double-antibody radioimmunoassay capable of detecting nanogram quantities of plasma OCA was developed. In a preliminary study of ovarian cancer patients, OCA appeared to be a more sensitive marker for ovarian cancer than CEA. There was virtually no correlation (r 2 = 0.1) between OCA and CEA levels in these patients, as determined by radioimmunoassay

  19. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

    Science.gov (United States)

    Montanaro, Jacqueline; Inic-Kanada, Aleksandra; Ladurner, Angela; Stein, Elisabeth; Belij, Sandra; Bintner, Nora; Schlacher, Simone; Schuerer, Nadine; Mayr, Ulrike Beate; Lubitz, Werner; Leisch, Nikolaus; Barisani-Asenbauer, Talin

    2015-01-01

    To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

  20. Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

    Science.gov (United States)

    Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

    2017-08-01

    To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple

  1. Broad-Range Bacterial Capture from Fluid-Samples: Implications for Amplification-Free Contamination Detection

    Directory of Open Access Journals (Sweden)

    Monika WEBER

    2016-08-01

    Full Text Available Fluid-Screen, Inc. presents a bacterial concentration and filtration method based on dielectrophoresis and alternating current kinetics. Dielectrophoresis has been previously shown to induce particle motion; however, bacterial capture efficiency and reproducibility have consistently been low, reducing its potential for practical applications. In this study, we introduce a novel, patent-pending electrode system optimized to simultaneously capture a wide range of bacterial species from a variety of aqueous solutions. Specifically, we show that the method of dielectrophoresis used induces responses in both characteristic Gram- negative Escherichia coli and Gram-positive Enterococcus faecalis bacteria, as well as with Bacillus subtilis and Aestuariimicrobium kwangyangense. We have adapted the electrode design to create a bacterial sample preparatio unit, termed the sample sorter, that is able to capture multiple bacterial species and release them simultaneously for bacterial concentration and exchange from complex matrices to defined buffer media. This technology can be used on its own or in conjunction with standard bacterial detection methods such as mass spectroscopy. The Fluid-Screen product will dramatically improve testing and identification of bacterial contaminants in various industrial settings by eliminating the need for amplification of samples and by reducing the time to identification.

  2. Detection of mastitis pathogens by analysis of volatile bacterial metabolites

    NARCIS (Netherlands)

    Hettinga, K.A.; Valenberg, van H.J.F.; Lam, T.J.G.M.; Hooijdonk, van A.C.M.

    2008-01-01

    The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In

  3. Real-time qPCR improves meningitis pathogen detection in invasive bacterial-vaccine preventable disease surveillance in Fiji

    OpenAIRE

    Dunne, Eileen M.; Mantanitobua, Silivia; Singh, Shalini P.; Reyburn, Rita; Tuivaga, Evelyn; Rafai, Eric; Tikoduadua, Lisi; Porter, Barbara; Satzke, Catherine; Strachan, Janet E.; Fox, Kimberly K.; Jenkins, Kylie M.; Jenney, Adam; Baro, Silo; Mulholland, E. Kim

    2016-01-01

    As part of the World Health Organization Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance in Suva, Fiji, cerebrospinal fluid (CSF) samples from suspected meningitis patients of all ages were examined by traditional methods (culture, Gram stain, and latex agglutination for bacterial antigen) and qPCR for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Of 266 samples tested, pathogens were identified in 47 (17.7%). S. pneumoniae was the most co...

  4. Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens.

    Directory of Open Access Journals (Sweden)

    Ana Maria Fonseca

    Full Text Available Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens.We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87. IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106 than in Mozambican men (n = 102 and Spanish individuals (n = 101; p<0.05. Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003-2005 than during 2010-2012, in accordance with the levels of malaria transmission reported for these years in Mozambique.The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among pregnant women and dependent on the

  5. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti...

  6. Development of an Immunochromatographic Test for Diagnosis of Visceral Leishmaniasis Based on Detection of a Circulating Antigen.

    Directory of Open Access Journals (Sweden)

    Chun-hua Gao

    Full Text Available Visceral leishmaniasis (VL is a life-threatening disease caused by protozoan parasites of the Leishmania donovani complex. Early case detection followed by adequate treatment is essential to the control of VL. However, the available diagnostic tests are either invasive and require considerable expertise (parasitological demonstration of the parasite in tissue smears or unable to distinguish between past and active infection (serological methods. Therefore, we aimed to develop a lateral flow assay in the form of an immunochromatographic test (ICT device based on the detection of a circulating Leishmania antigen using monoclonal antibodies (mAbs.mAbs were produced by fusion of murine myeloma cells with splenocytes isolated from a mouse immunized with L. donovani soluble crude antigen. Out of 12 cloned hybridoma cell lines, two secreted mAbs recognizing the same leishmanial protein. These mAbs were used to produce an ICT as a sandwich assay for the detection of circulating antigen in serum and blood samples. The ICT was evaluated with 213 serum samples from VL patients living in VL endemic areas in China, and with 156 serum samples from patients with other diseases as well as 78 serum samples from healthy donors. Sensitivity, specificity and diagnostic efficiency of the new ICT was 95.8%, 98.7% and 97.3%, respectively. Compared with a commercially available antibody detecting ICT, our antigen-based ICT performed slightly better.The newly developed ICT is an easy to use and more accurate diagnostic tool which fulfils the performance and operational characteristics required for VL case detection under field and laboratory conditions. As our ICT detects a circulating antigen, it will also be useful in monitoring treatment success and diagnosing VL in immunocompromised patients.

  7. Evaluation of antigen detection and polymerase chain reaction for diagnosis of amoebic liver abscess in patients on anti-amoebic treatment

    Directory of Open Access Journals (Sweden)

    Jaiswal Virendra

    2012-08-01

    Full Text Available Abstract Background Diagnosis of amoebic liver abscess (ALA in patients on anti-amoebic drugs is difficult. There is scanty data on this issue using Entamoeba histolytica (E. histolytica lectin antigen and polymerase chain reaction (PCR. We studied utility of lectin antigen, PCR, and IgG antibody in diagnosis of liver abscess in patients on anti-amoebic treatment. Liver aspirate of 200 patients, of which 170 had anti-amoebic drug prior to drainage, was tested for E. histolytica lectin antigen by (ELISA, PCR, bacterial culture, and serum IgG antibody by (ELISA. Classification of abscesses was based on result of anti-amoebic IgG antibody and bacterial culture, E. histolytica PCR and bacterial culture, and E. histolytica lectin antigen and bacterial culture. Findings Using anti-amoebic IgG antibody and bacterial culture, 136/200 (68.0% were classified as ALA, 12/200 (6.0% as pyogenic liver abscess (PLA, 29/200 (14.5% as mixed infection, and 23/200 (11.5% remained unclassified. Using amoebic PCR and bacterial culture 151/200 (75.5% were classified as ALA, 25/200 (12.5% as PLA, 16/200 (8.0% as mixed infection, and 8/200 (4.0% remained unclassified. With E. histolytica lectin antigen and bacterial culture, 22/200 (11.0% patients were classified as ALA, 39/200 (19.5% as PLA, 2/200 (1.0% as mixed infection, and 137/200 (68.5% remained unclassified. Conclusions E. histolytica lectin antigen was not suitable for classification of ALA patients who had prior anti-amoebic treatment. However, PCR may be used as alternative test to anti-amoebic antibody in diagnosis of ALA.

  8. Counterpoint: Prostate-specific antigen velocity is not of value for early detection of cancer.

    Science.gov (United States)

    Vickers, Andrew J

    2013-03-01

    Firm evidence shows that prostate-specific antigen (PSA) velocity is statistically associated with many prostate cancer outcomes, including those related to early detection. However, the clinical use of a marker depends on clinical and statistical significance. Before PSA velocity is used to inform decisions such as whether to perform a biopsy, evidence should be clear that doing so would improve clinical outcome. A systematic review on PSA velocity found that almost no studies had evaluated whether PSA velocity aids in clinical decision-making. Since that time, several reports have indicated that including PSA in a statistical model alongside standard predictors (eg, PSA, digital rectal examination) does not improve predictive accuracy. Specifically, performing a biopsy on men with high PSA velocity in the absence of other indications, as recommended by the NCCN Clinical Practice Guidelines in Oncology for Prostate Cancer Early Detection, would lead to many millions of unnecessary biopsies, without a corresponding number of aggressive cancers being detected. Advocates of PSA velocity have been reduced to citing a single article claiming that PSA velocity aids in clinical decision-making. The article involves selective reporting of an unusual subgroup analysis based on an extremely limited number of events. This is not to say that, in clinical practice, urologists should ignore prior PSA values: clinical judgment can be aided by careful longitudinal evaluation of PSA changes, interpreted in the context of symptoms and treatments. However, the literature clearly shows that simplistic application of PSA velocity cutoffs is not of value for early detection of prostate cancer.

  9. Prostate Cancer Detection and Prognosis: From Prostate Specific Antigen (PSA) to Exosomal Biomarkers.

    Science.gov (United States)

    Filella, Xavier; Foj, Laura

    2016-10-26

    Prostate specific antigen (PSA) remains the most used biomarker in the management of early prostate cancer (PCa), in spite of the problems related to false positive results and overdiagnosis. New biomarkers have been proposed in recent years with the aim of increasing specificity and distinguishing aggressive from non-aggressive PCa. The emerging role of the prostate health index and the 4Kscore is reviewed in this article. Both are blood-based tests related to the aggressiveness of the tumor, which provide the risk of suffering PCa and avoiding negative biopsies. Furthermore, the use of urine has emerged as a non-invasive way to identify new biomarkers in recent years, including the PCA3 and TMPRSS2:ERG fusion gene. Available results about the PCA3 score showed its usefulness to decide the repetition of biopsy in patients with a previous negative result, although its relationship with the aggressiveness of the tumor is controversial. More recently, aberrant microRNA expression in PCa has been reported by different authors. Preliminary results suggest the utility of circulating and urinary microRNAs in the detection and prognosis of PCa. Although several of these new biomarkers have been recommended by different guidelines, large prospective and comparative studies are necessary to establish their value in PCa detection and prognosis.

  10. Metal-organic gel enhanced fluorescence anisotropy for sensitive detection of prostate specific antigen

    Science.gov (United States)

    Zhao, Ting Ting; Peng, Zhe Wei; Yuan, Dan; Zhen, Shu Jun; Huang, Cheng Zhi; Li, Yuan Fang

    2018-03-01

    In this contribution, we demonstrated that Cu-based metal-organic gel (Cu-MOG) was able to serve as a novel amplification platform for fluorescence anisotropy (FA) assay for the first time, which was confirmed by the sensitive detection of a common cancer biomarker, prostate specific antigen (PSA). The dye-labeled probe aptamer (PA) product was adsorbed onto the benzimidazole derivative-containing Cu-MOG via electrostatic incorporation and strong π-π stacking interactions, which significantly increased the FA value due to the enlargement of the molecular volume of the PA/Cu-MOG complex. With the introduction of target PSA, the FA value was obviously decreased on account of the specific recognition between PSA and PA which resulted in the detachment of PA from the surface of MOG. The linear range was from 0.5-8 ng/mL, with a detection limit of 0.33 ng/mL. Our work has thus helped to demonstrate promising application of MOG material in the fields of biomolecules analysis and disease diagnosis.

  11. Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

    Science.gov (United States)

    Okabayashi, Tamaki; Sasaki, Tadahiro; Masrinoul, Promsin; Chantawat, Nantarat; Yoksan, Sutee; Nitatpattana, Narong; Chusri, Sarunyou; Morales Vargas, Ronald E; Grandadam, Marc; Brey, Paul T; Soegijanto, Soegeng; Mulyantno, Kris Cahyo; Churrotin, Siti; Kotaki, Tomohiro; Faye, Oumar; Faye, Ousmane; Sow, Abdourahmane; Sall, Amadou Alpha; Puiprom, Orapim; Chaichana, Panjaporn; Kurosu, Takeshi; Kato, Seiji; Kosaka, Mieko; Ramasoota, Pongrama; Ikuta, Kazuyoshi

    2015-02-01

    Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Comparison of direct and indirect immunofluorescence staining of clinical specimens for detection of respiratory syncytial virus antigen.

    OpenAIRE

    Minnich, L L; Ray, C G

    1982-01-01

    Immunofluorescence staining methods for respiratory syncytial virus antigen detection were compared. Of 50 specimens originally positive for respiratory syncytial virus by direct immunofluorescence and culture, 49 were positive by repeat direct immunofluorescence and 32 were positive by indirect immunofluorescence. Additional results obtained on specimens originally negative for respiratory syncytial virus by direct immunofluorescence, culture, or both indicate that direct immunofluorescence ...

  13. Detection and partial characterization of a midlamina lucida-hemidesmosome-associated antigen (19-DEJ-1) present within human skin

    DEFF Research Database (Denmark)

    Fine, J D; Horiguchi, Y; Jester, J

    1989-01-01

    immunoelectron microscopy demonstrated localization of 19-DEJ-1 to the level of the midlamina lucida, directly underneath hemidesmosomes; absent staining was noted beneath melanocytes. 19-DEJ-1 antigen was detectable in unfixed A431 cells grown on coverslips. After radioincorporation of 35S-methionine into A431...

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

    Science.gov (United States)

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

    2017-04-01

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

  15. A functional gene array for detection of bacterial virulence elements

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C

    2007-11-01

    We report our development of the first of a series of microarrays designed to detect pathogens with known mechanisms of virulence and antibiotic resistance. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. To validate our approach, we developed a first generation array targeting genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for microorganism detection and discrimination, measured the required target concentration, and assessed tolerance for mismatches between probe and target sequences. Mismatch tolerance is a priority for this application, due to DNA sequence variability among members of gene families. Arrays were created using the NimbleGen Maskless Array Synthesizer at Lawrence Livermore National Laboratory. Purified genomic DNA from combinations of one or more of the four target organisms, pure cultures of four related organisms, and environmental aerosol samples with spiked-in genomic DNA were hybridized to the arrays. Based on the success of this prototype, we plan to design further arrays in this series, with the goal of detecting all known virulence and antibiotic resistance gene families in a greatly expanded set of organisms.

  16. 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...

  17. Development of a lateral flow immunoassay strip for rapid detection of CagA antigen of Helicobacter pylori.

    Science.gov (United States)

    Karakus, Cebrail

    2015-01-01

    About half of the world populations are known to be infected with Helicobacter pylori. The CagA antigen secreting strains provoke severe mucosal damages and act as a risk factor for the development of peptic ulceration and gastric cancer. A lateral flow immunoassay (LFIA) strip was developed based on sandwich format for rapid detection of CagA antigen of H. pylori using gold conjugated monoclonal antibody. This LFIA strip will provide a good aid in the diagnosis of CagA-secreting H. pylori within 10 min instead of time consuming, expensive and laborious invasive approaches.

  18. Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections

    NARCIS (Netherlands)

    van den Bos, I. C.; Khanolkar-Young, S.; Das, P. K.; Lockwood, D. N.

    1999-01-01

    A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid

  19. Fabrication of graphene/gold-modified screen-printed electrode for detection of carcinoembryonic antigen

    Energy Technology Data Exchange (ETDEWEB)

    Chan, K.F. [Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, UPM Serdang, 43400 Selangor (Malaysia); Lim, H.N., E-mail: janetlimhn@gmail.com [Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, UPM Serdang, 43400 Selangor (Malaysia); Shams, N. [Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, UPM Serdang, 43400 Selangor (Malaysia); Jayabal, S.; Pandikumar, A.; Huang, N.M. [Low Dimensional Materials Research Centre (LDMRC), Physics Department, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2016-01-01

    Immunosensors based on gold nanoparticles and reduced graphene oxide (AuNPs/rGO)-modified screen-printed electrodes (SPEs) were successfully synthesized using an electrochemical deposition method. The modified SPEs were characterized using a field emission scanning electron microscope (FESEM) and Raman spectroscopy to analyze the morphology and composition of AuNPs and rGO. Both the FESEM and Raman spectroscopy revealed that the AuNPs were successfully anchored on the thin film of rGO deposited on the surface of the SPEs. Characterization with a ferri–ferrocyanide couple [Fe(CN){sub 6}{sup 3−/4−}] showed that the electron transfer kinetic between the analyte and electrode was enhanced after the modification with the AuNPs/rGO composite on the electrode surface, in addition to increasing the effective surface area of the electrode. The modified SPE was immobilized with a sandwich type immunosensor to mimic the ELISA (enzyme-linked immunosorbent assay) immunoassay. The modified SPE that was fortified with the sandwich type immunosensor exhibited double electrochemical responses in the detection of carcinoembryonic antigen (CEA), with linear ranges of 0.5–50 ng/mL and 250–2000 ng/mL and limits of detection of 0.28 ng/mL and 181.5 ng/mL, respectively. - Highlights: • An AuNP/rGO-modified SPE is prepared via an in-situ electrodeposition method. • It is introduced in a sandwich-type immunoassay for the detection of CEA. • The LODs for CEA are 0.28 ng/mL for 0.5–25 ng/mL, and 181.5 ng/mL for 250–2000 ng/mL.

  20. Role of serum carcinoembryonic antigen in the detection of colorectal cancer before and after surgical resection

    Science.gov (United States)

    Su, Bin-Bin; Shi, Hui; Wan, Jun

    2012-01-01

    AIM: To determine whether serum levels of carcinoembryonic antigen (CEA) correlate with the presence of primary colorectal cancer (CRC), and/or recurrent CRC following radical resection. METHODS: A total of 413 patients with CRC underwent radical surgery between January 1998 and December 2002 in our department and were enrolled in this study. The median follow-up period was 69 mo (range, 3-118 mo), and CRC recurrence was experienced by 90/413 (21.8%) patients. Serum levels of CEA were assayed preoperatively, and using a cutoff value of 5 ng/mL, patients were divided into two groups, those with normal serum CEA levels (e.g., ≤ 5 ng/mL) and those with elevated CEA levels (> 5 ng/mL). RESULTS: The overall sensitivity of CEA for the detection of primary CRC was 37.0%. The sensitivity of CEA according to stage, was 21.4%, 38.9%, and 41.7% for stages I-III, respectively. Moreover, for stage II and stage III cases, the 5-year disease-free survival rates were reduced for patients with elevated preoperative serum CEA levels (P < 0.05). The overall sensitivity of CEA for detecting recurrent CRC was 54.4%, and sensitivity rates of 36.6%, 66.7%, and 75.0% were associated with cases of local recurrence, single metastasis, and multiple metastases, respectively. In patients with normal serum levels of CEA preoperatively, the sensitivity of CEA for detecting recurrence was reduced compared with patients having a history of elevated CEA prior to radical resection (32.6% vs 77.3%, respectively, P < 0.05). CONCLUSION: CRC patients with normal serum CEA levels prior to resection maintained these levels during CRC recurrence, especially in cases of local recurrence vs cases of metastasis. PMID:22563201

  1. Aptamer-based microchip electrophoresis assays for amplification detection of carcinoembryonic antigen.

    Science.gov (United States)

    Pan, Li; Zhao, Jingjin; Huang, Yong; Zhao, Shulin; Liu, Yi-Ming

    2015-10-23

    Carcinoembryonic antigen (CEA) as one of the most widely used tumor markers is used in the clinical diagnosis of colorectal, pancreatic, gastric, and cervical carcinomas. We developed an aptamer-based microchip electrophoresis assay technique for assaying CEA in human serum for cancer diagnosis. The magnetic beads (MBs) are employed as carriers of double strand DNA that is formed by an aptamer of the target and a complementary DNA of the aptamer. After the aptamer in the MB-dsDNA conjugate binds with the target, the complementary DNA was released from the MB-dsDNA conjugate. The released complementary DNA hybridizes with a fluorescein amidite (FAM) labeled DNA, and forms a DNA duplex, which triggers the selective cleavage of FAM labeled DNA by nicking endonuclease Nb.BbvCI, and generating a FAM labeled DNA segment. The released complementary DNA hybridizes with another FAM labeled DNA, resulting in a continuous cleavage of FAM labeled DNA, and the generation of large numbers of FAM labeled DNA segments. In MCE laser induced fluorescence detection (LIF), the FAM labeled DNA segment is separated and detected. The linear range for CEA was 130 pg/ml-8.0 ng/ml with a correlation coefficient of 0.9916 and a detection limit of 68 pg/ml. The CEA concentration in the serum samples from healthy subjects was found to be in the range 1.3 ng/ml to 3.2 ng/ml. The CEA concentration in the samples from cancer patients was found to be >15 ng/ml. This method may become a useful tool for rapid analysis of CEA and other tumor markers in biomedical analysis and clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice.

    Science.gov (United States)

    Cui, Z; Ojaghian, M R; Tao, Z; Kakar, K U; Zeng, J; Zhao, W; Duan, Y; Vera Cruz, C M; Li, B; Zhu, B; Xie, G

    2016-05-01

    The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae. Specific primers were designed through a bioinformatics pipeline. Sensitivity of detection was established using both traditional PCR and quantitative real-time PCR on isolated DNA and on bacterial cells both in vitro and in simulated diseased seeds and the parameters were optimized for an mPCR assay. A total of 150 bacterial strains were tested for specificity. The mPCR assay accurately predicted the presence of pathogens among 44 symptomatic and asymptomatic rice seed, sheath and leaf samples. This study confirmed that this mPCR assay is a rapid, reliable and simple tool for the simultaneous detection of six important rice bacterial pathogens. This study is the first report of a method allowing simultaneous detection of six major rice pathogens. The ability to use crude extracts from plants without bacterial isolation or DNA extraction enhances the value of this mPCR technology for rapid detection and aetiological/epidemiological studies. © 2016 The Society for Applied Microbiology.

  3. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

    Directory of Open Access Journals (Sweden)

    Montanaro J

    2015-07-01

    Full Text Available Jacqueline Montanaro,1 Aleksandra Inic-Kanada,1 Angela Ladurner,1 Elisabeth Stein,1 Sandra Belij,1 Nora Bintner,1 Simone Schlacher,1 Nadine Schuerer,1 Ulrike Beate Mayr,2 Werner Lubitz,2 Nikolaus Leisch,3 Talin Barisani-Asenbauer11Laura Bassi Centres of Expertise, OCUVAC – Centre of Ocular Inflammation and Infection, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria; 2BIRD-C GmbH & Co KG, Kritzendorf, Austria; 3Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaAbstract: To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN, whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results

  4. Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping.

    Science.gov (United States)

    Hayashi, Tomoya; Hirayama, Fumiya

    2015-07-01

    Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia.

  5. Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe

    Directory of Open Access Journals (Sweden)

    Wang S

    2016-01-01

    Full Text Available Shuo Wang, Wanming Li, Dezheng Yuan, Jindan Song, Jin Fang Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, People’s Republic of China Abstract: The large external antigen (LEA is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605 conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05, indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of

  6. Brucella abortus RB51 and hot saline extract from Brucella ovis as antigens in a complement fixation test used To detect sheep vaccinated with Brucella abortus RB51.

    Science.gov (United States)

    Adone, R; Ciuchini, F

    2001-01-01

    The efficacy of Brucella abortus RB51 and hot saline extract (HSE) from Brucella ovis as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with B. abortus strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 x 10(9) CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with RB51 as antigen, previously deprived of anticomplementary activity, and with HSE antigen, which already used as the official antigen to detect B. ovis-infected sheep. The results showed that vaccinated sheep developed antibodies which reacted weakly against HSE antigen and these antibodies were detectable for 30 days after vaccination. However, antibodies to RB51 could be detected for a longer period after vaccination by using homologous RB51 antigen in CF tests. In fact, high titers were still present at 110 days postvaccination with RB51 antigen. Sera from sheep naturally infected with B. ovis also reacted to RB51 but gave lower titers than those detected by HSE antigen. As expected, all sera from RB51-vaccinated sheep remained negative when tested with standard S-type Brucella standard antigens.

  7. Simultaneous Detection of Three Bacterial Seed-Borne Diseases in Rice Using Multiplex Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    In Jeong Kang

    2016-12-01

    Full Text Available Burkholderia glumae (bacterial grain rot, Xanthomonas oryzae pv. oryzae (bacterial leaf blight, and Acidovorax avenae subsp. avenae (bacterial brown stripe are major seedborne pathogens of rice. Based on the 16S and 23S rDNA sequences for A. avenae subsp. avenae and B. glumae, and transposase A gene sequence for X. oryzae pv. oryzae, three sets of primers had been designed to produce 402 bp for B. glumae, 490 bp for X. oryzae, and 290 bp for A. avenae subsp. avenae with the 63°C as an optimum annealing temperature. Samples collected from naturally infected fields were detected with two bacteria, B. glumae and A. avenae subsp. avenae but X. oryzae pv. oryzae was not detected. This assay can be used to identify pathogens directly from infected seeds, and will be an effective tool for the identification of the three pathogens in rice plants.

  8. Simultaneous Detection of Three Bacterial Seed-Borne Diseases in Rice Using Multiplex Polymerase Chain Reaction.

    Science.gov (United States)

    Kang, In Jeong; Kang, Mi-Hyung; Noh, Tae-Hwan; Shim, Hyeong Kwon; Shin, Dong Bum; Heu, Suggi

    2016-12-01

    Burkholderia glumae (bacterial grain rot), Xanthomonas oryzae pv. oryzae (bacterial leaf blight), and Acidovorax avenae subsp. avenae (bacterial brown stripe) are major seedborne pathogens of rice. Based on the 16S and 23S rDNA sequences for A. avenae subsp. avenae and B. glumae , and transposase A gene sequence for X. oryzae pv. oryzae , three sets of primers had been designed to produce 402 bp for B. glumae , 490 bp for X. oryzae , and 290 bp for A. avenae subsp. avenae with the 63°C as an optimum annealing temperature. Samples collected from naturally infected fields were detected with two bacteria, B. glumae and A. avenae subsp. avenae but X. oryzae pv. oryzae was not detected. This assay can be used to identify pathogens directly from infected seeds, and will be an effective tool for the identification of the three pathogens in rice plants.

  9. Detection of Peroxynitrite in Plants Exposed to Bacterial Infection.

    Science.gov (United States)

    Bellin, Diana; Delledonne, Massimo; Vandelle, Elodie

    2016-01-01

    Peroxynitrite is a highly reactive derivative of nitric oxide (NO) which is gaining attention in the plant biology community because it may play a role in NO signaling during biotic stress. Peroxynitrite can react with many different biomolecules, but its ability to nitrate the tyrosine residues of proteins is particularly important because this may regulate defense signaling in response to pathogens. The analysis of peroxynitrite levels in the context of its proposed defense role requires an accurate and specific detection method. Here, we describe a photometric assay using the fluorescent dye Hong Kong Green 2 as a specific and quantitative probe for peroxynitrite in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the preparation of plant samples, the assay procedure, the measurement of peroxynitrite-specific fluorescence, and data presentation.

  10. 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.

  11. Association between use of rapid antigen detection tests and adherence to antibiotics in suspected streptococcal pharyngitis.

    Science.gov (United States)

    Llor, Carl; Hernández, Silvia; Sierra, Nuria; Moragas, Ana; Hernández, Marta; Bayona, Carolina

    2010-03-01

    Few studies have analysed adherence to antibiotic treatment in pharyngitis. The aim of this study was to evaluate the association of rapid antigen detection tests (RADT) and treatment adherence among patients 18 years of age or over with pharyngitis treated with different antibiotic regimens. Prospective study from 2003 to 2008. Office-based physician practices. Intervention. The adherence of patients prior to the use of RADTs - no test was available until mid-2006 - was compared with the adherence associated with the use of RADTs. Patients with suspected streptococcal pharyngitis. Patient adherence was assessed by electronic monitoring. The adherence outcomes considered were antibiotic-taking adherence, correct dosing, and good timing adherence during at least 80% of the antibiotic course. A total of 196 patients were recruited. The percentage of container openings was 77.9%+/-17.7%, being significantly higher for patients in whom the RADTs were performed compared with those in whom this test was not undertaken (80.1% vs. 70.8% for thrice-daily antibiotic regimens and 88.1% vs. 76.5% for twice-daily regimens; p pills (71.3% vs. 42.2%; p dosing was always greater when the patient had undergone an RADT. Adherence to antibiotic treatment is higher when an RADT is carried out at the consultation prior to administration of antibiotic treatment.

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

    Science.gov (United States)

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

    2015-08-25

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

  13. Lectin inhibits antigen-antibody reaction in a glycoform-specific manner: Application for detecting α2,6sialylated-carcinoembryonic antigen.

    Science.gov (United States)

    Ito, Hiromi; Hoshi, Kyoka; Osuka, Fumihiko; Gotoh, Mitsukazu; Saito, Takuro; Hojo, Hiroshi; Suzuki, Rei; Ohira, Hiromasa; Honda, Takashi; Hashimoto, Yasuhiro

    2016-12-01

    Carcinoembryonic antigen (CEA) is a glycoprotein marker, which is widely used for diagnosing various cancers, especially colon adenocarcinoma. In addition, CEA mediates homotypic adhesion of colon adenocarcinoma cells, which appears to favor hematogenous metastasis. CEA carries α2,6sialyl residues on its N-glycans whereas a normal counterpart, normal fecal antigen-2, does α2,3sialyl residues, suggesting that cancer-specific  α2,6sialylation on CEA may play a role for cell invasion and metastasis. A simple and rapid estimation of α2,6sialyled CEA in detergent extracts from formalin-fixed colon adenocarcinoma by "lectin inhibition" is reported. In the lectin inhibition method, Sambucus sieboldiana Agglutinin (SSA) lectin, an α2,6sialic acid binder, was used as a glycoform-specific inhibitor for antigen-antibody reaction in ELISA. Detergent extracts from colon adenocarcinoma showed a fair amount of ELISA signal in the absence of SSA whereas the signal was markedly reduced (45≈74%) in the presence of SSA, suggesting that the extracts contains α2,6sialyled CEA. The presence of α2,6sialyled CEA in the extracts was confirmed by lectin microarray, in which SSA, Sambucus nigra agglutinin, and Trichosanthes japonica agglutinin I lectins were used as α2,6sialyl binders. Thus lectin inhibition is a simple and rapid method for detecting α2,6sialyled CEA even in crude detergent extracts from formalin-fixed adenocarcinoma tissue. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. A novel label-free microfluidic paper-based immunosensor for highly sensitive electrochemical detection of carcinoembryonic antigen.

    Science.gov (United States)

    Wang, Yang; Xu, Huiren; Luo, Jinping; Liu, Juntao; Wang, Li; Fan, Yan; Yan, Shi; Yang, Yue; Cai, Xinxia

    2016-09-15

    In this work, a highly sensitive label-free paper-based electrochemical immunosensor employing screen-printed working electrode (SPWE) for detection of carcinoembryonic antigen (CEA) was fabricated. In order to raise the detection sensitivity and immobilize anti-CEA, amino functional graphene (NH2-G)/thionine (Thi)/gold nanoparticles (AuNPs) nanocomposites were synthesized and coated on SPWE. The principle of the immunosensor determination was based on the fact that the decreased response currents of Thi were proportional to the concentrations of corresponding antigens due to the formation of antibody-antigen immunocomplex. Experimental results revealed that the immunoassay enabled the determination of standard CEA solutions with linear working ranges of 50pgmL(-1) to 500ngmL(-1), the limit of detections for CEA is 10pgmL(-1) (S/N=3) and its corresponding correlation coefficients were 0.996. Furthermore, the proposed immunosensor could be used for the determination of clinical serum samples. A large number of clinical serum samples were detected and the relative errors between measured values and reference concentrations were calculated. Results showed that this novel paper-based electrochemical immunosensor could provide a new platform for low cost, sensitive, specific, and point-of-care diagnosis in cancer detection. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Bacterial gene expression detected in human faeces by reverse transcription-PCR

    NARCIS (Netherlands)

    Fitzsimons, N.A.; Akkermans, A.D.L.; Vos, de W.M.; Vaughan, E.E.

    2003-01-01

    A method to isolate and specifically detect bacterial messenger RNA (mRNA) in human faeces is presented. The surface layer protein gene slpA of Lactobacillus acidophilus ATCC 4356(T) was chosen as a model system because it is transcribed at a high level to a relatively stable mRNA (Boot et al.,

  16. Detection of bacterial blight resistant gene xa5 using linked marker ...

    African Journals Online (AJOL)

    USER

    2010-06-14

    Jun 14, 2010 ... Detection of bacterial blight resistant gene xa5 using linked marker approaches. Shahzad Amir Naveed2, Muhammad Babar3*, Ajuman Arif1, Yusaf Zafar1, Muhammad Sabar1,. Iftikhar Ali2, Muhammad Chragh1 and Muhammad Arif1. 1National Institute for Biotechnology and Genetic Engineering (NIBGE), ...

  17. Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils

    DEFF Research Database (Denmark)

    Gram, T.; Ahrens, Peter; Nielsen, J.P.

    1996-01-01

    strains of A. lignieresii. The lower detection limit of the PCR test was 10(3) A. pleuropneumoniae CFU/PCR test tube and was not affected by addition of 10(6) E. coli CFU/PCR test tube. Mixed bacterial cultures from tonsils of 101 pigs from 9 different herds were tested by culture and by PCR using four...

  18. 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.

  19. Effect of irradiation on the detection of bacterial DNA in contaminated food samples by DNA hybridization

    International Nuclear Information System (INIS)

    Rowe, T.F.; Towner, K.J.

    1994-01-01

    A membrane-based DNA hybridization technique was used in a model system to examine the effect of irradiation treatment on the detection of bacterial contamination in foodstuffs. Although hybridization signals were reduced compared with otherwise identical unirradiated food samples, artificial contamination levels in excess of 10 5 cfu per test could be distinguished in 12 of the 13 foods examined following the irradiation process. In no case were viable bacteria detected following irradiation treatment. (Author)

  20. Evaluation of Dirofilaria immitis antigen detection comparing heated and unheated serum in dogs with experimental heartworm infections

    Directory of Open Access Journals (Sweden)

    James Carmichael

    2017-11-01

    Full Text Available Abstract Background To evaluate whether heated serum allows for earlier detection of Dirofilaria immitis antigen, dogs with experimental D. immitis infections underwent weekly blood sampling to compare antigen results using both heated and unheated serum. Methods One of two isolates (JYD-34 or Big Head™ were used to infect naïve laboratory beagle dogs. Serum was collected from dogs weekly and divided into two aliquots, heated and unheated. The samples designated as heated were placed in a heat block at 104 °C for 10 min then centrifuged with collection of the resulting supernatant. Two commercial ELISAs, DiroCHEK® (Synbiotics Corporation, Zoetis and PetChek® (IDEXX Laboratories, Inc., were used to conduct D. immitis antigen testing on all serum samples. Results There was no statistical difference in the mean number of days from infection to positive D. immitis antigen status between the two commercial testing kits (DiroCHEK® versus PetChek® with either heated or unheated serum. When unheated serum was utilized, very strong agreement between the two assays was demonstrated using Lin’s concordance correlation coefficient (R c  = 0.98. However, when heated serum was compared, Lin’s concordance correlation coefficient was only R c  = 0.64, showing a lesser agreement. There was a statistical difference in the mean number of days from infection to a positive test result for unheated serum when compared to mean days to positive status with heated serum. For DiroCHEK® the heated serum yielded a positive result 126.9 ± 18.9 days postinfection while the unheated serum yielded a positive result 162.6 ± 23.0 days postinfection; this was a significant 35.7 ± 32.2 days longer, on average, compared with heated serum. With PetChek® the heated serum yielded a positive result 131.5 ± 11.7 days postinfection while the unheated serum yielded a positive result 162.8 ± 23.8 days postinfection; this was a significant 31.3

  1. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    Sofía Duque-Beltrán

    2002-12-01

    Full Text Available The present study developed and standardized an enzime-linked immunosorbent assay (ELISA to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate. One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%; specificity, 95% (95% CI: 88.6-97.6%; positive predictive value, 91% (95% CI: 81.4-95.9%; and negative predictive value, 100% (95% CI: 96.1-100%. This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

  2. Comparison of two indirect ELISA coating antigens for the detection of dairy cow antibodies against Pasteurella multocida.

    Science.gov (United States)

    Tankaew, Pallop; Srisawat, Wanwisa; Singhla, Tawatchai; Tragoolpua, Khajornsak; Kataoka, Yasushi; Sawada, Takuo; Sthitmatee, Nattawooti

    2018-02-01

    The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160μg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Measurement of serum carcinoembryonic antigen, carbohydrate antigen 19-9, cytokeratin-19 fragment and matrix metalloproteinase-7 for detecting cholangiocarcinoma: a preliminary case-control study.

    Science.gov (United States)

    Lumachi, Franco; Lo Re, Giovanni; Tozzoli, Renato; D'Aurizio, Federica; Facomer, Flavio; Chiara, Giordano B; Basso, Stefano M M

    2014-11-01

    Cholangiocarcinoma is a malignant tumor of the liver arising from the bile duct epithelium, accounting for 10-25% of all primary hepatic cancers. The clinical presentation of this tumor is not specific and the diagnosis of early cholangiocarcinoma is difficult, especially in patients with other biliary diseases. Measurement of serum carbohydrate antigen (CA) 19-9 and carcinoembryonic antigen (CEA) are commonly used to monitor response to therapy, but are also useful for confirming the presence of a cholangiocarcinoma. In this setting, other biomarkers have been previously tested, including cytokeratin-19 fragment (CYFRA 21-1) and the matrix metalloproteinase-7 (MMP7). The purpose of this retrospective study was to determine the clinical usefulness of the assay of serum CEA, CA 19-9, CYFRA 21-1 and MMP7, individually and together, as tumor markers for the diagnosis of cholangiocarcinoma. Twenty-four patients (14 men, 10 women, 62.6±8.2 years of age) with histologically-confirmed cholangiocarcinoma (cases) and 25 age- and sex-matched patients with benign liver disease (controls) underwent measurement of these biomarkers. The mean values of all serum markers of patients with cholangiocarcinoma were significantly higher (p<0.01) than that of the controls. No correlation was found between serum tumor markers and total bilirubin, aspartate aminotransferase (AST) and alkaline phosphatase (ALP). The sensitivity, specificity and accuracy were: CEA: 52%, 55%, and 58%; CA 19-9: 74%, 82% and 78%; CYFRA 21-1: 76%, 79% and 78%; MMP7: 78%, 77% and 80%, respectively. The combination of all serum markers afforded 92.0% sensitivity and 96% specificity in detecting cholangiocarcinoma, showing the highest diagnostic accuracy (94%). In conclusion, our preliminary results suggest that the measurement of all four biomarkers together can help in the early detection of cholangiocarcinoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights

  4. Cryopreservation of MHC Multimers: Recommendations for Quality Assurance in Detection of Antigen Specific T Cells

    Science.gov (United States)

    Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M; van der Burg, Sjoerd H; Walter, Steffen; Gouttefangeas, Cécile

    2015-01-01

    Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5–16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry PMID:25297339

  5. Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells.

    Science.gov (United States)

    Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M; van der Burg, Sjoerd H; Walter, Steffen; Gouttefangeas, Cécile

    2015-01-01

    Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry.

  6. Evaluation of Sofia Fluorescent immunoassay analyzer for pneumococcal urinary antigen detection in hospitalized patients with community-acquired pneumonia.

    Science.gov (United States)

    Vicente, Diego; López-Olaizola, Maddi; de la Caba, Idoia; Cilla, Gustavo

    2017-10-01

    The Sofia Streptococcus pneumoniae FIA® test was prospectively evaluated in non-concentrated urine samples of 106 hospitalized patients with community-acquired pneumonia. The test detected pneumococcal urinary antigen in 24/31 (77.4%) confirmed pneumococcal community-acquired pneumonia episodes. The specificity of the test was 86.7% (92% after urine heating). Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Comparison of clinical performance of antigen basedenzyme immunoassay (EIA and major outer membrane protein (MOMP-PCR for detection of genital Chlamydia trachomatis infection

    Directory of Open Access Journals (Sweden)

    Mahmoud Nateghi Rostami

    2016-06-01

    Full Text Available Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA and major outer membrane protein (MOMP-polymerase chain reaction (PCR for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV and negative predictive values (NPV were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14% cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMPPCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48. Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  8. The colitis-associated transcriptional profile of commensal Bacteroides thetaiotaomicron enhances adaptive immune responses to a bacterial antigen.

    Directory of Open Access Journals (Sweden)

    Jonathan J Hansen

    Full Text Available Inflammatory bowel diseases (IBD may be caused in part by aberrant immune responses to commensal intestinal microbes including the well-characterized anaerobic gut commensal Bacteroides thetaiotaomicron (B. theta. Healthy, germ-free HLA-B27 transgenic (Tg rats develop chronic colitis when colonized with complex gut commensal bacteria whereas non-transgenic (nTg rats remain disease-free. However, the role of B. theta in causing disease in Tg rats is unknown nor is much known about how gut microbes respond to host inflammation.Tg and nTg rats were monoassociated with a human isolate of B. theta. Colonic inflammation was assessed by histologic scoring and tissue pro-inflammatory cytokine measurement. Whole genome transcriptional profiling of B. theta recovered from ceca was performed using custom GeneChips and data analyzed using dChip, Significance Analysis of Microarrays, and Gene Set Enrichment Analysis (GSEA software. Western Blots were used to determine adaptive immune responses to a differentially expressed B. theta gene.B. theta monoassociated Tg rats, but not nTg or germ-free controls, developed chronic colitis. Transcriptional profiles of cecal B. theta were significantly different in Tg vs. nTg rats. GSEA revealed that genes in KEGG canonical pathways involved in bacterial growth and metabolism were downregulated in B. theta from Tg rats with colitis though luminal bacterial concentrations were unaffected. Bacterial genes in the Gene Ontology molecular function "receptor activity", most of which encode nutrient binding proteins, were significantly upregulated in B. theta from Tg rats and include a SusC homolog that induces adaptive immune responses in Tg rats.B. theta induces colitis in HLA-B27 Tg rats, which is associated with regulation of bacterial genes in metabolic and nutrient binding pathways that may affect host immune responses. These studies of the host-microbial dialogue may lead to the identification of novel microbial targets

  9. Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections

    DEFF Research Database (Denmark)

    Olesen, Cathrine Holm; Brahimi, Karima; Vandahl, Brian

    2010-01-01

    then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies. METHODS: Naturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P....... falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high...... groups of individuals with different levels of exposure to P. falciparum infections. RESULTS: IgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic Ig...

  10. An integrated microfluidic analysis microsystems with bacterial capture enrichment and in-situ impedance detection

    Science.gov (United States)

    Liu, Hai-Tao; Wen, Zhi-Yu; Xu, Yi; Shang, Zheng-Guo; Peng, Jin-Lan; Tian, Peng

    2017-09-01

    In this paper, an integrated microfluidic analysis microsystems with bacterial capture enrichment and in-situ impedance detection was purposed based on microfluidic chips dielectrophoresis technique and electrochemical impedance detection principle. The microsystems include microfluidic chip, main control module, and drive and control module, and signal detection and processing modulet and result display unit. The main control module produce the work sequence of impedance detection system parts and achieve data communication functions, the drive and control circuit generate AC signal which amplitude and frequency adjustable, and it was applied on the foodborne pathogens impedance analysis microsystems to realize the capture enrichment and impedance detection. The signal detection and processing circuit translate the current signal into impendence of bacteria, and transfer to computer, the last detection result is displayed on the computer. The experiment sample was prepared by adding Escherichia coli standard sample into chicken sample solution, and the samples were tested on the dielectrophoresis chip capture enrichment and in-situ impedance detection microsystems with micro-array electrode microfluidic chips. The experiments show that the Escherichia coli detection limit of microsystems is 5 × 104 CFU/mL and the detection time is within 6 min in the optimization of voltage detection 10 V and detection frequency 500 KHz operating conditions. The integrated microfluidic analysis microsystems laid the solid foundation for rapid real-time in-situ detection of bacteria.

  11. Electrochemical immunosensor based on ZnO nanorods-Au nanoparticles nanohybrids for ovarian cancer antigen CA-125 detection.

    Science.gov (United States)

    Gasparotto, Gisane; Costa, João Paulo C; Costa, Paulo I; Zaghete, Maria A; Mazon, Talita

    2017-07-01

    In this research, ZnO nanorods - Au nanoparticles nanohybrids have been fabricated and employed to sensitive electrochemical strategy for the specific detection of the ovarian cancer antigen CA-125/MUC126. The microdevice was developed in our lab based on gold and silver electrodes sputtered on glass substrate. The ZnO nanorods arrays were grown on working electrode using assisted microwave hydrothermal synthesis than gold nanoparticles (Au NPs) were deposited by sputtering. The Au NPs onto ZnO nanorods surface provides a favorable platform for efficient loading of anti-CA-125 antibody via binding with cystamine and glutaraldehyde. The effective loading of the biological material (CA-125 antibody and antigen) on the matrix was observed by SEM images. The electrochemical immunosensor shows a sensitive response to ovarian cancer antigen recombinant human CA-125/MUC126 with detection of 2.5ng/μL, 100 times lower than immunoblot system. Due to high specificity, reproducibility and noteworthy stability, the developed sensor will provide a sensitive, selective and convenient approach to be used to detect CA-125/MUC126. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection.

    Science.gov (United States)

    Higgins, Owen; Clancy, Eoin; Forrest, Matthew S; Piepenburg, Olaf; Cormican, Martin; Boo, Teck Wee; O'Sullivan, Nicola; McGuinness, Claire; Cafferty, Deirdre; Cunney, Robert; Smith, Terry J

    2018-01-30

    Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Broad-spectrum biosensor capable of detecting and identifying diverse bacterial and Candida species in blood.

    Science.gov (United States)

    Metzgar, David; Frinder, Mark; Lovari, Robert; Toleno, Donna; Massire, Christian; Blyn, Lawrence B; Ranken, Raymond; Carolan, Heather E; Hall, Thomas A; Moore, David; Hansen, Christian J; Sampath, Rangarajan; Ecker, David J

    2013-08-01

    We describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter. Over 99% of all detections yielded correct identifications, whether they were obtained at concentrations well above the limit of detection or at the lowest detectable concentrations. This study demonstrates the ability of broad-spectrum PCR/ESI-MS assays to detect and identify diverse organisms in complex natural matrices that contain high levels of background DNA.

  14. Detection antigen virus den on monocyts by streptavidin biotin test as early diagnostic for dengue fever hemorrhagic

    Directory of Open Access Journals (Sweden)

    Y NINING SRI WURYANINGSIH

    2007-07-01

    Full Text Available Dengue virus infection is the main cause of morbidity and mortality in the tropical and sub-tropical countries of the world. Clinically it may manifest as asymtomastic,undifferentiated fever,dengue ever,dengue haemorrhagic fever and dengue shock syndrome cases. The mechanism underlying the disease with severe complication is not clear yet,however it has been previosus reported that primary and secondary infections of dengue virus play an important role in the patogenesis of this diseases. Early diagnosis of dengue virus infection has a great contribution for appropriate management of the disease, especialy for the prognosis of the patient. Laboratory investigations for such cases will be methods on serological investigation as well as virus isolation and identification.of dengue virus infection could be made by detection of specific virus ,viral antigen,genomic sequence and or detection of antibodies. These methods are sensitive and precise for detecting dengue virus infection,but there need special equipment,costly and detection of IgM and IgG often positive or negative false the dengue virus in the blood stream There for, this study was performed in order to develop a method to detect dengue virus antigen on the monocytes using Streptavidin biotin technique. The result of Streptavidin biotin study demonstrated that 32 sera from patient suspected with DHF 78,1% were positive DHF,and 21,9% were negative DHF. These results are consistent with the result from WHO criteria as standard .The Chi Square analysis showed that the presentage of sensitivity and specificity of Streptavidin biotin methode were 88% and 87,7% respectively. In conclusions, immunocytochemistry method using streptavidin biotin technique could be used as a method to detect antigen dengue virus on monocytes in the serum patient suspected with DHF. This technique has high sensitivity and specivicity and consistent with the clinical WHO criteria for DHF.

  15. [Agrobacterium-mediated transformation of lettuce (Lactuca sativa L.) with vectors bearing genes of bacterial antigenes from Mycobacterium tuberculosis].

    Science.gov (United States)

    Marveeva, N A; Vasilenko, M Iu; Shakhovskiĭ, A M; Kuchuk, N V

    2009-01-01

    Transgenic plants of lettuce Lactuca sativa L. cv. Eralash, Sniezinka, Rubinovoje kruzevo with genes coding synthesis of tuberculosis antigenes have been obtained by Agrobacterium-mediated transformation. Cotyledons of in vitro seedlings were used as the initial material for transformation with plasmids pCB063 (genes ESAT6, nptII) and pCB064 (genes ESAT6:AG85B(-TMD), nptII). PCR-analysis has shown the presence both selective and target genes in all plants analyzed. At the same time, the RT-PCR has shown that both the presence and the absence of a transcription of gene ESAT6 at a stable transcription of a gene nptII is possible.

  16. Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics.

    Science.gov (United States)

    Rocco, C J; Davey, M E; Bakaletz, L O; Goodman, S D

    2017-04-01

    Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Impact of sensitivity of human leucocyte antigen antibody detection by Luminex technology on graft loss at 1 year.

    Science.gov (United States)

    Szatmary, Peter; Jones, James; Hammad, Abdul; Middleton, Derek

    2013-06-01

    The clinical relevance of the detection of human leucocyte antigen (HLA) antibodies in sera of renal transplant recipients by highly sensitive methods such as Luminex alone is uncertain and a matter of debate. The choice of output thresholds affects antibody detection and thus organ allocation, yet there are no internationally agreed threshold levels. This study aims at evaluating our current practice of using an MFI threshold of 1000 in antibody detection. We carried out a case-control study by looking at 761 renal transplant recipients at one unit between 2000 and 2010. Of these, there were 93 cases of graft loss within 1 year and stored serum samples of 40 cases were available for testing. Controls were selected (graft function >2 years) and individually matched according to age, sex, number of transplants and date of transplant. All 40 cases and 40 controls had negative crossmatch by complement-dependent cytotoxicity (CDC) at the time of transplant, and pre-transplant sera were re-analysed for the presence of detectable HLA and donor-specific antibodies (DSAs) using Luminex screen and single-antigen beads and MFI threshold values of 1000, 2000 and 4000. In nearly 48% of cases with graft loss within a year, HLA antibodies were detectable by Luminex when using a 1000 MFI threshold. This was 25% greater than in controls (P = 0.017). There was also a 15% increase in detected DSAs; however, statistical significance depends on the inclusion or exclusion of one specific case. Using MFI thresholds of 2000 and 4000, no DSAs were found in any long-term surviving grafts. Selection of appropriate MFI cut-off values influences the detection of DSAs and, thus, organ allocation. Using a threshold of 1000 led to the detection of DSAs in 5% of long-term graft survivors in our population and should be considered too sensitive. Using a detection threshold of 2000 is sufficiently sensitive and leads to clinically relevant detection of DSA.

  18. Real-time detection of antibiotic activity by measuring nanometer-scale bacterial deformation

    Science.gov (United States)

    Iriya, Rafael; Syal, Karan; Jing, Wenwen; Mo, Manni; Yu, Hui; Haydel, Shelley E.; Wang, Shaopeng; Tao, Nongjian

    2017-12-01

    Diagnosing antibiotic-resistant bacteria currently requires sensitive detection of phenotypic changes associated with antibiotic action on bacteria. Here, we present an optical imaging-based approach to quantify bacterial membrane deformation as a phenotypic feature in real-time with a nanometer scale (˜9 nm) detection limit. Using this approach, we found two types of antibiotic-induced membrane deformations in different bacterial strains: polymyxin B induced relatively uniform spatial deformation of Escherichia coli O157:H7 cells leading to change in cellular volume and ampicillin-induced localized spatial deformation leading to the formation of bulges or protrusions on uropathogenic E. coli CFT073 cells. We anticipate that the approach will contribute to understanding of antibiotic phenotypic effects on bacteria with a potential for applications in rapid antibiotic susceptibility testing.

  19. Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

    DEFF Research Database (Denmark)

    Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen....... There is a particular interest in developing robust high-throughput assays as chicken vaccine trials usually comprise many individuals. In many respects, the avian immune system differs from the mammalian, and T cell assessment protocols must be adjusted accordingly to account for, e.g., differences in leukocyte...... in the cells even throughout division. This leads to daughter cells containing half the fluorescence of their parents. When lymphocytes are loaded with CFSE prior to ex vivo stimulation with specific antigen, the measurement of serial halving of its fluorescence by flow cytometry identifies the cells...

  20. Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

    Energy Technology Data Exchange (ETDEWEB)

    Pinzon, NM; Aukema, KG; Gralnick, JA; Wackett, LP

    2011-06-28

    A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high

  1. Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix®RT-PCR.

    Science.gov (United States)

    Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

    2018-04-01

    Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was

  2. Comparison of enterovirus detection in cerebrospinal fluid with Bacterial Meningitis Score in children

    OpenAIRE

    Pires, Frederico Ribeiro; Franco, Andréia Christine Bonotto Farias; Gilio, Alfredo Elias; Troster, Eduardo Juan

    2017-01-01

    ABSTRACT Objective To measure the role of enterovirus detection in cerebrospinal fluid compared with the Bacterial Meningitis Score in children with meningitis. Methods A retrospective cohort based on analysis of medical records of pediatric patients diagnosed as meningitis, seen at a private and tertiary hospital in São Paulo, Brazil, between 2011 and 2014. Excluded were patients with critical illness, purpura, ventricular shunt or recent neurosurgery, immunosuppression, concomitant bact...

  3. The use of antigen-detection ELISA for the diagnosis of bovine trypanosomosis in Cote d'Ivoire

    International Nuclear Information System (INIS)

    Kone, P.; Komoin-Oka, C.; N'Depo, A.

    1997-01-01

    An Antigen ELISA (Ag-ELISA) detecting circulating antigens of trypanosomes was evaluated in the central region of Cote d'Ivoire for the serodiagnosis of cattle trypanosomosis. Of 1423 sera examined, only 43 were positive in the MHCT/BCT, 105 (7%) were detected using stained blood smears, and 74 (5%) were found positive using the Ag-ELISA. The predominant trypanosome species was T. brucei, being present in 84% of the positive samples as detected by the BCT, in 96% using stained bloodsmears, and in 72% by Ag-ELISA. T.vivax was detected less frequently. The serological (ELISA) test did not detect all positive animals as found by the haematological techniques. However, the two techniques should be used in a complementary way to improve the diagnosis of the disease. The results confirm that the prevalence of trypanosomes in cattle is low in the study area. The low prevalence can be due to prophylaxis and therapy of livestock in combination with successful tsetse trapping. (author). 4 refs, 2 figs, 4 tabs

  4. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes...... on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  5. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    Directory of Open Access Journals (Sweden)

    Gottfried H. Kellermann

    2013-09-01

    Full Text Available Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™ to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

  6. Detection and Antigenic Profiling of Undeclared Peanut in Imported Garlic Using an xMAP Multiplex Immunoassay for Food Allergens.

    Science.gov (United States)

    Pedersen, Ronnie O; Peters, Tim; Panda, Rakhi; Wehling, Paul; Garber, Eric A E

    2017-07-01

    A shipment of imported garlic powder was suspected of containing peanut. Samples (subs) collected from the shipment displayed considerable variability in peanut antigenicity when analyzed by enzyme-linked immunosorbent assay (ELISA). This raised questions regarding whether peanut was actually present, the amount present, and the basis for the variability in antigenic content. Analyses that used an xMAP multiplex assay for the detection of peanut and additional food allergens generated responses that were characteristic of peanut. Specifically, the relative intensities of two different peanut-specific antibodies coupled to beads (peanut-37 and -38) and the antigen profiles were identical to garlic controls spiked with peanut. In addition, the xMAP data did not indicate the presence of other allergens. Quantitative analyses indicated an approximately fivefold variation in peanut concentration among different subs. In contrast, within a sub, the apparent peanut concentration appeared constant. Particle size analyses of the garlic powder subs indicated a single distribution profile, with a peak at 380 μm. ELISA analysis of sieve-fractionated garlic powder from one of the subs indicated that slightly less than half of the detectable peanut was smaller than 212 μm, with the remainder almost evenly split between 212 and 300 μm and >300 μm. Modeling to predict possible oral exposure levels of peanut other than those directly measured requires additional research on the physicochemical properties of peanut and garlic, along with information on the production of the garlic powder.

  7. Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

    Science.gov (United States)

    Rahmadane, Ibnu; Certoma, Andrea F; Peck, Grantley R; Fitria, Yul; Payne, Jean; Colling, Axel; Shiell, Brian J; Beddome, Gary; Wilson, Susanne; Yu, Meng; Morrissy, Chris; Michalski, Wojtek P; Bingham, John; Gardner, Ian A; Allen, John D

    2017-11-01

    Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller's stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0

  8. Rapid detection of bacterial meningitis using a point-of-care glucometer.

    Science.gov (United States)

    Rousseau, Geoffroy; Asmolov, Romain; Grammatico-Guillon, Leslie; Auvet, Adrien; Laribi, Said; Garot, Denis; Jouan, Youenn; Dequin, Pierre-François; Guillon, Antoine

    2017-08-10

    In case of acute bacterial meningitis, a decision on the need for intensive care admission should be made within the first hour. The aim of this study was to assess the ability of a point-of-care glucometer to determine abnormal cerebrospinal fluid (CSF) glucose concentration at the bedside that contributes toward bacterial meningitis diagnosis. We carried out a prospective study and simultaneously measured the glucose concentrations in CSF and blood using a central laboratory and a point-of-care glucometer. We compared CSF/blood glucose ratios obtained at the bedside with a glucometer versus those obtained by the central laboratory. We determined the performance characteristics of the CSF/blood glucose ratio provided by a glucometer to detect bacterial infection in the CSF immediately after CSF sampling. We screened 201 CSF collection procedures during the study period and included 172 samples for analysis. Acute bacterial meningitis was diagnosed in 17/172 (9.9%) of CSF samples. The median turnaround time for a point-of-care glucometer analysis was 5 (interquartile range 2-10) min versus 112 (interquartile range 86-154) min for the central laboratory (P<0.0001). The optimal cut-off of the CSF/blood glucose ratio calculated from a bedside glucometer was 0.46, with a sensitivity of 94.1% (95% confidence interval: 71.3-99.9%), a specificity of 91% (95% confidence interval: 85.3-95%), and a positive likelihood ratio of 10. A glucometer accurately detects an abnormal CSF/blood glucose ratio immediately after the lumbar puncture. This cheap point-of-care method has the potential to speed up the diagnostic process of patients with bacterial meningitis.

  9. [Development of immunochromatographic test system for the rapid detection of lipopolysaccharide antigen and cells of causative agent of bovine brucellosis].

    Science.gov (United States)

    Byzova, N A; Zherdev, A V; Eskendirova, S Z; Baltin, K K; Unysheva, G B; Mukanov, K K; Ramankulov, E M; Dzantiev, B B

    2012-01-01

    A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.

  10. Detection and localization of rabbit hepatitis e virus and antigen in systemic tissues from experimentally intraperitoneally infected rabbits.

    Directory of Open Access Journals (Sweden)

    Jingjing Mao

    Full Text Available Rabbit hepatitis E virus (HEV is a novel genotype of HEV, and is considered to pose a risk of zoonotic transmission. Research into the systemic distribution of rabbit HEV in rabbits during different periods of infection has rarely been reported. To better understand this virus, we infected rabbits with second-passage rabbit HEV via an intraperitoneal route. After inoculation, the infection showed two types, temporary and constant infection. The detection of HEV RNA in the feces varied with time, and serum antigen correlated with fecal HEV RNA. Viremia only appeared 72 days after inoculation. The rabbits remained antibody negative throughout the experimental period. When HEV was localized, several organs besides the liver were HEV RNA positive. Tissue antigen was observed immunohistochemically in the different cells of various organs, especially in parts of the small intestine and the characteristic rabbit gut-associated lymphoid tissue. These data provide valuable information for future research into the pathogenesis of HEV.

  11. Dynamic laser speckle to detect motile bacterial response of Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Sendra, H; Murialdo, S; Passoni, L

    2007-01-01

    This proposal deals with the technique for detection of motile response of Pseudomonas aeruginosa using dynamic laser speckle or biospeckle as an alternative method. The study of bacterial displacement plays an essential role in biocatalysts processes and biodegradation. Hence, some biodegrading enzymes are benign catalytic that could be used for the production of industrially useful compounds as well as in wastewater treatments. This work presents an experimental set up and a computational process using frame sequences of dynamic laser speckle as a novel application. The objective was the detection of different levels of motility in bacteria. The encouraging results were achieved through a direct and non invasive observation method of the phenomenon

  12. CD1c tetramers detect ex vivo T cell responses to processed phosphomycoketide antigens

    NARCIS (Netherlands)

    Ly, Dalam; Kasmar, Anne G.; Cheng, Tan-Yun; de Jong, Annemieke; Huang, Shouxiong; Roy, Sobhan; Bhatt, Apoorva; van Summeren, Ruben P.; Altman, John D.; Jacobs, William R.; Adams, Erin J.; Minnaard, Adriaan J.; Porcelli, Steven A.; Moody, Branch; Jacobs Jr., William R.

    2013-01-01

    CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32

  13. A sensitive immunoradiometric assay for the detection of hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Cameron, C.H.; Combridge, B.S.; Howell, D.R.; Barbara, J.A.J.

    1980-01-01

    A solid-phase immunoradiometric assay for hepatitis B surface antigen is described which has been in use since 1972. Initially it was used for reference laboratory work, but from 1974 it has also been used for screening blood and blood products. Methods for the production of reagents and their use in blood transfusion and reference work, are outlined. (Auth.)

  14. DETECTION OF A PUTATIVE 30-KDA LIGAND OF THE CLUSTER-2 ANTIGEN

    NARCIS (Netherlands)

    HELFRICH, W; VANGEEL, M; THE, TH; DELEIJ, L

    1994-01-01

    The cluster-2 antigen, also called EGP-2, is a 38-kDa trans-membrane glycoprotein with a distribution that is largely confined to human epithelial cells and their derived carcinomas. Monoclonal antibodies (MAbs) directed against EGP-2 have been extensively studies as anti-tumors agents, yet the

  15. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

    DEFF Research Database (Denmark)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-01-01

    microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 oC without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies...

  16. Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections

    NARCIS (Netherlands)

    P. Koraka (Penelope); C.P. Burghoorn-Maas; A. Falconar; T.E. Setiati (Tatty); K. Djamiatun; J. Groen (Jan); A.D.M.E. Osterhaus (Albert)

    2003-01-01

    textabstractAccurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot

  17. Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

    NARCIS (Netherlands)

    Koraka, P.; Burghoorn-Maas, C.P.; Falconar, A.; Setiati, T.E.; Djamiatun, K.; Groen, J.; Osterhaus, A.D.

    2003-01-01

    Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot

  18. Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

    International Nuclear Information System (INIS)

    Decho, Alan W; Beckman, Erin M; Chandler, G Thomas; Kawaguchi, Tomohiro

    2008-01-01

    An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae

  19. Detection of the circulating antigen 14-3-3 protein of Schistosoma japonicum by time-resolved fluoroimmunoassay in rabbits

    Directory of Open Access Journals (Sweden)

    Wang Jie

    2011-05-01

    Full Text Available Abstract Background Schistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum. Results The detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection. Conclusions A sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.

  20. Collection of corneal impression cytology directly on a sterile glass slide for the detection of viral antigen: An inexpensive and simple technique for the diagnosis of HSV epithelial keratitis – A pilot study

    Directory of Open Access Journals (Sweden)

    Bandlapally Sesha

    2001-09-01

    Full Text Available Abstract Background Herpes simplex keratitis (HSK is a sight threatening ocular infection and occurs worldwide. A prompt laboratory diagnosis is often very useful. Conventional virology techniques are often expensive and time consuming. We describe here a highly economical, simple, rapid and sensitive technique for the collection of impression cytology, for the laboratory diagnosis of HSK. Methods Fifteen patients with a clinical diagnosis of HSK (either dendritic or geographic ulcers and five patients with other corneal infections (Mycotic keratitis, n = 3, Bacterial keratitis, n = 2 were included in the study. Corneal impression cytology specimens were collected using a sterile glass slide with polished edges instead of a membrane, by pressing the surface of one end of the slide firmly, but gently on the corneal lesion. Additionally, corneal scrapings were collected following the impression cytology procedure. Impression cytology and corneal scrapings were stained by an immunoperoxidase or immunofluorescence assay for the detection of HSV-1 antigen using a polyclonal antibody to HSV-1. Corneal scrapings were processed for viral cultures by employing a shell vial assay. Results This simple technique allowed the collection of adequate corneal epithelial cells for the detection of HSV-1 antigen in a majority of the patients. HSV-1 antigen was detected in 12/15 (80% cases while virus was isolated from 5/15 (33.3% patients with HSK. All the patients with a clinical diagnosis of HSK (n = 15 were confirmed by virological investigations (viral antigen detection and/or viral cultures. HSV-1 antigen was detected in the impression cytology smears and corneal scrapings in 11/15 (73.3% and 12/15 (80% of the patients, respectively (P = 1.00. None of the patients in the control group were positive for viral antigen or virus isolation. Minimal background staining was seen in impression cytology smears, while there was some background staining in corneal

  1. Using a genomic assay for the detection of SNPs of Knops blood group antigens leads to the identification of two caucasians homozygous for the SNP associated with the knops SL3 antigen

    DEFF Research Database (Denmark)

    Jakobsen, M. A.; Sprogoe, U.

    2015-01-01

    Background/Case Studies: The antigens of the Knops (Kn) blood group system are associated with SNPs located on exon 29 and (to lesser extent) on exon 26 of the complement receptor 1 (CR1) gene. Because of a lack of proper typing antibodies, serologic detection of Kn antigens is not feasible. We...... previously described to be Sl3- (personal communication from the International Blood Group Reference Laboratory in Bristol). * Number indicates the nucleotide position of the CR1 gene. Conclusion: In this study, we have set up a genomic assay for identifying the antigens in the Knops system. We found...... designed a genomic assay based on sequencing targeting the SNPs underlying the antigens of the Knops system. Study Design/Methods: Samples from a total of 105 blood donors and 2 patients were examined for polymorphisms in CR1 exon 29 by using PCR and subsequent Sanger sequencing. Results...

  2. Detection of human bacterial pathogens in ticks collected from Louisiana black bears (Ursus americanus luteolus).

    Science.gov (United States)

    Leydet, Brian F; Liang, Fang-Ting

    2013-04-01

    There are 4 major human-biting tick species in the northeastern United States, which include: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. The black bear is a large mammal that has been shown to be parasitized by all the aforementioned ticks. We investigated the bacterial infections in ticks collected from Louisiana black bears (Ursus americanus subspecies luteolus). Eighty-six ticks were collected from 17 black bears in Louisiana from June 2010 to March 2011. All 4 common human-biting tick species were represented. Each tick was subjected to polymerase chain reaction (PCR) targeting select bacterial pathogens and symbionts. Bacterial DNA was detected in 62% of ticks (n=53). Rickettsia parkeri, the causative agent of an emerging spotted fever group rickettsiosis, was identified in 66% of A. maculatum, 28% of D. variabilis, and 11% of I. scapularis. The Lyme disease bacterium, Borrelia burgdorferi, was detected in 2 I. scapularis, while one A. americanum was positive for Borrelia bissettii, a putative human pathogen. The rickettsial endosymbionts Candidatus Rickettsia andeanae, rickettsial endosymbiont of I. scapularis, and Rickettsia amblyommii were detected in their common tick hosts at 21%, 39%, and 60%, respectively. All ticks were PCR-negative for Anaplasma phagocytophilum, Ehrlichia spp., and Babesia microti. This is the first reported detection of R. parkeri in vector ticks in Louisiana; we also report the novel association of R. parkeri with I. scapularis. Detection of both R. parkeri and B. burgdorferi in their respective vectors in Louisiana demands further investigation to determine potential for human exposure to these pathogens. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy.

    Science.gov (United States)

    Amemiya, Yosuke; Tanaka, Tsuyoshi; Yoza, Brandon; Matsunaga, Tadashi

    2005-11-21

    A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.

  4. Species Specific Bacterial Spore Detection Using Lateral-Flow Immunoassay with DPA-Triggered Tb Luminescence

    Science.gov (United States)

    Ponce, Adrian

    2003-01-01

    A method of detecting bacterial spores incorporates (1) A method of lateral-flow immunoassay in combination with (2) A method based on the luminescence of Tb3+ ions to which molecules of dipicolinic acid (DPA) released from the spores have become bound. The present combination of lateral-flow immunoassay and DPA-triggered Tb luminescence was developed as a superior alternative to a prior lateral-flow immunoassay method in which detection involves the visual observation and/or measurement of red light scattered from colloidal gold nanoparticles. The advantage of the present combination method is that it affords both (1) High selectivity for spores of the species of bacteria that one seeks to detect (a characteristic of lateral-flow immunoassay in general) and (2) Detection sensitivity much greater (by virtue of the use of DPA-triggered Tb luminescence instead of gold nanoparticles) than that of the prior lateral-flow immunoassay method

  5. Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen: Involvement of marginal metallophilic macrophages in the uptake of immune-stimulating complexes

    NARCIS (Netherlands)

    Claassen, I.J.T.M.; Osterhaus, A.D.M.E.; Claassen, E.

    1995-01-01

    Several mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune-stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system. However, until

  6. Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen: involvement of marginal metallophilic macrophages in the uptake of immune-stimulating complexes.

    NARCIS (Netherlands)

    I.J.Th.M. Claassen (Ivo); A.D.M.E. Osterhaus (Albert); H.J.H.M. Claassen (Eric)

    1995-01-01

    textabstractSeveral mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune-stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system.

  7. Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease.

    Science.gov (United States)

    Bell, Colin J; Blackburn, Paul; Elliott, Mark; Patterson, Tony I A P; Ellison, Sean; Lahuerta-Marin, Angela; Ball, Hywel J

    2014-09-01

    Bovine respiratory disease (BRD) causes severe economic losses to the cattle farming industry worldwide. The major bacterial organisms contributing to the BRD complex are Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, Pasteurella multocida, and Trueperella pyogenes. The postmortem detection of these organisms in pneumonic lung tissue is generally conducted using standard culture-based techniques where the presence of therapeutic antibiotics in the tissue can inhibit bacterial isolation. In the current study, conventional and real-time polymerase chain reaction (PCR) assays were used to assess the prevalence of these 5 organisms in grossly pneumonic lung samples from 150 animals submitted for postmortem examination, and the results were compared with those obtained using culture techniques. Mannheimia haemolytica was detected in 51 cases (34%) by PCR and in 33 cases (22%) by culture, H. somni was detected in 35 cases (23.3%) by PCR and in 6 cases (4%) by culture, Myc. bovis was detected in 53 cases (35.3%) by PCR and in 29 cases (19.3%) by culture, P. multocida was detected in 50 cases (33.3%) by PCR and in 31 cases (20.7%) by culture, and T. pyogenes was detected in 42 cases (28%) by PCR and in 31 cases (20.7%) by culture, with all differences being statistically significant. The PCR assays indicated positive results for 111 cases (74%) whereas 82 cases (54.6%) were culture positive. The PCR assays have demonstrated a significantly higher rate of detection of all 5 organisms in cases of pneumonia in cattle in Northern Ireland than was detected by current standard procedures. © 2014 The Author(s).

  8. Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.

    Science.gov (United States)

    Crompot, Emerence; Van Damme, Michael; Duvillier, Hugues; Pieters, Karlien; Vermeesch, Marjorie; Perez-Morga, David; Meuleman, Nathalie; Mineur, Philippe; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

    2015-01-01

    Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

  9. Current advances in aptamer-assisted technologies for detecting bacterial and fungal toxins.

    Science.gov (United States)

    Alizadeh, N; Memar, M Y; Mehramuz, B; Abibiglou, S S; Hemmati, F; Samadi Kafil, H

    2018-03-01

    Infectious diseases are among the common leading causes of morbidity and mortality worldwide. Associated with the emergence of new infectious diseases, the increasing number of antimicrobial-resistant isolates presents a serious threat to public health and hospitalized patients. A microbial pathogen may elicit several host responses and use a variety of mechanisms to evade host defences. These methods and mechanisms include capsule, lipopolysaccharides or cell wall components, adhesions and toxins. Toxins inhibit phagocytosis, cause septic shock and host cell damages by binding to host surface receptors and invasion. Bacterial and fungal pathogens are able to apply many different toxin-dependent mechanisms to disturb signalling pathways and the structural integrity of host cells for establishing and maintaining infections Initial techniques for analysis of bacterial toxins were based on in vivo or in vitro assessments. There is a permanent demand for appropriate detection methods which are affordable, practical, careful, rapid, sensitive, efficient and economical. Aptamers are DNA or RNA oligonucleotides that are selected by systematic evolution of ligands using exponential enrichment (SELEX) methods and can be applied in diagnostic applications. This review provides an overview of aptamer-based methods as a novel approach for detecting toxins in bacterial and fungal pathogens. © 2017 The Society for Applied Microbiology.

  10. Comparison of individual and pooled sampling methods for detecting bacterial pathogens of fish

    Science.gov (United States)

    Mumford, Sonia; Patterson, Chris; Evered, J.; Brunson, Ray; Levine, J.; Winton, J.

    2005-01-01

    Examination of finfish populations for viral and bacterial pathogens is an important component of fish disease control programs worldwide. Two methods are commonly used for collecting tissue samples for bacteriological culture, the currently accepted standards for detection of bacterial fish pathogens. The method specified in the Office International des Epizooties Manual of Diagnostic Tests for Aquatic Animals permits combining renal and splenic tissues from as many as 5 fish into pooled samples. The American Fisheries Society (AFS) Blue Book/US Fish and Wildlife Service (USFWS) Inspection Manual specifies the use of a bacteriological loop for collecting samples from the kidney of individual fish. An alternative would be to more fully utilize the pooled samples taken for virology. If implemented, this approach would provide substantial savings in labor and materials. To compare the relative performance of the AFS/USFWS method and this alternative approach, cultures of Yersinia ruckeri were used to establish low-level infections in groups of rainbow trout (Oncorhynchus mykiss) that were sampled by both methods. Yersinia ruckeri was cultured from 22 of 37 groups by at least 1 method. The loop method yielded 18 positive groups, with 1 group positive in the loop samples but negative in the pooled samples. The pooled samples produced 21 positive groups, with 4 groups positive in the pooled samples but negative in the loop samples. There was statistically significant agreement (Spearman coefficient 0.80, P methods to permit detection of low-level bacterial infections of rainbow trout.

  11. Comparison of enterovirus detection in cerebrospinal fluid with Bacterial Meningitis Score in children.

    Science.gov (United States)

    Pires, Frederico Ribeiro; Franco, Andréia Christine Bonotto Farias; Gilio, Alfredo Elias; Troster, Eduardo Juan

    2017-01-01

    To measure the role of enterovirus detection in cerebrospinal fluid compared with the Bacterial Meningitis Score in children with meningitis. A retrospective cohort based on analysis of medical records of pediatric patients diagnosed as meningitis, seen at a private and tertiary hospital in São Paulo, Brazil, between 2011 and 2014. Excluded were patients with critical illness, purpura, ventricular shunt or recent neurosurgery, immunosuppression, concomitant bacterial infection requiring parenteral antibiotic therapy, and those who received antibiotics 72 hours before lumbar puncture. The study included 503 patients. Sixty-four patients were excluded and 94 were not submitted to all tests for analysis. Of the remaining 345 patients, 7 were in the Bacterial Meningitis Group and 338 in the Aseptic Meningitis Group. There was no statistical difference between the groups. In the Bacterial Meningitis Score analysis, of the 338 patients with possible aseptic meningitis (negative cultures), 121 of them had one or more points in the Bacterial Meningitis Score, with sensitivity of 100%, specificity of 64.2%, and negative predictive value of 100%. Of the 121 patients with positive Bacterial Meningitis Score, 71% (86 patients) had a positive enterovirus detection in cerebrospinal fluid. Enterovirus detection in cerebrospinal fluid was effective to differentiate bacterial from viral meningitis. When the test was analyzed together with the Bacterial Meningitis Score, specificity was higher when compared to Bacterial Meningitis Score alone. Avaliar o papel da pesquisa de enterovírus no líquido cefalorraquidiano em comparação com o Escore de Meningite Bacteriana em crianças com meningite. Coorte retrospectiva, realizada pela análise de prontuários, incluindo pacientes pediátricos, com diagnóstico de meningite e atendidos em um hospital privado e terciário, localizado em São Paulo, entre 2011 e 2014. Foram excluídos os pacientes com doença crítica, púrpura, deriva

  12. Towards One-Step Quantitation of Prostate-Specific Antigen (PSA) in Microfluidic Devices: Feasibility of Optical Detection with Nanoparticle Labels

    NARCIS (Netherlands)

    Barbosa, Ana I.; Wichers, J.H.; Amerongen, van A.; Reis, Nuno M.

    2017-01-01

    Rapid and quantitative prostate-specific antigen (PSA) biomarker detection would be beneficial to cancer diagnostics, improving early detection and therefore increasing chances of survival. Nanoparticle-based detection is routinely used in one-step nitrocellulose-based lateral flow (LF)

  13. Comparison between a soluble antigen-based ELISA and IFAT in detecting antibodies against Babesia canis in dogs.

    Science.gov (United States)

    Furuta, Patrícia Iriê; Oliveira, Tricia Maria Ferreira de Sousa; Theixeira, Márcia Cristina Alves; Rocha, Artur Gouveia; Machado, Rosangela Zacarias; Tinucci-Costa, Mirela Gouveia

    2009-01-01

    An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-B. canis antibodies in the sera of dogs using, indirect fluorescent antibody test (IFAT) as a reference test. ELISA uses a soluble antigenic preparation of B. canis and the optimal dilutions of the antigen, serum and conjugate were determined by check board titration, using positive and negative reference serum. The soluble antigen preparation of B. canis merozoites was 10 microg x mL(-1), with reference sera from positive and negative in a single dilution of 1:100, and conjugated to 1:4.000. A total of 246 serum samples were collected from dogs during the rabies vaccination campaign in Jaboticabal, São Paulo, Brazil and examined for the presence of antibodies against B. canis by ELISA and IFAT. Under these conditions, the average absorbance of negative serum was 0.129 + or - 0.025, resulting in a cut-of value of 0.323 (ELISA level 3) and the average absorbance of positive reference serum was 2.156 + or - 1.187. The serological positive samples tested for B. canis by ELISA and IFAT were 67.89% (n = 167) and 59.35% (n = 146), respectively. These results suggest that ELISA described may prove to be an effective serological test to diagnose canine babesiosis.

  14. 'Bioluminescent' reporter phage for the detection of Category A bacterial pathogens.

    Science.gov (United States)

    Schofield, David A; Molineux, Ian J; Westwater, Caroline

    2011-07-08

    Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively. Although the natural occurrence of both diseases' is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant "light-tagged" reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens. For demonstration purposes, here we describe the method for the phage

  15. Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

    Science.gov (United States)

    Halpern, Micah D.; Molins, Claudia R.; Schriefer, Martin

    2014-01-01

    A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. PMID:24899074

  16. Highly sensitive and selective lateral flow immunoassay based on magnetic nanoparticles for quantitative detection of carcinoembryonic antigen.

    Science.gov (United States)

    Liu, Fangming; Zhang, Honglian; Wu, Zhenhua; Dong, Haidao; Zhou, Lin; Yang, Dawei; Ge, Yuqing; Jia, Chunping; Liu, Huiying; Jin, Qinghui; Zhao, Jianlong; Zhang, Qiqing; Mao, Hongju

    2016-12-01

    Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

    Science.gov (United States)

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

  18. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations

    Directory of Open Access Journals (Sweden)

    Law eJodi Woan-Fei

    2015-01-01

    Full Text Available The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR, multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases.

  19. Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

    OpenAIRE

    Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

    2016-01-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1...

  20. Prostate-specific membrane antigen PET/MRI validation of MR textural analysis for detection of transition zone prostate cancer.

    Science.gov (United States)

    Bates, Anthony; Miles, Kenneth

    2017-12-01

    To validate MR textural analysis (MRTA) for detection of transition zone (TZ) prostate cancer through comparison with co-registered prostate-specific membrane antigen (PSMA) PET-MR. Retrospective analysis was performed for 30 men who underwent simultaneous PSMA PET-MR imaging for staging of prostate cancer. Thirty texture features were derived from each manually contoured T2-weighted, transaxial, prostatic TZ using texture analysis software that applies a spatial band-pass filter and quantifies texture through histogram analysis. Texture features of the TZ were compared to PSMA expression on the corresponding PET images. The Benjamini-Hochberg correction controlled the false discovery rate at detection of TZ prostate cancer. • Prostate transition zone (TZ) MR texture analysis may assist in prostate cancer detection. • Abnormal transition zone PSMA expression correlates with altered texture on T2-weighted MR. • TZ with abnormal PSMA expression demonstrates significantly reduced MI, SD and MPP.

  1. Development of Recombinant Flagellar Antigens for Serological Detection of Salmonella enterica Serotypes Enteritidis, Hadar, Heidelberg, and Typhimurium in Poultry

    Directory of Open Access Journals (Sweden)

    Charles L. Hofacre

    2013-07-01

    Full Text Available Accurate and fast detection of harmful Salmonella is a major concern of food safety. Common Salmonella serotypes responsible for human associated foodborne outbreaks are S. Enteritidis, S. Hadar, S. Heidelberg, and S. Typhimurium are also commonly isolated from poultry. Serology is commonly used to monitor disease in poultry, therefore application of Salmonella serotype-specific test will have added value in Salmonella surveillance or monitoring vaccine efficacy. Recombinant flagellins were purified to be used as antigens in an ELISA. In this study, an ELISA was developed for the serological detection of S. Enteritidis. Once optimized, 500 ng of purified recombinant S. Enteritidis flagellin and a 1:64 dilution were determined to be optimal for testing sera. A negative baseline cutoff was calculated to be an optical density (OD of 0.35. All sera from birds with history of S. Enteritidis exposure tested positive and all sera from chickens with no exposure tested negative to this Salmonella serotype. Current ELISA for serological detection of Salmonella suffers from cross reactivity inherent in lipopolysaccharide (LPS or whole cell antigen based serological tests. This new ELISA eliminates common cross reactivity by focusing specifically on the flagellins of the Salmonella serotypes common in poultry and associated with foodborne outbreaks.

  2. [Detection of stool antigens of Echinococcus granulosus in dogs belonging to slaughterhouse workers and offal merchants in Metropolitan Lima].

    Science.gov (United States)

    Merino, Veronika; Falcón, Néstor; Morel, Noelia; González, Gualberto

    2017-04-20

    To demonstrate the presence of Echinoccocus granulosus in the definitive host in the city of Lima, Perú, by detecting parasite antigens in the stool of dogs belonging to offal handlers and merchants in authorized slaughterhouses in Metropolitan Lima. Stool samples were collected from 58 dogs and examined using the coproELISA technique for the detection of secretory/excretory antigens of E. granulosus. A survey was conducted to obtain information on pet feeding and handling practices. Positivity to E. granulosus was detected in 13.8% (8/58) of the dogs. In 27.8% (5/18) of the homes, at least one animal showed positivity, and in families that had more than four dogs the chances of finding positivity in at least one dog were higher (P dog tested positive the pets were fed on offal. Of study participants, 94.4% (17) knew nothing about the routes of transmission of hydatid disease. Results show the presence of definitive hosts in the urban area of Lima and underscore the need to more widely disseminate practices for the prevention of parasite transmission.

  3. Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

    Directory of Open Access Journals (Sweden)

    Gema Álvarez García

    2006-08-01

    Full Text Available A Neospora caninum 17 kDa protein fraction (p17 has been described as an immunodominant antigen (IDA under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17 was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86.

  4. Comparison of the Sofia and Veritor direct antigen detection assay systems for identification of influenza viruses from patient nasopharyngeal specimens.

    Science.gov (United States)

    Leonardi, G P; Wilson, A M; Mitrache, I; Zuretti, A R

    2015-04-01

    Influenza antigen detection assays (Sofia fluorescent immunoassay [FIA] and Veritor) yield objective results, which are potentially useful for point-of-care testing. The assays were evaluated with reverse transcriptase PCR (RT-PCR) using 411 nasopharyngeal swab specimens. Sensitivity and specificity values (percentages) of 79.0/99.0 and 64.0/99.4 for influenza A and 92.9/96.7 and 78.6/98.7 for influenza B were obtained for the Sofia and Veritor assays, respectively. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample.

    Science.gov (United States)

    Saidin, Syazwan; Yunus, Muhammad Hafiznur; Othman, Nurulhasanah; Lim, Yvonne Ai-Lian; Mohamed, Zeehaida; Zakaria, Nik Zairi; Noordin, Rahmah

    2017-05-01

    Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml -1 . Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml -1 . The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.

  6. Comparison of counter-current immunoelectrophoresis, latex agglutination, and radioimmunoassay in detection of soluble capsular polysaccharide antigens of Haemohpilus influenzae type b and Neisseria meningitidis of groups A or C

    International Nuclear Information System (INIS)

    Leinonen, M.; Kaeythy, H.

    1978-01-01

    Three serological methods, radioimmunoassay (RIA), latex agglutination (LX), and counter-current immunoelectrophoresis (CIEP), for sensitivity in the detection of the capsular polysaccharide antigen of Haemophilus influenzae type b or Neisseria meningitidis groups A and C were compared. RIA was consistently the most sensitive, LX the next, and CIEP the least sensitive. When RIA and LX were used to test cerebrospinal fluid (CSF) samples of patients with meningitis, they gave very similar results. In only two out of 47 samples, in which RIA detected one of the three antigens, was the amount of the specific polysaccharide too low to be detected by LX. By the serological methods evidence of specific pathogen could be detected in 49 samples, including nine from patients who had received intensive antimicrobial treatment for up to three days and from whom specimens yielded no bacteria on culture. The reactions were specific in all cases except two out of 47 tests positive to LX. From these two CSF samples N. meningitidis group B could be cultivated, whereas the LX was recorded positive for N. meningitidis of group A in one case, and of group C in the other. The nonspecific reactions could be due to antibodies to bacterial components other than the capsular polysaccharide. (author)

  7. Generation of monoclonal antibodies against prostate specific antigen (PSA) for the detection of PSA and its purification

    International Nuclear Information System (INIS)

    Acevedo Castro, Boris Ernesto

    2012-01-01

    The prostate cancer in Cuba is a problem of health (2672 diagnosed cases and 2769 deaths in 2007). Various diagnostic methods have been implemented for the detection and management of this disease, emphasizing among them (PSA) prostate-specific antigen serological determination. At this work was generated and characterized a panel of 11 antibodies (AcMs) monoclonal IgG1 detected with high affinity described major epitopes of the PSA, both in solution and attached to the test plate. From the panel obtained AcMs was the standardization of an essay type ELISA for the detection of serum total PSA (associated and free) equimolar, based on antibody monoclonal CB-PSA.4 in the coating and the CB-PSA.9 coupled with biotin as liner, with a detection limit of 0.15 ng/mL. Similarly, standardized system for detection in serum free PSA, based on the AcMs CB-PSA.4 (coating) and CB-PSA.2 coupled with biotin (liner), with a detection limit of 0.5 ng/mL. Finally, with the purpose of using PSA as standard in trials type ELISA, developed a simple method of inmunopurificación based on the AcM, CB-PSA.2, which was obtained the PSA with a purity exceeding 90%. Immunoassay Centre on the basis of the AcMs panel and the results of this study, developed and recorded two diagnostic systems for the detection of PSA in human serum. (author)

  8. Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis.

    Science.gov (United States)

    Jiang, Lu Xi; Ren, Hong Yu; Zhou, Hai Jian; Zhao, Si Hong; Hou, Bo Yan; Yan, Jian Ping; Qin, Tian; Chen, Yu

    2017-08-01

    Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  9. Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

    Science.gov (United States)

    Memon, Atta Muhammad; Bhuyan, Anjuman Ara; Chen, Fangzhou; Guo, Xiaozhen; Menghwar, Harish; Zhu, Yinxing; Ku, Xugang; Chen, Shuhua; Li, Zhonghua; He, Qigai

    2017-05-01

    Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 10 2.5 TCID 50 /mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

  10. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung' u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.

    2010-04-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  11. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Directory of Open Access Journals (Sweden)

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  12. Detection of soluble antigen and DNA of Trypanosoma cruzi in urine is independent of renal injury in the guinea pig model.

    Directory of Open Access Journals (Sweden)

    Yagahira E Castro-Sesquen

    Full Text Available The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA. T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa were detected in the acute phase (67.5% and the chronic phase (45%. Parasite DNA in urine was detected only in the acute phase (45%. Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1 and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.

  13. Development of a immunochromatographic test with avidin-biotin for the detection of antibodies against antigen e of hepatitis B in human plasma

    International Nuclear Information System (INIS)

    Mainet Gonzalez, Damian; Palenzuela Gardon, Daniel O; Diaz Argudin, Tamara

    2007-01-01

    The disappearance of antigen e of hepatitis B in the presence of the plasmatic antibodies against antigen e may indicate a satisfactory therapeutic response in patients with chronic hepatitis B. The immuno-chromatographic test carried out in the diagnosis of diseases use different antibody combinations and may employ the avidin or streptavidin-biotin technology to develop a rapid immuno-chromatographic test for the detection of antibodies anti-antigen e in the plasma. They were detected in the laboratory by means of two fast immuno-chromatographic tests when using in one of them the avidin-biotin technology. These tests are carried out with a one-step competitive inhibition format and amplified or not with avidin-biotin. Monoclonal antibodies against antigen e obtained by cellular hybridization were used. Forty-six plasmatic samples classified as positive and negative to the anti-antigen antibodies were evaluated with a reference immunochromatographic test Advanced QualityTM. The possible expiry time of the biological reagents forming part of these tests were studied with accelerated thermal-stability experiments. The possible interference in the plasma of some of the biochemical compounds used in these trials was analyzed. Four murine monoclonal antibodies anti-antigen e were obtained and only one of them was used in these immunochromatographic tests with an anti-antigen polyclonal antibody conjugated with gold. Both tests and their stable biological reagents discriminated the positive and negative samples to the antibodies anti-antigen e, as well as the commercial test. There was no interference in the biochemical compounds studied in these tests. Both immuno-chromatographic tests made in the laboratory are useful to detect antibodies anti-antigen e in the plasma. The avidin-biotin increased the analytical sensitivity of this type of fast immuno-chromatographic test without altering its performance features. (Author)

  14. Disposable bioluminescence-based biosensor for detection of bacterial count in food.

    Science.gov (United States)

    Luo, Jinping; Liu, Xiaohong; Tian, Qing; Yue, Weiwei; Zeng, Jing; Chen, Guangquan; Cai, Xinxia

    2009-11-01

    A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10(3) to 10(8) colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n=22) within 5min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.

  15. Detection of Antineutrophil Autoantibodies by Flow Cytometry: Use of Unfixed Neutrophils as Antigenic Targets

    Science.gov (United States)

    1993-01-01

    idiopathic thrombocytopenic purpura have used unfixed cells as antigenic targets for these studies. (ITP), human immunodeficiency virus disease (HIV...chronic neutropenia 8/29 (28%) 4:4 I (0-2) 330(50-800) Immune thrombocytopenic purpura 6/12 (50%) 5:1 10 (1-17) 270 (0-880) Other diagnosis; includes HIV...Boxer LA. Baehner RL: The use and limitation of staph- fant with autoimmune neutropenia. Am J Dis Child 136:718-722, 1982. ylococcal protein A for

  16. Polymicrobial subdural empyema: involvement of Streptococcus pneumoniae revealed by lytA PCR and antigen detection

    DEFF Research Database (Denmark)

    Greve, Thomas; Clemmensen, Dorte; Ridderberg, Winnie

    2011-01-01

    The authors report a case of a subdural empyema (SDE) caused by a coinfection with Streptococcus intermedius and Streptococcus pneumoniae, initially considered a S. intermedius infection only. An otherwise healthy 11-year-old female was admitted to the hospital after 5 days of illness. Symptoms....... The empyema was evacuated twice, day 8 and 18, with good results. Primary samples showed growth of S. intermedius only. The severity of the clinical picture elicited supplementary samples, which were additionally positive for S. pneumoniae by an in-house specific lytA PCR and/or a commercial antigen test....

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    into the lipid A part and the polysaccharide part. The polysaccharide was conjugated regiospecifically to a photochemically active compound, anthraquinone, resulting in a polysaccharide-anthraquinone conjugate. Anthraquinones forms active radicals when exposed to soft UV irradiation (350 nm) permitting...... the formation of stable covalent bonds to polymers e.g, microtiter plates. By this technique the polysaccharides are bound through the anthraquinone part of the polysaccharide-anthraquinone conjugates to the microtiter plates. This minimizes denaturation of O-antigen epitopes during binding to the microtiter...

  18. Evaluation of an x-ray microprobe technique as a possible aid to detect salmonellae

    International Nuclear Information System (INIS)

    Richter, E.R.; Banwart, G.J.

    1982-01-01

    Specific bacterial antigen (Salmonella) increased in phosphorus and sqlfur after reaction with the flukrescain isothiocyanate-tagged anti-Salmonella antibody, while nonspecific antigen (Escherichia coli) did not. X-ray microprobe analysis may be useful in detecting salmonellae or other bacteria by determining increases in the elemental constituents of bacterial cells when reacted with elemental-tagged antibodies

  19. Shear horizontal surface acoustic wave microsensor for Class A viral and bacterial detection.

    Energy Technology Data Exchange (ETDEWEB)

    Branch, Darren W.; Huber, Dale L.; Brozik, Susan Marie; Edwards, Thayne L.

    2008-10-01

    The rapid autonomous detection of pathogenic microorganisms and bioagents by field deployable platforms is critical to human health and safety. To achieve a high level of sensitivity for fluidic detection applications, we have developed a 330 MHz Love wave acoustic biosensor on 36{sup o} YX Lithium Tantalate (LTO). Each die has four delay-line detection channels, permitting simultaneous measurement of multiple analytes or for parallel detection of single analyte containing samples. Crucial to our biosensor was the development of a transducer that excites the shear horizontal (SH) mode, through optimization of the transducer, minimizing propagation losses and reducing undesirable modes. Detection was achieved by comparing the reference phase of an input signal to the phase shift from the biosensor using an integrated electronic multi-readout system connected to a laptop computer or PDA. The Love wave acoustic arrays were centered at 330 MHz, shifting to 325-328 MHz after application of the silicon dioxide waveguides. The insertion loss was -6 dB with an out-of-band rejection of 35 dB. The amplitude and phase ripple were 2.5 dB p-p and 2-3{sup o} p-p, respectively. Time-domain gating confirmed propagation of the SH mode while showing suppression of the triple transit. Antigen capture and mass detection experiments demonstrate a sensitivity of 7.19 {+-} 0.74{sup o} mm{sup 2}/ng with a detection limit of 6.7 {+-} 0.40 pg/mm{sup 2} for each channel.

  20. Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response

    International Nuclear Information System (INIS)

    Rodak, L.

    1975-01-01

    An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125 I-labelled IgG 2 anti CSA (for demonstration of antigen) or with 125 I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection

  1. Development of a bacterial bioassay for atrazine and cyanuric acid detection

    Directory of Open Access Journals (Sweden)

    Anna eHUA

    2015-03-01

    Full Text Available The s-triazine herbicides are compounds which can disseminate into soils and water. Due to their toxic effects on living organisms, their concentrations in drinking water are legislated by WHO recommendations. Here we have developed for the first time, to the best of our knowledge, an alternative method for physicochemical quantification using two bioluminescent bacterial biosensors: E. coli SM003 for cyanuric acid detection and E. coli SM004 for both atrazine and cyanuric acid detection. The concentration of cyanuric acid detection for E. coli SM003 ranges from 7.83 µM to 2.89 mM, and for E. coli SM004 ranges from 0.22 µM to 15 µM. Moreover, atrazine detection by E. coli SM004 ranges from 1.08 µM to 15 µM. According to WHO recommendations, the cyanuric acid detection range is sensitive enough to discriminate between polluted and drinking water. Nevertheless, the detection of atrazine by E. coli SM004 is only applicable for high concentrations of contaminants.

  2. 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

  3. Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections

    Directory of Open Access Journals (Sweden)

    Højrup Peter

    2010-10-01

    Full Text Available Abstract Background In endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies. Methods Naturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa or low molecular weight (P. falciparum infections. Results IgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic IgG subclasses in plasma samples from individuals with higher levels of exposure to P. falciparum infections distinctly detected higher numbers of low molecular weight parasite antigens. Conclusions In the present study, there was no evidence for switching of antibody responses from non-cytophilic to cytophilic subclasses against blood-stage parasite antigens as a likely mechanism for induction of protective immunity against malaria.

  4. Serodiagnosis of bovine cysticercosis by detecting live Taenia saginata cysts using a monoclonal antibody-based antigen-ELISA

    Directory of Open Access Journals (Sweden)

    W. Wanzala

    2002-07-01

    Full Text Available An ante mortem antigen-ELISA-based diagnosis of Taenia saginata cysticercosis was studied in artificially (n = 24 and naturally (n = 25 infected cattle with the objective of further validating the assay as a field diagnostic test. Based on total dissection as the definitive method of validity, the assay minimally detected 14 live cysticerci in artificially infected calves and 2 in naturally infected steers. In natural infections, the minimum number of live cysticerci consistently detected by Ag-ELISA was 5 while in artificial infections it was above 14. However, other animals with 12 and 17 live cysticerci in artificially infected calves, and 1 and 2 live cysticerci in naturally infected steers, escaped detection for unknown reasons. Animals harbouring dead cysticerci gave negative reactions in the assay as was the case in non-infected experimental control calves. There was a statistically significant positive linear correlation between Ag-ELISA optical density values and burdens of live cysticerci as obtained by total dissection of both artificially infected calves (r = 0.798, n = 24 ; P < 0.05 and naturally infected steers (r = 0.631, n = 25 ; P < 0.05. These results clearly show the potential effectiveness of ante mortem monoclonal antibody-based antigen detection ELISA in the diagnosis of bovine cysticercosis in cattle. Its value lies in the diagnosis of infection in cattle as a screening test in a herd, rather than as a diagnostic test at the individual level, due to false positive and negative reactions. In a herd of heavily infected cattle, the assay may, however, provide for individual diagnosis. Nevertheless, more work is recommended to increase its sensitivity so as to be able to diagnose light infections consistently in the field.

  5. Oxygen measurements in platelet fluids - a new non-invasive method to detect bacterial contaminations in platelets.

    Science.gov (United States)

    Mueller, M M; Hourfar, M K; Huber, E; Sireis, W; Weichert, W; Seifried, E; Tonn, T; Schmidt, M

    2012-06-01

    The residual risk for bacterial contamination in blood components especially in platelets is one to two orders of magnitude higher than for transfusion relevant viral infections. The majority of all bacterial transmitted fatalities occurred at the end of platelet shelf life. Therefore, the maximum shelf life of platelet concentrates (PC) was reduced to 4 days after blood donation in Germany in 2008. A new continuous non-invasive bacterial detection method was developed by O(2) measurements in the platelet fluids and tested with 10 transfusion relevant bacteria species. The bacterial concentration at the time point of a positive signal of PreSense O(2) ranged between 10(2) and 10(5) CFU mL(-1) . Harmful transfusion-transmitted bacterial infection would have probably been prevented by this novel technology. Only strict anaerobic bacteria strains like Clostridium perfringens were not detected within the study period of 72 h. The described non-invasive bacterial detection method represents a new approach to prevent transmission of bacterial infection in platelets. The method is characterised by the advantage that all investigations can be performed until right up to the time of transfusion, and therefore, reduce the risk for sample errors to a minimum. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

  6. Detection of Antileukocytic Antibodies in Blood Serum using Lymphocytes and Latex Microspheres Carrying HLA-Antigens upon Alloimmunization of Women with Recurrent Pregnancy Loss.

    Science.gov (United States)

    Stepanova, E O; Nikolaeva, M A; Golubeva, E L; Vtorushina, V V; Van'ko, L V; Khodzhaeva, Z S; Krechetova, L V

    2016-03-01

    Anti-HLA-antibodies were detected using cross-reaction of blood serum with allogenic T and B cells and latex microspheres coated with HLA-I and HLA-II antigens. HLA+ and HLA-sera obtained from women before and after allogeneic immunization were tested. The results obtained by these methods significantly differed. The test with latex microspheres detected antibodies to HLA-I and HLA-II antigens with high sensitivity and specificity and can be used for assessment of clinical significance of alloantibody detection when using alloimmunization in the therapy of gestation disorders.

  7. Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

    Science.gov (United States)

    Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

    2017-12-20

    Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

  8. Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators.

    Science.gov (United States)

    Scheler, Ott; Kindt, Jared T; Qavi, Abraham J; Kaplinski, Lauris; Glynn, Barry; Barry, Thomas; Kurg, Ants; Bailey, Ryan C

    2012-01-01

    A label-free biosensing method for the sensitive detection and identification of bacterial transfer-messenger RNA (tmRNA) is presented employing arrays of silicon photonic microring resonators. Species specific tmRNA molecules are targeted by complementary DNA capture probes that are covalently attached to the sensor surface. Specific hybridization is monitored in near real-time by observing the resonance wavelength shift of each individual microring. The sensitivity of the biosensing platform allowed for detection down to 53 fmol of Streptococcus pneumoniae tmRNA, equivalent to approximately 3.16×10(7) CFU of bacteria. The simplicity and scalability of this biosensing approach makes it a promising tool for the rapid identification of different bacteria via tmRNA profiling. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Bacterial and viral pathogens detected in sea turtles stranded along the coast of Tuscany, Italy.

    Science.gov (United States)

    Fichi, G; Cardeti, G; Cersini, A; Mancusi, C; Guarducci, M; Di Guardo, G; Terracciano, G

    2016-03-15

    During 2014, six loggerhead turtles, Caretta caretta and one green turtle, Chelonia mydas, found stranded on the Tuscany coast of Italy, were examined for the presence of specific bacterial and viral agents, along with their role as carriers of fish and human pathogens. Thirteen different species of bacteria, 10 Gram negative and 3 Gram positive, were identified. Among them, two strains of Vibrio parahaemolyticus and one strain of Lactococcus garviae were recovered and confirmed by specific PCR protocols. No trh and tdh genes were detected in V. parahaemolyticus. The first isolation of L. garviae and the first detection of Betanodavirus in sea turtles indicate the possibility for sea turtles to act as carriers of fish pathogens. Furthermore, the isolation of two strains of V. parahaemolyticus highlights the possible role of these animals in human pathogens' diffusion. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Analysis of bacterial metagenomes from the Southwestern Gulf of Mexico for pathogens detection.

    Science.gov (United States)

    Escobedo-Hinojosa, Wendy; Pardo-López, Liliana

    2017-07-31

    Little is known about the diversity of bacteria in the Southwestern Gulf of Mexico. The aim of the study illustrated in this perspective was to search for the presence of bacterial pathogens in this ecosystem, using metagenomic data recently generated by the Mexican research group known as the Gulf of Mexico Research Consortium. Several genera of bacteria annotated as pathogens were detected in water and sediment marine samples. As expected, native and ubiquitous pathogenic bacteria genera such as Burkolderia, Halomonas, Pseudomonas, Shewanella and Vibrio were highly represented. Surprisingly, non-native genera of public health concern were also detected, including Borrelia, Ehrlichia, Leptospira, Mycobacterium, Mycoplasma, Salmonella, Staphylococcus, Streptococcus and Treponema. While there are no previous metagenomics studies of this environment, the potential influences of natural, anthropogenic and ecological factors on the diversity of putative pathogenic bacteria found in it are reviewed. The taxonomic annotation herein reported provides a starting point for an improved understanding of bacterial biodiversity in the Southwestern Gulf of Mexico. It also represents a useful tool in public health as it may help identify infectious diseases associated with exposure to marine water and ingestion of fish or shellfish, and thus may be useful in predicting and preventing waterborne disease outbreaks. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Heteroassembled gold nanoparticles with sandwich-immunoassay LSPR chip format for rapid and sensitive detection of hepatitis B virus surface antigen (HBsAg).

    Science.gov (United States)

    Kim, Jinwoon; Oh, Seo Yeong; Shukla, Shruti; Hong, Seok Bok; Heo, Nam Su; Bajpai, Vivek K; Chun, Hyang Sook; Jo, Cheon-Ho; Choi, Bong Gill; Huh, Yun Suk; Han, Young-Kyu

    2018-06-01

    This study aimed to develop a more sensitive method for the detection of hepatitis B surface antigen (HBsAg) using heteroassembled gold nanoparticles (AuNPs). A single layered localized surface plasmon resonance (LSPR) chip format was developed with antigen-antibody reaction-based detection symmetry using AuNPs, which detected HBsAg at 10 pg/mL. To further improve the detection limit, a modified detection format was fabricated by fixing a secondary antibody (to form a heteroassembled sandwich format) to the AuNP monolayer, which enhanced the detection sensitivity by about 100 times. The developed heteroassembled AuNPs sandwich-immunoassay LSPR chip format was able to detect as little as 100 fg/mL of HBsAg within 10-15 min. In addition, the heteroassembled AuNPs sandwich-immunoassay LSPR chip format did not show any non-specific binding to other tested antigens, including alpha fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). These findings confirm that the proposed detection strategy of heteroassembled AuNPs sandwich-immunoassay LSPR chip format may provide a new platform for early diagnosis of various human diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-15

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

  13. A novel sandwiched electrochemiluminescence immunosensor for the detection of carcinoembryonic antigen based on carbon quantum dots and signal amplification.

    Science.gov (United States)

    Li, Nian-Lu; Jia, Li-Ping; Ma, Rong-Na; Jia, Wen-Li; Lu, Yi-Yang; Shi, Sha-Shan; Wang, Huai-Sheng

    2017-03-15

    In this study, a novel sandwiched electrochemiluminescence (ECL) immunosensor for the detection of carcinoembryonic antigen (CEA) was developed. The nanocomposite of polydopamine and Ag nanoparticles (PDA-AgNPs) was prepared by the redox reaction between Ag + and dopamine. This nanocomposite not only provided an effective matrix for the immobilization of primary antibody (Ab 1 ) but also enhanced the conductivity of the electrode. Carbon quantum dots (CQDs) were immobilized on the poly(ethylenimine) functionalized graphene oxide (PEI-GO) through amido-bond. Then Au nanoparticles were decorated on the CQDs modified PEI-GO matrix, and the resulted complex AuNPs/CQDs-PEI-GO was introduced to link secondary antibody (Ab 2 ). The CQDs can be connected to the electrode surface through the combination of CEA with Ab 1 and Ab 2 , and then the amplified electrochemiluminescence signal of CQDs was obtained with the synergistic effect of AgNPs, polydopamine, AuNPs and PEI-GO. Under the optimal conditions, the ECL intensity was proportional to the logarithm value of CEA concentration in the linear range from 5pgmL -1 to 500ngmL -1 with a detection limit of 1.67pgmL -1 for CEA detection. The immunosensor was applied for the CEA detection in real samples with satisfactory results. The proposed ECL immunosensor showed good performance with high sensitivity, specificity, reproducibility, stability and will be potential in clinical detection. Copyright © 2016. Published by Elsevier B.V.

  14. Dual Immunomagnetic Nanobeads-Based Lateral Flow Test Strip for Simultaneous Quantitative Detection of Carcinoembryonic Antigen and Neuron Specific Enolase

    Science.gov (United States)

    Lu, Wenting; Wang, Kan; Xiao, Kun; Qin, Weijian; Hou, Yafei; Xu, Hao; Yan, Xinyu; Chen, Yanrong; Cui, Daxiang; He, Jinghua

    2017-01-01

    A novel immunomagnetic nanobeads -based lateral flow test strip was developed for the simultaneous quantitative detection of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA), which are sensitive and specific in the clinical diagnosis of small cell lung cancer. Using this nanoscale method, high saturation magnetization, carboxyl-modified magnetic nanobeads were successfully synthesized. To obtain the immunomagnetic probes, a covalent bioconjugation of the magnetic nanobeads with the antibody of NSE and CEA was carried out. The detection area contained test line 1 and test line 2 which captured the immune complexes sensitively and formed sandwich complexes. In this assay, cross-reactivity results were negative and both NSE and CEA were detected simultaneously with no obvious influence on each other. The magnetic signal intensity of the nitrocellulose membrane was measured by a magnetic assay reader. For quantitative analysis, the calculated limit of detection was 0.094 ng/mL for NSE and 0.045 ng/mL for CEA. One hundred thirty clinical samples were used to validate the test strip which exhibited high sensitivity and specificity. This dual lateral flow test strip not only provided an easy, rapid, simultaneous quantitative detection strategy for NSE and CEA, but may also be valuable in automated and portable diagnostic applications. PMID:28186176

  15. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  16. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases

    Directory of Open Access Journals (Sweden)

    Seyyed Javad SEYYEDTABAEI

    2017-06-01

    Full Text Available Background: Visceral leishmaniasis (VL and cutaneous leishmaniasis (CL are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB compared with indirect immunofluorescence test (IFAT to serodiagnosis of leishmaniasis.Methods: This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. Results: WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups.Conclusion: WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

  17. Evaluation of an immunoblot methodology for the detection of relevant Entamoeba histolytica antigens by antibodies induced in human amebiasis.

    Science.gov (United States)

    Argüello-García, R; Sánchez-Guillén, M C; Garduño, G; Valadez-Salazar, A; Martínez-García, M C; Muñoz, O; Ortega-Pierres, M G

    1990-01-01

    The complexity of the clinical spectrum in human amebiasis and the high variability in laboratory methods used to detect Entamoeba histolytica infections have impeded the collection and evaluation of reliable epidemiological data. Thus, more sensitive, specific and standardized methods are needed in order to accurately identify infections with this parasite. An important step in the development of serological diagnostic methods is the identification and isolation of specific parasite antigens which are immunogenic in the host. In this work, we have standardized an electroimmunotransfer blot technique to characterize E. histolytica antigens recognized by antibodies present during human amebic infections. An important aspect was an investigation of technical variations in the preparation of cell lysates including the use of different protease inhibitors and solubilizing agents. The highest yield of protein was achieved by homogenization of trophozoites in the presence of 10 mM p-hydroxymercuribenzoate (pHMB) as a protease inhibitor and by lysis using Triton X-100 and a mixture of protease inhibitors. Recovery of degraded vs non-degraded proteins in the cell extracts was evaluated by gradient polyacrylamide gel electrophoresis. Both quantitative and qualitative differences were noted between the different methods of preparing soluble cell extracts. A more complete set of antigenic components was obtained by homogenization and use of pHMB. Thus parasite extracts prepared by this method were selected for protein transfer. In this, the optimal protein concentration was of 120 micrograms of protein per cm of gel width and efficient transfer of proteins to nitrocellulose sheets was achieved at 100 V for 2 hrs and at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen

    Science.gov (United States)

    Lee, I.-K.; Jeun, M.; Jang, H.-J.; Cho, W.-J.; Lee, K. H.

    2015-10-01

    Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor

  19. Detection rate of prostate cancer using prostate specific antigen in patients presenting with lower urinary tract symptoms: A retrospective study

    Directory of Open Access Journals (Sweden)

    Chavan P

    2009-01-01

    Full Text Available Background: Need for undertaking prostate biopsies for detection of prostate cancer is often decided on the basis of serum levels of prostate specific antigen (PSA. Aim: To evaluate the case detection rate of prostate cancer among patients presenting with lower urinary tract symptoms (LUTS on the basis of PSA levels and to assess the scope of prostate biopsy in these patients. Setting and Design: A retrospective study from a tertiary care center. Materials and Methods: The clinical and histopathological data of 922 patients presenting with LUTS in the last five years was obtained from the medical record section. They had been screened for prostate cancer using PSA and /or digital rectal examination examination followed by confirmation with prostate biopsy. Statistical Analysis Used: Detection rate and receiver operating characteristic curve were performed using SPSS 16 and Medcalc softwares. Results: The detection rate of prostate cancer according to the PSA levels was 0.6%, 2.3%, 2.5%, 34.1% and 54.9% in the PSA range of 0-4, 4-10, 10-20, 20-50 and> 50 ng/ml, respectively. Maximum prostate cancer cases were detected beyond a PSA value of 20 ng/ml whereas no significant difference in the detection rate was observed in the PSA range of 0-4, 4-10 and 10-20 ng/ml. Conclusion: A low detection rate of prostate cancer observed in the PSA range of 4-20 ng/ml in LUTS patients indicates the need for use of higher cutoff values of PSA in such cases. Therefore we recommend a cutoff of 20 ng/ml of PSA for evaluation of detection rate of prostate cancer among patients presenting with LUTS.

  20. Carcinoembryonic antigen detection with "Handing"-controlled fluorescence spectroscopy using a color matrix for point-of-care applications.

    Science.gov (United States)

    Qin, Weijian; Wang, Kan; Xiao, Kun; Hou, Yafei; Lu, Wenting; Xu, Hao; Wo, Yan; Feng, Shaoqing; Cui, Daxiang

    2017-04-15

    In this study, we developed a power-free, accurate, and portable diagnostic platform called Handing based on embedded technology, which can rapidly detect fluorescent signals from quantum dots (QDs) on lateral flow test strips (LFTSs). The Handing system has three components: a hand-held test terminal, LFTS cartridge, and data server. The hand-held test terminal is the primary system component and it is integrated with tiny components using printed circuit board packaging for image acquisition, processing, and data handling by a specific program. A black smooth shell and three-dimensional printed test cartridge are used to facilitate the portability of the detection terminal, which provides a closed detection process as well as enhancing the anti-interference capacity. The functions of the data server comprise pre-editing, storage, range querying, and sharing with an international network. Multiple hand-held terminal devices can be linked simultaneously to the same data server. In this study, we detected the tumor marker carcinoembryonic antigen (CEA) using the QD-LFTS system, which allowed quantitative analysis in the range of 1-100ng/mL with an ideal detection limit of 0.049ng/mL. Thus, the system is suitable for detecting CEA in the clinically accepted range. We also detected 70 positive and 30 negative serum samples using the Handing system, which exhibited good specificity and sensitivity. Thus, Handing has the capacity for rapid quantitative detection with high stability and repeatability, so it could be used for in vitro diagnostics in the laboratory and in other conditions for various applications, e.g., food evaluation, disease screening, environmental monitoring, and drug testing. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Evaluation of a Direct Immunofluorescent Assay and/or Conjunctival Cytology for Detection of Canine Distemper Virus Antigen.

    Science.gov (United States)

    Athanasiou, Labrini V; Kantere, Maria C; Kyriakis, Constantinos S; Pardali, Dimitra; Adamama Moraitou, Katerina; Polizopoulou, Zoe S

    2017-11-29

    Canine distemper is a common and potentially lethal multisystemic disease caused by the Canine distemper virus (CDV). We evaluated the diagnostic performance of direct immunofluorescent assay (FA) and cytology to detect CDV antigen in conjunctival cells compared with an established polymerase chain reaction (PCR) detection assay used as a gold standard for CDV diagnosis. Samples were collected from 57 young dogs presenting with central nervous system signs compatible with distemper disease. Exfoliative epithelial cells were collected from the right and left conjunctiva of each animal using nylon-bristled cytobrushes for cytology and cotton swabs for FA and PCR. For the direct FA, samples were stained with anti-CDV polyclonal antiserum conjugated to fluorescein isothiocyanate and imaged using a fluorescent microscope. Out of 57 dogs tested, 19 were PCR positive (15 positive in direct FA and 4 positive in cytology, including one that was negative by PCR), whereas 37 dogs were negative in all methods. A good agreement was observed between the FA and PCR, with a κ-value of 0.833 (95% CI: 0.678-0.989). Meanwhile, there was poor agreement between cytology and PCR (κ-value of 0.164; 95% CI: -0.045 to 0.373) and a fair agreement between FA and cytology (κ-value of 0.231; 95% CI: -0.026 to 0.487). Our results indicated a poor performance of cytology for the detection of CDV antigen. In contrast, FA is a 100% specific and an adequately sensitive assay (sensitivity: 78.95%, negative likelihood ratio: 0.21, 95% CI: 0.09-0.50) for antemortem diagnosis of canine distemper.

  2. Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

    Directory of Open Access Journals (Sweden)

    Yuting Guo

    2017-07-01

    Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

  3. Bacterial wetwood detection in Fagus grandifolia and Prunus serotina sapwood using a conducting polymer electronic-nose device

    Science.gov (United States)

    A.D. Wilson

    2014-01-01

    New electronic gas-detection methods were developed and tested for the diagnosis of bacterial wetwood disease in Fagus grandifolia (American beech) and Prunus serotina (black cherry) using a Conducting Polymer (CP)-type electronic nose (e-nose), the Aromascan A32S, based on detection of headspace...

  4. Label-free electrochemical immunosensor based on Nile blue A-reduced graphene oxide nanocomposites for carcinoembryonic antigen detection.

    Science.gov (United States)

    Gao, Yan-Sha; Zhu, Xiao-Fei; Xu, Jing-Kun; Lu, Li-Min; Wang, Wen-Min; Yang, Tao-Tao; Xing, Hua-Kun; Yu, Yong-Fang

    2016-05-01

    In this article, a novel, label-free, and inherent electroactive redox immunosensor for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB-ERGO) is proposed. The composite of NB-graphene oxide (NB-GO) was prepared by π-π stacking interaction. Then, chronoamperometry was adopted to simultaneously reduce HAuCl4 and nanocomposites of NB-GO for synthesizing AuNPs/NB-ERGO. The immunosensor was fabricated by capturing CEA antibody (anti-CEA) at this nanocomposite modified electrode. The immunosensor determination was based on the fact that, due to the formation of antigen-antibody immunocomplex, the decreased response currents of NB were directly proportional to the concentrations of CEA. Under optimal conditions, the linear range of the proposed immunosensor was estimated to be from 0.001 to 40 ng ml(-1) and the detection limit was estimated to be 0.00045 ng ml(-1). The proposed immunosensor was used to determine CEA in clinical serum samples with satisfactory results. The proposed method may provide promising potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity, and selectivity. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Detection of prostate-specific antigen with biomolecule-gated AlGaN/GaN high electron mobility transistors

    International Nuclear Information System (INIS)

    Li, Jia-dong; Miao, Bin; Wei, Xiao-wei; Xie, Jie; Wu, Dong-min; Cheng, Jun-jie; Zhang, Jin-cheng; Zhang, Zhi-qiang

    2014-01-01

    In order to improve the sensitivity of AlGaN/GaN high electron mobility transistor (HEMT) biosensors, a simple biomolecule-gated AlGaN/GaN HEMT structure was designed and successfully fabricated for prostate specific antigen (PSA) detection. UV/ozone was used to oxidize the GaN surface and then a 3-aminopropyl trimethoxysilane (APTES) self-assembled monolayer was bound to the sensing region. This monolayer serves as a binding layer for attachment of the prostate specific antibody (anti-PSA). The biomolecule-gated AlGaN/GaN HEMT sensor shows a rapid and sensitive response when the target prostate-specific antigen in buffer solution was added to the antibody-immobilized sensing area. The current change showed a logarithm relationship against the PSA concentration from 0.1 pg/ml to 0.993 ng/ml. The sensitivity of 0.215% is determined for 0.1 pg/ml PSA solution. The above experimental result of the biomolecule-gated AlGaN/GaN HEMT biosensor suggested that this biosensor might be a useful tool for prostate cancer screening. (paper)

  6. Comparative study of sequence aligners for detecting antibiotic resistance in bacterial metagenomes.

    Science.gov (United States)

    McCall, C; Xagoraraki, I

    2018-03-01

    We aim to compare the performance of Bowtie2, bwa-mem, blastn and blastx when aligning bacterial metagenomes against the Comprehensive Antibiotic Resistance Database (CARD). Simulated reads were used to evaluate the performance of each aligner under the following four performance criteria: correctly mapped, false positives, multi-reads and partials. The optimal alignment approach was applied to samples from two wastewater treatment plants to detect antibiotic resistance genes using next generation sequencing. blastn mapped with greater accuracy among the four sequence alignment approaches considered followed by Bowtie2. blastx generated the greatest number of false positives and multi-reads when aligned against the CARD. The performance of each alignment tool was also investigated using error-free reads. Although each aligner mapped a greater number of error-free reads as compared to Illumina-error reads, in general, the introduction of sequencing errors had little effect on alignment results when aligning against the CARD. Given each performance criteria, blastn was found to be the most favourable alignment tool and was therefore used to assess resistance genes in sewage samples. Beta-lactam and aminoglycoside were found to be the most abundant classes of antibiotic resistance genes in each sample. Antibiotic resistance genes (ARGs) are pollutants known to persist in wastewater treatment plants among other environments, thus methods for detecting these genes have become increasingly relevant. Next generation sequencing has brought about a host of sequence alignment tools that provide a comprehensive look into antimicrobial resistance in environmental samples. However, standardizing practices in ARG metagenomic studies is challenging since results produced from alignment tools can vary significantly. Our study provides sequence alignment results of synthetic, and authentic bacterial metagenomes mapped against an ARG database using multiple alignment tools, and the

  7. A Genoproteomic Approach to Detect Peptide Markers of Bacterial Respiratory Pathogens.

    Science.gov (United States)

    Wang, Honghui; Drake, Steven K; Yong, Chen; Gucek, Marjan; Lyes, Matthew A; Rosenberg, Avi Z; Soderblom, Erik; Arthur Moseley, M; Dekker, John P; Suffredini, Anthony F

    2017-08-01

    Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. In silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii , Moraxella catarrhalis , Pseudomonas aeruginosa , Stenotrophomonas maltophilia , and Klebsiella pneumoniae . Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously. Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa , when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 10 3 and 2.2 (0.6) × 10 3 colony-forming units, respectively, by targeted LC-MS/MS. This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL. © 2017 American Association for Clinical Chemistry.

  8. Evaluation of vaginal pH for detection of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    R Hemalatha

    2013-01-01

    Full Text Available Background & objectives : Bacterial vaginosis (BV is highly prevalent among women in reproductive age group. Little information exists on routine vaginal p H measurement in women with BV. We undertook this study to assess the utility of vaginal p H determination for initial evaluation of bacterial vaginosis. Methods : In this cross-sectional study vaginal swabs were collected from women with complaints of white discharge, back ache and pain abdomen attending a government hospital and a community health clinic, and subjected to vaginal p H determination, Gram stain, wet mount and whiff test. Nugent score and Amsel criteria were used for BV confirmation. Results : Of the 270 women included in the analysis, 154 had BV based on Nugents′ score. The mean vaginal p H in women with BV measured by p H strips and p H glove was 5 and 4.9, respectively. The vaginal p H was significantly higher in women with BV. Vaginal discharge was prevalent in 84.8 per cent women, however, only 56.8 per cent of these actually had BV by Nugent score (NS. Presence of clue cells and positive whiff test were significant for BV. Vaginal p H >4.5 by p H strips and p H Glove had a sensitivity of 72 and 79 per cent and specificity of 60 and 53 per cent, respectively to detect BV. Among the combination criteria, clue cells and glove p H >4.5 had highest sensitivity and specificity to detect BV. Interpretation & conclusions : Vaginal p H determination is relatively sensitive, but less specific in detecting women with BV. Inclusion of whiff test along with p H test reduced the sensitivity, but improved specificity. Both, the p H strip and p H glove are equally suitable for screening women with BV on outpatient basis.

  9. KAtex antigen-detection test as a diagnostic tool for latent visceral ...

    African Journals Online (AJOL)

    tests have disadvantages. Parasitological diagnosis tools are accurate but invasive, while molecular tools are expensive and not commonly used. We report that the newly developed kAtex test is a rapid, non invasive and simple tool for the detection of visceral leishmaniasis (VL), capable of detecting and distinguishing ...

  10. Detection of survivin, carcinoembryonic antigen and ErbB2 level in oral squamous cell carcinoma patients.

    Science.gov (United States)

    Li, Shu-Xia; Yang, Yan-Qi; Jin, Li-Jian; Cai, Zhi-Gang; Sun, Zheng

    2016-01-01

    The aim of this study was to detect the survivin, carcinoembryonic antigen (CEA) and ErbB2 in the saliva, serum and local tumor-exfoliated cells of oral squamous cell carcinoma (OSCC) patients, for providing reliable tumor markers for the early detection of oral malignant cancer. The saliva, serum, and local tumor-exfoliated cell samples of 26 OSCC patients without chemotherapy and 10 non-cancer patients were collected in Department of Oral and Maxillofacial Surgery, School of Stomatology, Peking University. The contents of survivin, CEA and ErbB2 using were detected usingenzyme-linked immunosorbent assay. The survivin and CEA levels in saliva and local tumor-exfoliated cells of OSCC patients were significantly higher than those in the non-cancer patients (P < 0.05), but there was no significant difference in the content of the above factors in the serum sample between two groups. There was no significant difference in the ErbB2 content in the saliva, serum or local tumor-exfoliated cells between two groups. Survivin and CEA levels are significantly increased in the saliva and local tumor-exfoliated cells in OSCC patients, and they can be used as reliable markers for the early detection of oral malignant cancer.

  11. Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products.

    Science.gov (United States)

    Bolden, Jay; Smith, Kelly

    2017-01-01

    Recombinant Factor C (rFC) is non-animal-derived reagent used to detect bacterial endotoxins in pharmaceutical products. Despite the fact that the reagent was first commercially available nearly 15 years ago, the broad use of rFC in pharmaceutical industry has long been lagging, presumably due to historical single-source supplier concerns and the lack of inclusion in worldwide pharmacopeias. Commercial rFC reagents are now available from multiple manufacturers, thus single sourcing is no longer an issue. We report here the successful validation of several pharmaceutical products by an end-point florescence-based endotoxin method using the rFC reagent. The method is equivalent or superior to the compendia bacterial endotoxins test method. Based on the comparability data and extenuating circumstances, the incorporation of the end point fluorescence technique and rFC reagent in global compendia bacterial endotoxins test chapters is desired and warranted. LAY ABSTRACT: Public health has been protected for over 30 years with the use of a purified blood product of the horseshoe crab, limulus amebocyte lysate. More recently, this blood product can be produced in biotech manufacturing processes, which reduces potential impacts to the horseshoe crab and related species dependent upon the crab, for example, migrating shorebirds. The pharmaceutical industry has been slow to adopt the use of this reagent, Recombinant Factor C (rFC), for various reasons. We evaluated the use of rFC across many pharmaceutical products, and in other feasibility demonstration experiments, and found rFC to be a suitable alternative to the animal-derived limulus amebocyte lysate. Incorporation of rFC and its analytical method into national testing standards would provide an equivalent or better test while continuing to maintain patient safety for those who depend on medicines and while securing pharmaceutical supply chains. In addition, widespread use of this method would benefit existing animal

  12. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker; Bakker, Arnold H; Shu, Chengyi J

    2009-01-01

    The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses...... to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells...

  13. Beetroot-Pigment-Derived Colorimetric Sensor for Detection of Calcium Dipicolinate in Bacterial Spores

    Science.gov (United States)

    Gonçalves, Letícia Christina Pires; Da Silva, Sandra Maria; DeRose, Paul C.; Ando, Rômulo Augusto; Bastos, Erick Leite

    2013-01-01

    In this proof-of-concept study, we describe the use of the main red beet pigment betanin for the quantification of calcium dipicolinate in bacterial spores, including Bacillus anthracis. In the presence of europium(III) ions, betanin is converted to a water-soluble, non-luminescent orange 1∶1 complex with a stability constant of 1.4×105 L mol–1. The addition of calcium dipicolinate, largely found in bacterial spores, changes the color of the aqueous solution of [Eu(Bn)+] from orange to magenta. The limit of detection (LOD) of calcium dipicolinate is around 2.0×10–6 mol L–1 and the LOD determined for both spores, B. cereus and B. anthracis, is (1.1±0.3)×106 spores mL–1. This simple, green, fast and low cost colorimetric assay was selective for calcium dipicolinate when compared to several analogous compounds. The importance of this work relies on the potential use of betalains, raw natural pigments, as colorimetric sensors for biological applications. PMID:24019934

  14. A Feasibility Study for Microwave Breast Cancer Detection Using Contrast-Agent-Loaded Bacterial Microbots

    Directory of Open Access Journals (Sweden)

    Yifan Chen

    2013-01-01

    Full Text Available We propose a new approach to microwave breast tumor sensing and diagnosis based on the use of biocompatible flagellated magnetotactic bacteria (MTB adapted to operate in human microvasculature. It has been verified experimentally by Martel et al. that externally generated magnetic gradients could be applied to guide the MTB along preplanned routes inside the human body, and a nanoload could be attached to these bacterial microbots. Motivated by these useful properties, we suggest loading a nanoscale microwave contrast agent such as carbon nanotubes (CNTs or ferroelectric nanoparticles (FNPs onto the MTB in order to modify the dielectric properties of tissues near the agent-loaded bacteria. Subsequently, we propose a novel differential microwave imaging (DMI technique to track simultaneously multiple swarms of MTB microbots injected into the breast. We also present innovative strategies to detect and localize a breast tissue malignancy and estimate its size via this DMI-trackable bacterial microrobotic system. Finally, we use an anatomically realistic numerical breast phantom as a platform to demonstrate the feasibility of this tumor diagnostic method.

  15. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Kui Zhu

    2014-04-01

    Full Text Available Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv, as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs, magnetic nanoparticles (MNPs, quantum dots (QDs and carbon nanomaterials (graphene and carbon nanotube, for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC, which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT, will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  16. Toxocara canis: Analysis of the kinetics of antigen release and antibody production in an in vivo model for the detection of past or present infection.

    Science.gov (United States)

    Rodríguez-Caballero, Aarón; Martínez-Gordillo, Mario Noé; Caballero-Salazar, Silvia; Rufino-González, Yadira; Ponce-Macotela, Martha

    2017-08-30

    Worldwide, Toxocara canis is an important zoonotic nematode of public health concern. This soil-transmitted helminth causes visceral larva and ocular larva migrans in paratenic hosts. The detection of T. canis larva migrans is complicated because current immunological tests detect only IgG antibodies, which can cross-react with antigens from other parasites and cannot distinguish between the past and present infection. Analysis of antigen release and antibody production could help improve the detection of larva migrans. Here, we report the kinetics of antigen release, IgM and IgG production in an in vivo model for the detection of past or present infection. We used four groups of seven mice: two groups infected orally with 50 or 100 embryonated eggs, and the other two infected intraperitoneally with 50 or 100 live larvae. We obtained blood samples at 0, 3, 7, and 14days and, then, every two weeks until day 140. Sandwich ELISA and indirect ELISA were performed for antigen capture and the detection of immunoglobulins, respectively. Mice inoculated with larvae developed an immune response faster than those inoculated with eggs. In all groups, antigen capture was positive starting at 3days until 140days post-inoculation (dpi). Detection of immunoglobulins was at 14 or 28dpi in mice inoculated with larvae or eggs, respectively. Negative IgM values were detected at days 98 and 112. The samples remained positive for IgG until the last day of the experiment. Data suggest that in mice inoculated with T canis eggs, some larvae did not hatch, others died or never reached the bloodstream. Based on our model, we propose that there is early infection when only antigens are present, and active larva migrans when antigen and immunoglobulins are detected, implying an immune response of the host against the antigen. Our study offers a view into the parasite-host relationship and enables us to infer if there are live larvae. Additionally, these findings provide a foundation for the

  17. Specific Antibodies for the Detection of Alternaria Allergens and the Identification of Cross-Reactive Antigens in Other Fungi

    Science.gov (United States)

    Twaroch, Teresa E.; Curin, Mirela; Sterflinger, Katja; Focke-Tejkl, Margit; Swoboda, Ines; Valenta, Rudolf

    2017-01-01

    Background The mould Alternaria alternata is an important source of respiratory allergens. A. alternata extracts show great variations regarding allergenic potency. The aim of this study was to generate antibody probes specific for important Alternaria allergens and to use them to study allergen expression, depending on different culture conditions, as well as to search for cross-reactive allergens in other mould species. Methods Synthetic peptides from antigenic regions of A. alternata allergens (Alt a 1, Alt a 2, Alt a 3, Alt a 6 and Alt a 8) were used to raise highly specific rabbit antibodies. These antibodies and IgE from allergic patients were used to detect allergens by immunoblotting in extracts of 4 A. alternata strains grown under varying culturing conditions, in commercial skin-prick extracts and in closely (Cladosporium herbarum and Aureobasidium pullulans) or distantly related (Aspergillus niger and Penicillium chrysogenum) mould species. Results There was a wide variation of expression of the individual A. Alternata allergens, depending on the strain and culture conditions, but the antibody probes allowed us to distinguish strains and culture conditions with low and high allergen expression. In the commercial skin-prick solutions, varying levels of Alt a 1 were found, but no other allergens were detectable. Alt a 1 was identified as species-specific A. Alternata allergen, whereas Alt a 3, 6- and Alt a 8-cross-reactive antigens were found in C. herbarum and/or A. pullulans. Conclusions and Clinical Relevance Peptide-specific antibodies are useful to analyze diagnostic and therapeutic mould extracts, to study the presence of A. Alternata allergens in biological samples and to search for cross-reactive allergens in other mould species. PMID:27780168

  18. Evolution of MHC-based technologies used for detection of antigen-responsive T cells

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Hadrup, Sine Reker

    2017-01-01

    detection of 1-2 different T cell specificities per cell sample, to include more than 1000 evaluable pMHC molecules using novel technologies. Here, we provide an overview of MHC multimer-based detection technologies developed over two decades, focusing primarily on MHC class I interactions.......T cell-mediated recognition of peptide-major histocompatibility complex (pMHC) class I and II molecules is crucial for the control of intracellular pathogens and cancer, as well as for stimulation and maintenance of efficient cytotoxic responses. Such interactions may also play a role...... class I molecules provided sufficient stability to T cell receptor (TCR)-pMHC interactions, allowing detection of MHC multimer-binding T cells using flow cytometry. Since this breakthrough the scientific community has aimed for expanding the capacity of MHC multimer-based detection technologies...

  19. Comparative study of methods of detection of hepatitis ′B′ surface antigen (HBsAg.

    Directory of Open Access Journals (Sweden)

    Parab V

    1989-04-01

    Full Text Available The serum samples were collected from 52 patients of acute viral hepatitis and 235 hospital staff from Kasturba Hospital for Infectious Diseases. HBsAg was detected in their sera by counter-immuno-electrophoresis (CIEP, reverse passive hemogglutination (RPHA and by micro-enzyme-linked-immunosorbent assay (ELISA technique. Among the patients, HBsAg was detected in 12 cases (23% by CIEP, in 18 cases (34% by RPHA and in 23 patients (45% by ELISA. In the hospital staff, HBsAg was detected in 4 samples (1.7% by CIEP, in 8 samples (3.5% by RPHA and in 32 samples (13.5% by ELISA. Thus ELISA was found to be the most sensitive technique in detecting HBsAg.

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

    Directory of Open Access Journals (Sweden)

    Rafaella Fortini Queiroz Grenfell

    2012-08-01

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

  1. A new technique to detect antibody-antigen reaction (biological interactions) on a localized surface plasmon resonance (LSPR) based nano ripple gold chip

    Energy Technology Data Exchange (ETDEWEB)

    Saleem, Iram, E-mail: iiram.qau@gmail.com [Department of Physics and Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Widger, William, E-mail: widger@uh.edu [Department of Biology and Biochemistry and Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Chu, Wei-Kan, E-mail: wkchu@uh.edu [Department of Physics and Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States)

    2017-07-31

    Highlights: • The nano ripple LSPR chip has monolayer molecule-coating sensitivity and specific selectivity. • Gold nano-ripple sensing chip is a low cost, and a label-free method for detecting the antibody-antigen reaction. • The plasmonic resonance shift depends upon the concentration of the biomolecules attached on the surface of the nano ripple pattern. - Abstract: We demonstrate that the gold nano-ripple localized surface plasmon resonance (LSPR) chip is a low cost and a label-free method for detecting the presence of an antigen. A uniform stable layer of an antibody was coated on the surface of a nano-ripple gold pattern chip followed by the addition of different concentrations of the antigen. A red shift was observed in the LSPR spectral peak caused by the change in the local refractive index in the vicinity of the nanostructure. The LSPR chip was fabricated using oblique gas cluster ion beam (GCIB) irradiation. The plasmon-resonance intensity of the scattered light was measured by a simple optical spectroscope. The gold nano ripple chip shows monolayer scale sensitivity and high selectivity. The LSPR substrate was used to detect antibody-antigen reaction of rabbit X-DENTT antibody and DENTT blocking peptide (antigen).

  2. DETECTION OF HELICOBACTER ANTIGEN IN STOOL SAMPLES AND ITS RELATION TO H. PYLORI POSITIVE CHOLECYSTITIS IN EGYPTIAN PATIENTS WITH CHRONIC CALCULAR CHOLECYSTITIS.

    Science.gov (United States)

    Hassan, Ehsan H; Gerges, Shawkat S; Ahmed, Rehab; Mostafa, Zeinab M; Al-Hamid, Hager Abd; Abd El-Galil, Heba; Thabet, Suzan

    2015-12-01

    Evidences supporting the association between H. pylori infection and chronic cholecystitis could be found by using direct culture or staining of H. pylori in gallbladder tissues as well as indirect techniques. Stool antigen test has been widely used due to its noninvasive nature. Various stool antigen tests were developed to detect H. pylori using an enzyme immunoassay (EIA) based on monoclonal or polyclonal antibodies This study evaluated the frequency of H. pylori antigen in stool samples of patients with chronic calcular cholecystitis as regard gall bladder histopathological changes. Fifty patients were included presented with symptomatic qholecystolithiasis recruited from the outpatient clinic of National Hepatology and Tropical Medicine Research Institute during 2014-2015. Full history and clinical examination and abdominal ultrasonography were performed. Stool samples were collected, prepared and examined for detection of H. pylori antigen. Cholecystectomy was done for all patients; 45 patients (90%) by laparoscopic Cholecystectomy and 5 patients (10%) by open surgery and removed gallbladders were submitted to pathology department for detection of H. pylori in tissue under microscope using Giemsa stain. The results showed that (82%) were females with mean age (42.6 +/- 1 years). The mean BMI was (29 + 7.2) H. pylori-specific antigen in stool samples was detected in 40% of patients and 38% were detected in patients; tissue, with significant correlation between H. pylori-specific antigen in stool and in tissue. Histopathological pictures infection in tissue were 68.4% mucosal erosions, 63.2% mucosal atrophy, 57.9% mucosal hyperplasia, 26.3% metaplasia, 42.1% musculosa hypertrophy, 26.3% fibrosis, but lymphoid aggregates were in 42.1% of cases.

  3. A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

    Science.gov (United States)

    Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

    2014-01-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

  4. A novel label-free electrochemical immunosensor based on functionalized nitrogen-doped graphene quantum dots for carcinoembryonic antigen detection.

    Science.gov (United States)

    Yang, Yuying; Liu, Qing; Liu, Yan; Cui, Jianjian; Liu, Hui; Wang, Ping; Li, Yueyun; Chen, Lei; Zhao, Zengdian; Dong, Yunhui

    2017-04-15

    A novel and ultrasensitive label-free electrochemical immunosensor was fabricated for quantitative detection of carcino-embryonic antigen (CEA). The nitrogen-doped graphene quantum dots (N-GQDs) supported PtPd bimetallic nanoparticles (PtPd/N-GQDs) were synthesized by a simple and green hydrothermal procedure. Subsequently, PtPd/N-GQDs functionalized Au nanoparticles (PtPd/N-GQDs@Au) were prepared successfully via a self-assembly approach. Because of the synergetic effect present in PtPd/N-GQDs@Au, this novel nanocomposites has shown excellent electrocatalytic activity towards hydrogen peroxide (H 2 O 2 ) reduction. Featuring good biocompatibility, excellent conductivity and large surface area, PtPd/N-GQDs@Au was applied as transducing materials to efficiently conjugate capture antibodies and amplify electrochemical signal. Under the optimal conditions, the proposed immunosensor was used for the detection of CEA with wide dynamic range in the range from 5 fg/mL to 50ng/mL with a low detection limit of 2fg/mL (S/N=3). Furthermore, this label-free immunosensor possesses high sensitivity, special selectivity and long-term stability, which shows promising application in bioassay analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Good performance of rapid prostate-specific antigen test for detection of semen exposure in women: implications for qualitative research.

    Science.gov (United States)

    Hobbs, Marcia M; Steiner, Markus J; Rich, Kimberly D; Gallo, Maria F; Alam, Anadil; Rahman, Motiur; Menezes, Prema; Chipato, Tsungai; Warner, Lee; Macaluso, Maurizio

    2009-08-01

    Prostate-specific antigen (PSA) is a valid biomarker of semen exposure in women and has been used to assess reliability of self-reported sexual behavior as well as serve as a proxy measure for condom efficacy. Quantitative PSA tests are expensive and require specialized equipment. A simple, rapid, and inexpensive test for PSA would facilitate semen biomarker evaluation in a variety of research settings. This study evaluated the performance of a rapid PSA test compared with a quantitative assay to identify semen in vaginal swab specimens. We tested 581 vaginal swabs collected from 492 women participating in 2 separate research studies in Bangladesh and Zimbabwe. PSA in vaginal secretions was detected using the quantitative IMx (Abbott Laboratories) assay and the ABAcard p30 (Abacus Diagnostics) rapid immunochromatographic strip test. The ABAcard test was 100% sensitive (95% confidence interval [CI], 98%-100%) and 96% specific (95% CI, 93%-97%) compared with the quantitative test in detecting >1.0 ng PSA/mL vaginal swab eluate. Rapid PSA results were semiquantitative and correlated well with PSA concentrations (kappa = 0.88; 95% CI, 0.85-0.90). Rapid PSA detection requires no instrumentation and can be performed easily and economically. Having rapid PSA results available immediately following interview provides opportunities to explore discrepancies between the objective marker of recent semen exposure and self-reported behaviors.

  6. Rapid and quantitative detection of prostate specific antigen with a quantum dot nanobeads-based immunochromatography test strip.

    Science.gov (United States)

    Li, Xue; Li, Wenbin; Yang, Qiuhua; Gong, Xiaoqun; Guo, Weisheng; Dong, Chunhong; Liu, Junqing; Xuan, Lixue; Chang, Jin

    2014-05-14

    Convenient and fast testing using an immunochromatography test strip (ICTS) enables rapid yes/no decisions regarding a disease to be made. However, the fundamental limitations of an ICTS, such as a lack of quantitative and sensitive analysis, severely hampers its application in reliable medical testing for the early detection of cancer. Herein, we overcame these limitations by integrating an ICTS with quantum dot nanobeads (QD nanobeads), which were fabricated by encapsulating QDs within modified poly(tert-butyl acrylate-co-ethyl acrylate-co-methacrylic acid) and served as a robust signal-generating reagent for the ICTS. Prostate specific antigen (PSA) was used as a model analyte to demonstrate the performance of the QD nanobeads-based ICTS platform. Under optimized conditions, the concentration of PSA could be determined within 15 min with high sensitivity and specificity using only 40 μL of sample. The detection limit was enhanced by ∼12-fold compared with that of an ICTS that used QDs encapsulated by commercial 11-mercaptoundecanoic acid (QDs@MUA) as the signal-generating reagent. At the same time, the possible clinical utility of this approach was demonstrated by measurements recorded from PSA-positive patient specimens. Our data suggest that the QD nanobeads-based ICTS platform is not only rapid and low-cost but also highly sensitive and specific for use in quantitative point-of-care diagnostics; thus, it holds promise for becoming a part of routine medical testing for the early cancer of detection.

  7. Preparation of Pd/Bacterial Cellulose Hybrid Nanofibers for Dopamine Detection

    Directory of Open Access Journals (Sweden)

    Dawei Li

    2016-05-01

    Full Text Available Palladium nanoparticle-bacterial cellulose (PdBC hybrid nanofibers were synthesized by in-situ chemical reduction method. The obtained PdBC nanofibers were characterized by a series of analytical techniques. The results revealed that Pd nanoparticles were evenly dispersed on the surfaces of BC nanofibers. Then, the as-prepared PdBC nanofibers were mixed with laccase (Lac and Nafion to obtain mixture suspension, which was further modified on electrode surface to construct novel biosensing platform. Finally, the prepared electrochemical biosensor was employed to detect dopamine. The analysis result was satisfactory, the sensor showed excellent electrocatalysis towards dopamine with high sensitivity (38.4 µA·mM−1, low detection limit (1.26 µM, and wide linear range (5–167 µM. Moreover, the biosensor also showed good repeatability, reproducibility, selectivity and stability and was successfully used in the detection of dopamine in human urine, thus providing a promising method for dopamine analysis in clinical application.

  8. PRISM hepatitis B surface antigen detection of hepatits B virus minipool nucleic acid testing yield samples.

    Science.gov (United States)

    Linauts, Sandy; Saldanha, John; Strong, D Michael

    2008-07-01

    Hepatitis B virus (HBV) residual risk has been estimated at 1:63,000-1:205,000 and introduction of more sensitive serological tests and nucleic acid testing (NAT) would reduce that risk. Sensitivity of the recently licensed Abbott PRISM hepatitis B surface antigen (HBsAg) CLIA and minipool (MP) HBV NAT has been described as comparable and thus the need for HBV NAT has not been compelling. In this study, eight samples identified as yield samples with MP HBV NAT were tested using the PRISM test. Seven samples were identified using the Roche COBAS AmpliScreen HBV test and one additional sample was obtained from the clinical trial for the Roche cobas TaqScreen MPX test. Each of these samples was reactive by MP HBV NAT and nonreactive for HBsAg using one of three licensed enzyme immunoassay (EIA) tests. After licensure of the PRISM HBsAg, aliquots were tested with this assay, and DNA quantitation and genotyping were repeated where sample volume permitted. Three samples (2000, 2300, and 61,000 copies/mL) produced reactive results with PRISM. Four samples with viral loads less than 300 copies per mL produced nonreactive results. One sample, originally quantitated at 37,000 copies per mL (but 3850 copies/mL in repeat testing) was also nonreactive by PRISM. Genotyping of this sample indicated a type C genotype with no mutations. Adding serological sensitivity of PRISM CLIA reduced the NAT yield from the original 1: 385,555 to 1:610,488. However, MP HBV NAT still provides additional sensitivity over CLIA, even for a donation with a viral load of almost 4000 copies per mL.

  9. Redox cycling-based immunoassay for detection of carcinogenic embryonic antigen.

    Science.gov (United States)

    Lee, Ga-Yeon; Park, Jun-Hee; Chang, Young Wook; Cho, Sungbo; Kang, Min-Jung; Pyun, Jae-Chul

    2017-06-08

    Redox cycling based on an interdigitated electrode (IDE) was used as a highly sensitive immunoassay for carcinogenic embryonic antigen (CEA) through the quantification of 3,3',5,5'-tetramethylbenzidine (TMB). For the redox cycling process, one pair of interdigitated finger electrodes was used as the first working electrode (generator) for cyclic voltammetry of TMB, and another pair of interdigitated finger electrodes was used as the second working electrode (collector) for sequential application of potentials for reduction and oxidation of TMB. The reduction (and oxidation) products of TMB at the collector were supplied to the generator, and following sequential oxidization (and reduction) at the generator, again supplied to the collector. Such redox recycling processes between the generator and collector allowed signal amplification. In this work, the influences of the following factors on the redox cycling of TMB were analyzed: (1) the redox potential at the collector, (2) the gap between the interdigitated finger electrodes, and (3) the scan rate of the generator. The redox potential and electrode gap influences were simulated with COMSOL software and compared with empirical results. At the optimum redox potentials and electrode gap, redox cycling was estimated to be five-fold more sensitive for the quantification of TMB than conventional cyclic voltammetry using one pair of interdigitated finger electrodes as the working electrode. Finally, redox cycling was applied to a commercial immunoassay for CEA, and the sensitivity of redox cycling was three-fold higher than that of conventional cyclic voltammetry using a single set of interdigitated finger electrodes as the working electrode. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. A novel colloidal gold labeled antigen for the detection of Deoxynivalenol using an immunochromatographic assay method

    Science.gov (United States)

    Jin, Yu; Liu, Renrong; Zhu, Lixin; Chen, Zhenzhen

    2017-11-01

    In this paper, an immunochromatographic assay card was developed for the detection of DON in feed and cereals using a novel colloidal gold labeling method. For the colloidal gold immunochromatographic rapid detection (GICD) card, a monoclonal antibody DON-mAb and a goat anti-chicken IgY were drawn on NC membrane as the test line (T line) and the control line (C line) respectively. A gold labeled DON-CBSA conjugate and a gold labeled chicken IgY were sprayed onto the conjugate pad. The GICD card has cut-off levels of 50ng/mL for DON, which is invulnerable to matrix interference, and applicable to a wide range of samples. The GICD detecting results of feed and grain samples were compared with the results of ELISA testing, which showed good consistency.

  11. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Changhua; Mao, Mao [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Yuan, Hang [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Shen, Huaibin [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Wu, Feng; Ma, Lan, E-mail: malan@sz.tsinghua.edu.cn [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Li, Lin Song, E-mail: lsli@henu.edu.cn [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China)

    2013-09-15

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 Degree-Sign C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  12. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    Science.gov (United States)

    Zhou, Changhua; Mao, Mao; Yuan, Hang; Shen, Huaibin; Wu, Feng; Ma, Lan; Li, Lin Song

    2013-09-01

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 °C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

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

    Directory of Open Access Journals (Sweden)

    Kazuki Morioka

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

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

    Science.gov (United States)

    Morioka, Kazuki; Fukai, Katsuhiko; Sakamoto, Kenichi; Yoshida, Kazuo; Kanno, Toru

    2014-01-01

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

  15. Bench-Top Antigen Detection Technique that Utilizes Nanofiltration and Fluorescent Dyes which Emit and Absorb Light in the Near Infrared

    Science.gov (United States)

    Varaljay-Spence, Vanessa A.; Scardelletti, Maximilian C.

    2007-01-01

    This article discusses the development of a bench-top technique to detect antigens in fluids. The technique involves the use of near infrared NIR fluorescent dyes conjugated to antibodies, centrifugation, nanofilters, and spectrometry. The system used to detect the antigens utilizes a spectrometer, fiber optic cables, NIR laser, and laptop computer thus making it portable and ideally suited for desk top analysis. Using IgM as an antigen and the secondary antibody, anti-IgM conjugated to the near infrared dye, IRDye (trademark) 800, for detection, we show that nanofiltration can efficiently and specifically separate antibody-antigen complexes in solution and that the complexes can be detected by a spectrometer and software using NIR laser excitation at 778 nm and NIR dye offset emission at 804 nm. The peak power detected at 778 nm for the excitation emission and at 804 nm for the offset emission is 879 pW (-60.06 dBm) and 35.7 pW (-74.5 dBm), respectively.

  16. Enhanced detection sensitivity of carcinoembryonic antigen on a plasmonic nanoimmunosensor by transmission grating-based total internal reflection scattering microscopy.

    Science.gov (United States)

    Ahn, Sujin; Yu, Hyunung; Kang, Seong Ho

    2017-10-15

    Carcinoembryonic antigen (CEA) is a glycoprotein associated with colorectal carcinomas and is commonly used as a clinical tumor marker. Enhanced detection sensitivity for the assay of CEA molecules was achieved on a plasmonic nanoimmunosensor by wavelength-dependent transmission grating (TG)-based total internal reflection scattering microscopy (TIRSM). The plasmonic nanoparticles were placed in an evanescent field layer on a glass nanoimmunosensor that produced evanescent wave scattering by the total internal reflection of light from two lasers. The light scattered by target protein (CEA)-bound 20-nm silver nanoparticles (plasmonic nanoprobes) was collected and spectrally isolated in first-order spectral images (n=+1) by a TG (70 grooves/mm). The combination of evanescent wave scattering and TG ​significantly enhanced the detection sensitivity and selectivity due to the minimized spectroscopic interference and background noise. The TG-TIRSM method detected the CEA molecules at concentrations down to 19.75zM with a wide linear dynamic range of 19.75zM-39.50nM (correlation coefficient, R=0.9903), which was 45 to 1.25×10 9 times lower than the detection limits and 2×10 5 to 2×10 11 times wider than the dynamic ranges of previous assay methods. In particular, by simply changing the antibody of the target molecule, this technique can be used to detect various disease-related protein biomarkers directly in human biological samples at the single-molecule level. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Concurrent Detection of Human Norovirus and Bacterial Pathogens in Water Samples from an Agricultural Region in Central California Coast

    Directory of Open Access Journals (Sweden)

    Peng Tian

    2017-08-01

    Full Text Available Bacterial pathogens and human norovirus (HuNoV are major cause for acute gastroenteritis caused by contaminated food and water. Public waterways can become contaminated from a variety of sources and flood after heavy rain events, leading to pathogen contamination of produce fields. We initiated a survey of several public watersheds in a major leafy green produce production region of the Central California Coast to determine the prevalence of HuNoV as well as bacterial pathogens. Moore swabs were used to collect environmental samples bi-monthly at over 30 sampling sites in the region. High prevalence of HuNoV and bacterial pathogens were detected in environmental water samples in the region. The overall detection rates of HuNoV, O157 Shiga toxin-producing Escherichia coli (STEC, non-O157 STEC, Salmonella, and Listeria were 25.58, 7.91, 9.42, 59.65, and 44.30%, respectively. The detection rates of Salmonella and L. monocytogenes were significantly higher in the spring. Fall and spring had elevated detection rates of O157 STEC. The overall detection rates of non-O157 STEC in the fall were lower than the other seasons but not significant. The overall detection rates of HuNoV were highest in fall, followed by spring and winter, with summer being lowest and significantly lower than other seasons. This study presented the first study of evaluating the correlation between the detection rate of HuNoV and the detection rates of four bacterial pathogens from environmental water. Overall, there was no significant difference in HuNoV detection rates between samples testing positive or negative for the four bacterial pathogens tested. Pathogens in animal-impacted and human-impacted areas were investigated. There were significant higher detection rates in animal-impacted areas than that of human-impacted areas for bacterial pathogens. However, there was no difference in HuNoV detection rates between these two areas. The overall detection levels of generic E

  18. Emerging antigenic variants at the antigenic site Sb in pandemic A(H1N12009 influenza virus in Japan detected by a human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Mayo Yasugi

    Full Text Available The swine-origin pandemic A(H1N12009 virus, A(H1N1pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E in the hemagglutinin (HA protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently.

  19. Detection of antigen in sera of patients with invasive aspergillosis : Intra- and interlaboratory reproducibility

    NARCIS (Netherlands)

    Verweij, PE; Erjavec, Z; Sluiters, W; Goessens, W; Rozenberg-Arska, M; Debets-Ossenkopp, YJ; Guiot, HFL; Meis, JFGM

    The intra-and interlaboratory reproducibilities of a commercial sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Aspergillus galactomannan in serum (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) were evaluated in six laboratories of university

  20. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Solov'yov, Ilia A.; Balboni, Imelda M.

    2016-01-01

    molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best...

  1. A defect in epithelial barrier integrity is not required for a systemic response to bacterial antigens or intestinal injury in T cell receptor-alpha gene-deficient mice.

    Science.gov (United States)

    Sydora, Beate C; Tavernini, Michele M; Doyle, Jason; Fedorak, Richard N

    2006-08-01

    Genetically induced disruption of the intestinal epithelial barrier leads to development of intestinal inflammation. In the interleukin-10 gene-deficient inflammatory bowel disease (IBD) mouse model, for instance, a primary defect in intestinal epithelial integrity occurs before the development of enterocolitis. In humans, a causal role for epithelial barrier disruption is still controversial. Although studies with first-degree relatives of IBD patients suggests an underlying role of impaired barrier function, a primary epithelial barrier defect in IBD patients has not been confirmed. The purpose of this article is to examine whether a primary epithelial barrier disruption is a prerequisite for the development of intestinal inflammation or whether intestinal inflammation can develop in the absence of epithelial disruption. We examined the intestinal epithelial integrity of the T cell receptor (TCR)-alpha gene-deficient mouse model of IBD. In vivo colonic permeability, determined by mannitol transmural flux, was assessed in 6-week-, 12-week-, and 25-week-old TCR-alpha gene-deficient and wild-type control mice using a single-pass perfusion technique. Mice were scored for intestinal histological injury and intestinal cytokine levels measured in organ cultures. Systemic responses to bacterial antigens were determined through 48-h spleen cell cultures stimulated with sonicate derived from endogenous bacterial strains. In contrast with previous findings in the interleukin-10 gene-deficient IBD model, TCR-alpha gene-deficient mice did not demonstrate evidence of primary intestinal epithelial barrier disruption at any age, despite developing a moderate to severe colitis within 12 weeks. A rise in intestinal interferon (IFN)-gamma levels preceded the onset of mucosal inflammation and then correlated closely with the degree of intestinal inflammation and injury. Spleen cells from TCR-alpha gene-deficient mice released IFN-gamma in response to stimulation with endogenous

  2. Liquid-liquid extraction and liquid chromatography-mass spectrometry detection of curcuminoids from bacterial culture medium.

    Science.gov (United States)

    Tan, Suryani; Rupasinghe, Thusitha W T; Tull, Dedreia L; Augustin, Mary Ann; Gras, Sally L

    2015-04-15

    Liquid chromatography-mass spectrometry (LC-MS) has been used to detect polyphenolic curcuminoids found in turmeric but studies of metabolism by bacterial and mammalian cells in vitro are compromised by poor recovery from the culture medium. We report a liquid-liquid extraction procedure with ethyl acetate and use LC-MS to quantify extracted curcuminoids. Ethyl acetate allows recoveries of ∼ 80-86% of curcuminoids from the bacterial growth medium, bacterial cell lysate and combined bacterial cell and growth medium matrices; a clear improvement over acetonitrile where recoveries were ∼ 25-66%. This optimised method will enable studies of curcuminoid metabolism and may be applicable to other hydrophobic polyphenolic compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Comparison of PCR with Standard Method (MPN for detection of bacterial contamination in drinking water

    Directory of Open Access Journals (Sweden)

    Fatemeh Dehghan

    2014-11-01

    Full Text Available Background: Detection of bacterial contamination in drinking water by culture method is a time and cost consuming method and spends a few days depending on contamination degree. However, the people use the tap water during that time. Molecular methods are rapid and sensitive. In this study a rapid Multiplex PCR method was used for rapid analysis both coliform bacteria and E.coli, and probable detection of VBNC bacteria in drinking water, the experiments were performed in bacteriological lab of water and Wastewater Corporation in Markazi province. Material and Methods:Amplification of a fragment from each of lacZ and uidA genes in a Multiplex PCR was used for detection of coliforms. Eight samples was taken from Arak drinking water system including 36 samples of wells, 41 samples of water distribution network and 3 samples from water storages were examined by amplification of lacZ and uidA genes in a Multiplex PCR. Equivalently, the MPN test was applied as a standard method for all samples for comparison of results. Standard bacteria, pure bacteria isolated from positive MPN and CRM were examined by PCR and MPN method. Results: The result of most samples water network, water storages, and water well were same in both MPN and PCR method .The results of standard bacteria and pure cultures of bacteria isolated from positive MPN and CRM confirmed the PCR method. Five samples were positive in PCR but negative in MPN method. Duration time of PCR was decreased about 105 min by changing the PCR program and electrophoreses factors. Conclusion: The Multiplex PCR can detect coliform bacteria and E.coli synchronous in drinking water.

  4. Diagnostic Accuracy of Ascites Fluid Gross Appearance in Detection of Spontaneous Bacterial Peritonitis

    Directory of Open Access Journals (Sweden)

    Hamed Aminiahidashti

    2014-08-01

    Full Text Available Introduction: Spontaneous bacterial peritonitis (SBP as a monomicrobial infection of ascites fluid is one of the most important causes of morbidity and mortality in cirrhotic patients. This study was aimed to determine the diagnostic accuracy of ascites fluid color in detection of SBP in cirrhotic cases referred to the emergency department. Methods: Cirrhotic patients referred to the ED for the paracentesis of ascites fluid were enrolled. For all studied patients, the results of laboratory analysis and gross appearance of ascites fluid registered and reviewed by two emergency medicine specialists. The sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ration of the ascites fluid gross appearance in detection of SBP were measured with 95% confidence interval. Results: The present project was performed in 80 cirrhotic patients with ascites (52.5 female. The mean of the subjects’ age was 56.25±12.21 years (35-81. Laboratory findings revealed SBP in 23 (29% cases. Fifty nine (73% cases had transparent ascites fluid appearance of whom 17 (29% ones suffered from SBP. From 21 (26% cases with opaque ascites appearance, 15 (71% had SBP. The sensitivity and specificity of the ascites fluid appearance in detection of SBP were 46.88% (Cl: 30.87-63.55 and 87.50% (95% Cl: 75.3-94.14, respectively. Conclusion: It seems that the gross appearance of ascites fluid had poor diagnostic accuracy in detection of SBP and considering its low sensitivity, it could not be used as a good screening tool for this propose.

  5. Evaluation of a simple blood culture amplification and antigen detection method for diagnosis of Salmonella enterica serovar typhi bacteremia.

    Science.gov (United States)

    Castonguay-Vanier, Josée; Davong, Viengmon; Bouthasavong, Latsanyphone; Sengdetka, Davanh; Simmalavong, Manivone; Seupsavith, Amphayvanh; Dance, David A B; Baker, Stephen; Le Thi Phuong, Tu; Vongsouvath, Manivanh; Newton, Paul N

    2013-01-01

    In most areas where typhoid is endemic, laboratory diagnosis is not possible due to the lack of appropriate facilities. We investigated whether the combination of blood culture amplification of Salmonella enterica serovar Typhi with an S. Typhi antigen rapid diagnostic test (RDT) could be an accurate and inexpensive tool for the accelerated diagnosis of patients with acute typhoid in Laos. For a panel of 23 Gram-negative reference pathogens, the Standard Diagnostics (catalog no. 15FK20; Kyonggi-do, South Korea) RDT gave positive results for S. Typhi NCTC 8385, S. Typhi NCTC 786 (Vi negative), Salmonella enterica serovar Enteritidis (ATCC 13076), and Salmonella enterica serovar Ndolo NCTC 8700 (all group D). In a prospective study of 6,456 blood culture bottles from 3,028 patients over 15 months, 392 blood culture bottles (6.1%) from 221 (7.3%) patients had Gram-negative rods (GNRs) seen in the blood culture fluid. The sensitivity, negative predictive value, specificity, and positive predictive value were 96.7%, 99.5%, 97.9%, and 87.9%, respectively, for patients with proven S. Typhi bacteremia and 91.2%, 98.4%, 98.9%, and 93.9% for patients with group D Salmonella. The median (range) number of days between diagnosis by RDT and reference assays was 1 (-1 to +2) day for those with confirmed S. Typhi. The use of antigen-based pathogen detection in blood culture fluid may be a useful, relatively rapid, inexpensive, and accurate technique for the identification of important causes of bacteremia in the tropics.

  6. The clinical significance of detection of serum Pre-S1 antigen

    International Nuclear Information System (INIS)

    Jiang Xuehua; Huang Zhuqing; Han Yi; Gong Shoujun

    2003-01-01

    To study the clinical significance of detection of serum Pre-S 1 Ag, the serum Pre-S 1 Ag, HBV-marks and HBV-DNA were detected in 338 patients with hepatitis B. The positive rate and the relationship between them were analyzed and compared. In 338 patients, the positive rate of serum Pre-S 1 Ag, HBeAg, HBV-DNA was 63.02%, 48.52%, 68.05% respectively, and the co-positive rate of Pre-S 1 Ag with HBV-DNA, HBeAg was 78.56%, 81.17% respectively. There was a significant correlation between Pre-S 1 Ag, HBeAg and HBV-DNA (P 1 Ag could well reflect the reproductive status of hepatitis B virus, and so it could be used as the clinical marker of the reproductive status of hepatitis B virus

  7. Electrochemical Detecting Lung Cancer-Associated Antigen Based on Graphene-Gold Nanocomposite

    Directory of Open Access Journals (Sweden)

    Zheng Wei

    2017-03-01

    Full Text Available Using a Au nanoparticle/reduced graphene oxide composite (AuNP-RGO, a signal-enhanced electrochemical immunosensor without label was created to detect neuron-specific enolase (NSE. Furthermore, an environmentally-friendly method was developed to prepare AuNP-RGO by employing chitosan (CS, which served as reducing and stabilizing agent. We showed that the sensitivity of the immunosensor designed in this report was remarkably enhanced because of the numerous active sites in the sensor provided by the AuNP-RGO nanostructure. For the quantification of NSE, the immunosensor exhibited a positive linear relationship with the concentration in the range of 0.1 to 2000 ng/mL, where the limit of the detection was 0.05 ng/mL.

  8. A simple and inexpensive point-of-care test for hepatitis B surface antigen detection: serological and molecular evaluation.

    Science.gov (United States)

    Gish, R G; Gutierrez, J A; Navarro-Cazarez, N; Giang, K; Adler, D; Tran, B; Locarnini, S; Hammond, R; Bowden, S

    2014-12-01

    Early identification of chronic hepatitis B is important for optimal disease management and prevention of transmission. Cost and lack of access to commercial hepatitis B surface antigen (HBsAg) immunoassays can compromise the effectiveness of HBV screening in resource-limited settings and among marginalized populations. High-quality point-of-care (POC) testing may improve HBV diagnosis in these situations. Currently available POC HBsAg assays are often limited in sensitivity. We evaluated the NanoSign(®) HBs POC chromatographic immunoassay for its ability to detect HBsAg of different genotypes and with substitutions in the 'a' determinant. Thirty-seven serum samples from patients with HBV infection, covering HBV genotypes A-G, were assessed for HBsAg titre with the Roche Elecsys HBsAg II quantification assay and with the POC assay. The POC assay reliably detected HBsAg at a concentration of at least 50 IU/mL for all genotypes, and at lower concentrations for some genotypes. Eight samples with substitutions in the HBV 'a' determinant were reliably detected after a 1/100 dilution. The POC strips were used to screen serum samples from 297 individuals at risk for HBV in local clinical settings (health fairs and outreach events) in parallel with commercial laboratory HBsAg testing (Quest Diagnostics EIA). POC testing was 73.7% sensitive and 97.8% specific for detection of HBsAg. Although the POC test demonstrated high sensitivity over a range of genotypes, false negatives were frequent in a clinical setting. Nevertheless, the POC assay offers advantages for testing in both developed and resource-limited countries due to its low cost (0.50$) and immediately available results. © 2014 John Wiley & Sons Ltd.

  9. Top-down nanofabrication of silicon nanoribbon field effect transistor (Si-NR FET) for carcinoembryonic antigen detection.

    Science.gov (United States)

    Bao, Zengtao; Sun, Jialin; Zhao, Xiaoqian; Li, Zengyao; Cui, Songkui; Meng, Qingyang; Zhang, Ye; Wang, Tong; Jiang, Yanfeng

    2017-01-01

    Sensitive and quantitative detection of tumor markers is highly required in the clinic for cancer diagnosis and consequent treatment. A field-effect transistor-based (FET-based) nanobiosensor emerges with characteristics of being label-free, real-time, having high sensitivity, and providing direct electrical readout for detection of biomarkers. In this paper, a top-down approach is proposed and implemented to fulfill a novel silicon nano-ribbon FET, which acts as biomarker sensor for future clinical application. Compared with the bottom-up approach, a top-down fabrication approach can confine width and length of the silicon FET precisely to control its electrical properties. The silicon nanoribbon (Si-NR) transistor is fabricated on a Silicon-on-Insulator (SOI) substrate by a top-down approach with complementary metal oxide semiconductor (CMOS)-compatible technology. After the preparation, the surface of Si-NR is functionalized with 3-aminopropyltriethoxysilane (APTES). Glutaraldehyde is utilized to bind the amino terminals of APTES and antibody on the surface. Finally, a microfluidic channel is integrated on the top of the device, acting as a flowing channel for the carcinoembryonic antigen (CEA) solution. The Si-NR FET is 120 nm in width and 25 nm in height, with ambipolar electrical characteristics. A logarithmic relationship between the changing ratio of the current and the CEA concentration is measured in the range of 0.1-100 ng/mL. The sensitivity of detection is measured as 10 pg/mL. The top-down fabricated biochip shows feasibility in direct detecting of CEA with the benefits of real-time, low cost, and high sensitivity as a promising biosensor for tumor early diagnosis.

  10. Prostate cancer detection with digital rectal examination, prostate-specific antigen, transrectal ultrasonography and biopsy in clinical urological practice.

    Science.gov (United States)

    Ng, Tze Kiat; Vasilareas, Despina; Mitterdorfer, Andrew J; Maher, Peter O; Lalak, Andre

    2005-03-01

    To evaluate the utility of digital rectal examination (DRE), prostate specific antigen (PSA) and transrectal ultrasonography and biopsy (TRUSB) in detecting prostate cancer in one teaching-hospital urological practice. In all, 2800 consecutive patients had TRUSB as outpatients by one urologist, the indications for which were a raised or rising PSA level or an abnormal DRE. In addition, the indications for repeat TRUSB included previous abnormal histology, e.g. suspicious areas or atypia or high-grade prostatic intraepithelial neoplasia. All data were collected prospectively. Of 2800 TRUSB, 223 were known cases of prostate cancer (previously diagnosed from transurethral prostatectomy chips or after radical prostatectomy) and were excluded from the analysis. There were 2194 initial and 383 repeat TRUSB; of the former patients, 1129 were found to have prostate cancer, giving a cancer-detection rate of 52%. The positive predictive values (PPVs) for patients with a normal DRE and PSA of 10 ng/mL were 9%, 31% and 48%, respectively; the corresponding PPVs for patients with an abnormal DRE and the same PSA levels were 27%, 67% and 85%, respectively. Of the 383 repeat TRUSB, the cancer-detection rate was 31% for the first repeat and 28% for the second. The present values are higher than those reported previously, because these patients were within a clinical urological practice, and the indications for and methods of TRUSB have changed in recent years, such that more lateral areas were biopsied. These values are useful in helping clinicians to counsel patients about the probability of detecting cancer.

  11. Mite fauna and mite antigen detection in house dust found in residential areas in Los Baños, Laguna, Philippines.

    Science.gov (United States)

    Catanghal, Ronan Aldous M; Paller, Vachel Gay V

    2012-09-01

    Dust mites are a medically important group of animals commonly found in carpets and mattresses in houses. Antigens in their feces cause allergic reactions such as asthma and contact dermatitis. Dust samples were vacuum-collected in a special collecting bag from a one square meter area of living room floors of 100 randomly sampled houses in Los Baños, Laguna, Philippines for one minute. Chromato-immunoassay ELISA (Mitey Checker) was used to detect mite antigenicity. Twenty-three species of mites were identified belonging to 7 families. Of these, Blomia tropicalis (265 mites/g of dust in 87% of households) of Family Glycyphagidae and Dermatophagoides farinae (71 mites/g of dust in 58% of households) of Family Pyroglyphidae were the most prevalent and abundant species. Forty-eight percent of households were detected to have low levels of antigen (mite intensity and antigen level (r = 0.129). Mean Dermatophagoides intensity and antigen levels were also found to have a weak linear relationship. More mites were found in carpeted living rooms (822 mites/g) when compared to non-carpeted living rooms (645 mites/g). Different floor types did not show any difference in mean mite intensity. Likewise, mite intensity did not show correlation with household size.

  12. 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

  13. Effect of topical vaginal products on the detection of prostate-specific antigen, a biomarker of semen exposure, using ABAcards.

    Science.gov (United States)

    Snead, Margaret C; Kourtis, Athena P; Black, Carolyn M; Mauck, Christine K; Brown, Teresa M; Penman-Aguilar, Ana; Melendez, Johan H; Gallo, Maria F; Jamieson, Denise J; Macaluso, Maurizio

    2013-09-01

    Prostate-specific antigen (PSA) is a biomarker of recent semen exposure. There is currently only limited information on whether topical vaginal products affect PSA assays. We investigated this question using various dilutions of several vaginal products (lubricants and spermicides) and the Abacus ABAcard for PSA detection. Pooled semen controls and various dilutions of nonoxynol-9 (N9), carboxymethyl cellulose (CMC), Replens, Gynol 2, K-Y jelly, Astroglide, Surgilube, combined with pooled semen dilutions, were tested for PSA using the Abacus ABAcard. N9 (2% with saline) and CMC did not appear to affect the results of testing with the ABAcard, but not all semen dilutions were tested. The other products (including Replens and Gynol, which is 2% N9 with propylene glycol, K-Y, Astroglide and Surgilube) at some of the dilutions tested either affected or gave invalid results with PSA testing using the ABAcard. Both Gynol 2 and K-Y at 1:10 dilution gave false-positive results. Some vaginal products affect PSA results obtained by using the semiquantitative ABAcard. In vivo confirmation is necessary to further optimize PSA detection when topical vaginal products are present. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Prostate-specific membrane antigen PET/MRI validation of MR textural analysis for detection of transition zone prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bates, Anthony [Princess Alexandra Hospital, Brisbane (Australia); Miles, Kenneth [Princess Alexandra Hospital, Department of Diagnostic Radiology, Brisbane, QLD (Australia); University College London, Institute of Nuclear Medicine, London (United Kingdom)

    2017-12-15

    To validate MR textural analysis (MRTA) for detection of transition zone (TZ) prostate cancer through comparison with co-registered prostate-specific membrane antigen (PSMA) PET-MR. Retrospective analysis was performed for 30 men who underwent simultaneous PSMA PET-MR imaging for staging of prostate cancer. Thirty texture features were derived from each manually contoured T2-weighted, transaxial, prostatic TZ using texture analysis software that applies a spatial band-pass filter and quantifies texture through histogram analysis. Texture features of the TZ were compared to PSMA expression on the corresponding PET images. The Benjamini-Hochberg correction controlled the false discovery rate at <5%. Eighty-eight T2-weighted images in 18 patients demonstrated abnormal PSMA expression within the TZ on PET-MR. 123 images were PSMA negative. Based on the corrected p-value of 0.005, significant differences between PSMA positive and negative slices were found for 16 texture parameters: Standard deviation and mean of positive pixels for all spatial filters (p = <0.0001 for both at all spatial scaling factor (SSF) values) and mean intensity following filtration for SSF 3-6 mm (p = 0.0002-0.0018). Abnormal expression of PSMA within the TZ is associated with altered texture on T2-weighted MR, providing validation of MRTA for the detection of TZ prostate cancer. (orig.)

  15. Aluminum nanopyramid array with tunable ultraviolet-visible-infrared wavelength plasmon resonances for rapid detection of carbohydrate antigen 199.

    Science.gov (United States)

    Li, Wanbo; Qiu, Yongcai; Zhang, Li; Jiang, Lelun; Zhou, Zhangkai; Chen, Huanjun; Zhou, Jianhua

    2016-05-15

    Aluminum-based localized surface plasmon resonance (LSPR) holds attractive properties include low cost, high natural abundance, and ease of processing by a wide variety of methods including complementary metal oxide semiconductor process, making itself having an edge over conventional ones induced by noble metal. However, the inherent drawbacks of plasmonic mode limited on UV-green wavelength, low refractive index sensitivity, as well as heavy-shape-dependence greatly prevent aluminum plasmonics from real-life biosensing. Here, we demonstrated a uniform quasi-3-dimensional Al nanopyramid array (NPA) structure with tunable ultraviolet-visible-infrared (UV-vis-NIR) plasmon resonances for biosensing. By changing the reflection measuring angle, we could easily obtain typical peaks simultaneously exhibited on the reflectance spectrum across UV-vis-NIR wave region. The Al NPAs carried out high refractive index sensitivities which even comparable with that of noble metal, and can be used as a biosensor for directly detecting cytochrome c and carbohydrate antigen 199 in air after the sensing surface was washed cleanly and dried; the limits of detection were determined to be 800 nM and 29 ng/mL, respectively. Our proposed work therefore initiates the low-cost, high-performance biosensing using aluminum plasmonics, which would find wide applications in rapid diagnosis, mobile-healthcare and environmental monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Incorporation of antigens from whole cell lysates and purified virions from MP12 into fluorescence microsphere immunoassays for the detection of antibodies against Rift Valley fever virus

    Science.gov (United States)

    Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Vir...

  17. Evaluation of two real time PCR assays for the detection of bacterial DNA in amniotic fluid.

    Science.gov (United States)

    Girón de Velasco-Sada, Patricia; Falces-Romero, Iker; Quiles-Melero, Inmaculada; García-Perea, Adela; Mingorance, Jesús

    2018-01-01

    The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing. We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz. Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005). Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors.

    Science.gov (United States)

    Wynn, Daniel; Deo, Sapna; Daunert, Sylvia

    2017-01-01

    Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field. © 2017 Elsevier Inc. All rights reserved.

  19. Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes.

    Science.gov (United States)

    Scheler, Ott; Kaplinski, Lauris; Glynn, Barry; Palta, Priit; Parkel, Sven; Toome, Kadri; Maher, Majella; Barry, Thomas; Remm, Maido; Kurg, Ants

    2011-02-28

    We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

  20. Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

    Directory of Open Access Journals (Sweden)

    Toome Kadri

    2011-02-01

    Full Text Available Abstract Background We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification amplification, for sensitivity. Results We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

  1. Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

    LENUS (Irish Health Repository)

    Scheler, Ott

    2011-02-28

    Abstract Background We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. Results We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal\\/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

  2. Detection of polyomavirus major capsid antigen (VP-1) in human pilomatricomas.

    Science.gov (United States)

    Sanjuán, Norberto A; Símula, Silvina; Casas, José; Woscoff, Alberto

    2010-01-01

    The family Polyomaviridae is composed of small, non-enveloped, double-stranded DNA viruses widely used to study cell transformation in vitro and tumor induction in vivo. The development of pilomatricomas in mice experimentally infected with polyomavirus led us to detect the viral major capsid protein VP-1 in human pilomatricomas. This tumor, even uncommon, is one of the most frequent benign hair follicle tumors in humans and is composed of proliferating matrix cells that undergo keratinization, and form cystic neoplasms. The detection of VP-1 was performed using the peroxidase-antiperoxidase technique in paraffin-embedded slides with a specific primary serum. Adjacent slides treated with normal rabbit serum as a primary were employed as internal control. Positive and negative controls were also employed as well as slides of lesions caused by human papillomavirus to rule out any unspecific cross-reactivity. In 4 out of 10 cases polyomavirus VP-1 was clearly detected in nuclei of human pilomatricomas proliferating cells, in a patchy pattern of distribution. The controls confirmed the specificity of the immunocytochemical procedure. These results could indicate either an eventual infection of the virus in already developed tumors or alternatively, a direct involvement of polyomavirus in the pathogenesis of some pilomatricomas. The recent discovery of a new human polyomavirus associated with Merkel cell carcinomas has been a strong contribution to better understand the pathogenesis of some human uncommon skin cancers. Hopefully the results reported in this work will encourage further research on the role of polyomavirus in other human skin neoplasms.

  3. Detection of bacterial DNA in serial synovial samples obtained during antibiotic treatment from patients with septic arthritis

    NARCIS (Netherlands)

    van der Heijden, I. M.; Wilbrink, B.; Vije, A. E.; Schouls, L. M.; Breedveld, F. C.; Tak, P. P.

    1999-01-01

    The management of septic arthritis could benefit from sensitive tests that detect the persistence of microorganisms in the joint. The aim of this study was to determine the feasibility of monitoring the presence of bacterial DNA in synovial samples from septic arthritis patients during antibiotic

  4. Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia--a case-control study

    DEFF Research Database (Denmark)

    Helweg-Larsen, Jannik; Jensen, Jørgen Skov; Dohn, Birthe

    2002-01-01

    Pneumocystis jiroveci (formerly known as P. carinii f.sp. hominis) is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised individuals. Pneumocystis jiroveci can be detected by polymerase chain reaction (PCR). To investigate the clinical importance of a positive P...... Pneumocystis-PCR among HIV-uninfected patients suspected of bacterial pneumonia, a retrospective matched case-control study was conducted....

  5. Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia – a case-control study

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Jensen, JS; Dohn, G

    2002-01-01

    Pneumocystis jiroveci (formerly known as P. carinii f.sp. hominis) is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised individuals. Pneumocystis jiroveci can be detected by polymerase chain reaction (PCR). To investigate the clinical importance of a positive P...... Pneumocystis-PCR among HIV-uninfected patients suspected of bacterial pneumonia, a retrospective matched case-control study was conducted....

  6. Multiplex detection and identification of bacterial pathogens causing potato blackleg and soft rot in Europe, using padlock probes

    NARCIS (Netherlands)

    Slawiak, M.; Doorn, van R.; Szemes, M.; Speksnijder, A.G.C.L.; Waleron, M.; Wolf, van der J.M.; Lojkowska, E.; Schoen, C.D.

    2013-01-01

    The objective of this study was to develop a multiplex detection and identification protocol for bacterial soft rot coliforms, namely Pectobacterium wasabiae (Pw), Pectobacterium atrosepticum (Pba) and Dickeya spp., responsible for potato blackleg and tuber soft rot. The procedures were derived from

  7. Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

    Science.gov (United States)

    Chudley, Lindsey; McCann, Katy J; Coleman, Adam; Cazaly, Angelica M; Bidmon, Nicole; Britten, Cedrik M; van der Burg, Sjoerd H; Gouttefangeas, Cecile; Jandus, Camilla; Laske, Karoline; Maurer, Dominik; Romero, Pedro; Schröder, Helene; Stynenbosch, Linda F M; Walter, Steffen; Welters, Marij J P; Ottensmeier, Christian H

    2014-11-01

    Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

  8. Bacterial overgrowth can be detected by breath hydrogen measurement before clinical manifestations in suckling lambs

    Directory of Open Access Journals (Sweden)

    András Jávor

    2015-01-01

    Full Text Available Hydrogen breath test is a non-invasive and inexpensive method for estimation of small bowel transit time, detection of excess bacteria in the small intestine and demonstration of maldigestion or malabsorption. Until now, little has been known about breath hydrogen excretion in lambs. The aim of our study was to assess the patterns of breath hydrogen excretion in lambs before and after feeding ewe’s milk, and to evaluate pathological and/or physiological alterations in the lambs’ gastrointestinal function. We assumed that intestinal disorders may influence the breath hydrogen concentrations, which could be detected early in the subclinical stage. A total of 52 healthy black-headed Dorper lambs were included in the study. Breath hydrogen was measured after overnight fasting and at 30, 60 and 90 min after the start of feeding. There was a 2-week follow-up period after the measurements to assess the gastrointestinal health of lambs. During the follow-up period, clinical signs of diarrhoea developed in 6 lambs. Based on our results in healthy lambs, the median concentration of baseline breath hydrogen was 1.00 parts per million (minimum: 0.00, maximum: 2.00. We observed a significant elevation in breath hydrogen concentrations 60 min after feeding (P = 0.004, whereas the values detected 30 min after feeding were similar to the baseline values. Regarding the lambs in which clinical signs of diarrhoea developed, we revealed significantly higher baseline breath hydrogen concentrations compared to those which remained healthy (P < 0.001. Our observations underline that hydrogen breath test may be a useful tool for indicating potential bacterial overgrowth before any clinical signs of diarrhoea.

  9. Utility of MPT64 antigen detection for rapid confirmation of mycobacterium tuberculosis complex

    Directory of Open Access Journals (Sweden)

    Jyoti Arora

    2015-01-01

    Full Text Available Background: Rapid differentiation of the Mycobacterium tuberculosis complex (MTBC and mycobacteria other than tuberculosis (MOTT is crucial to facilitate early and effective treatment of the patients. Clinical presentation of MTBC and MOTT is not always very clear and routine conventional methods are time consuming. Materials and Methods: In the present study, the MPT64 protein detection-based immunochomatographic test (SD Bioline Kit, Standard Diagnostics, Inc., Korea was compared with the conventional biochemical method. Results: The sensitivity, specificity, positive predictive, and negative predictive values of the SD AgMPT64 kit were found to be 100, 96.4, 98.72, and 100%, respectively. Conclusions: Our results have demonstrated that the SD bioline kit is a rapid, reliable method and it can be used in the Revised National Tuberculosis Control Program (RNTCP of India, for the appropriate management of tuberculosis.

  10. The type-specific polysaccharide and the R protein antigens of the L-phase from a group B, type III Streptococcus.

    Science.gov (United States)

    Flores, A E; Ferrieri, P

    1985-04-01

    The type-specific polysaccharide and the R protein antigens from filtered culture supernatants of the bacterial phase and L-phase of the group B, type III streptococcal strain 76-043 were studied by several immunological methods. In the L-phase of growth, the two antigens were separate and distinct molecules which were found principally in the culture supernatant even on the 254th serial subculture in the cell-wall-defective state. Only trace amounts of these antigens were detected in extracts of L-phase cells. The type III polysaccharide antigens in the supernatant of cultures of the parent bacterium and the L-phase gave reactions of identity in immunodiffusion. Precipitin bands obtained by immunoelectrophoresis (IEP) revealed that the type-specific antigen of the bacterial phase of growth migrated toward the anode, whereas that of the L-phase remained near the antigen well. The R protein antigen in the L-phase supernatant was immunologically identical to the R protein of the supernatant and 1% trypsin-extracted antigens from whole cells of the parent bacterial strain, and other groups A, B and C streptococcal strains sharing a common R antigen. Immunologically, the R antigen appeared to be the species R4. The R protein of the L-phase and bacterial phase cultures was resistant to 5% trypsin but sensitive to 0.5% pepsin at 37 degrees C/2hr. Antiserum prepared in rabbits against L-phase cells contained an antibody reactive with the R protein antigens of the bacterial and L-phase cultures. The soluble, naturally released type III and R protein streptococcal antigens of the L-phase of growth permitted immunological confirmation of its bacterial origin.

  11. Combination of autoantibodies against NY-ESO-1 and viral capsid antigen immunoglobulin A for improved detection of nasopharyngeal carcinoma.

    Science.gov (United States)

    Peng, Yu-Hui; Xu, Yi-Wei; Qiu, Si-Qi; Hong, Chao-Qun; Zhai, Tian-Tian; Li, En-Min; Xu, Li-Yan

    2014-09-01

    Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China and Southeast Asia, and early detection remains a challenge. Autoantibodies have been found to precede the manifestations of symptomatic cancer by several months to years, making their identification of particular relevance for early detection. In the present study, the diagnostic value of serum autoantibodies against NY-ESO-1 in NPC patients was evaluated. The study included 112 patients with NPC and 138 normal controls. Serum levels of autoantibodies against NY-ESO-1 and classical Epstein-Barr virus marker, viral capsid antigen immunoglobulin A (VCA-IgA), were measured by enzyme-linked immunosorbent assay. Measurement of autoantibodies against NY-ESO-1 and VCA-IgA demonstrated a sensitivity/specificity of 42.9/94.9% [95% confidence interval (CI), 33.7-52.6/89.4-97.8%] and 55.4/95.7% (95% CI, 45.7-64.7/90.4-98.2%), respectively. The area under receiver operating characteristic curve for autoantibodies against NY-ESO-1 (0.821; 95% CI, 0.771-0.871) was marginally lower than that for VCA-IgA (0.860; 95% CI, 0.810-0.910) in NPC. The combination of autoantibodies against NY-ESO-1 and VCA-IgA yielded an enhanced sensitivity of 80.4% (95% CI, 71.6-87.0%) and a specificity of 90.6% (95% CI, 84.1-94.7%). Moreover, detection of autoantibodies against NY-ESO-1 could differentiate early-stage NPC patients from normal controls. Our results suggest that autoantibodies against NY-ESO-1 may serve as a potential biomarker, as a supplement to VCA-IgA, for the screening and diagnosis of NPC.

  12. Parallel reaction monitoring on a Q Exactive mass spectrometer increases reproducibility of phosphopeptide detection in bacterial phosphoproteomics measurements.

    Science.gov (United States)

    Taumer, Christoph; Griesbaum, Lena; Kovacevic, Alen; Soufi, Boumediene; Nalpas, Nicolas C; Macek, Boris

    2018-03-29

    Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms data-dependent acquisition (DDA) in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple heterogeneous phosphopeptide-enriched samples and conclude that PRM shows a great promise in bacterial phosphoproteomics analyses where reproducible detection and quantification of a relatively small set of phosphopeptides is desired. Bacterial phosphorylated peptides occur in low abundance compared to their unmodified counterparts, and are therefore rarely reproducibly detected in shotgun (DDA) proteomics measurements. Here we show that parallel reaction monitoring complements DDA analyses and makes detection of known, targeted phosphopeptides more reproducible. This will be of significance in replicated MS measurements that have a goal to reproducibly detect and quantify phosphopeptides of interest. Copyright

  13. 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.

  14. Electrochemical Immunosensor Based on Fe3O4/PANI/AuNP Detecting Interface for Carcinoembryonic Antigen Biomarker

    Science.gov (United States)

    Amarasiri, Chamali; Nguyen, Thanh Binh; Nguyen, Loc Thai; Thu, Vu Thi; Thuy, Nguyen Thi My; Dai Lam, Tran

    2017-10-01

    A low-cost screen-printed carbon electrode (SPCE) modified with Fe3O4, gold nanoparticles (AuNPs), and polyaniline (PANI) has been developed for rapid measurement of carcinoembryonic antigen (CEA) biomarker. The electrode surface was covered with Fe3O4 nanoparticles by drop coating then with PANI via electropolymerization. The resulting surface was further modified by AuNPs via electrodeposition. Key factors affecting the electrochemical behavior and sensing performance of the electrode were investigated. The results demonstrated that Fe3O4 mass loading of 2 mg/cm2 and 15 cycles of PANI polymerization were optimal for electrochemical measurement of CEA biomarker. In addition, compared with bare SPCE, coating the electrode surface with PANI, Fe3O4/PANI, and Fe3O4/PANI/AuNP significantly enhanced the peak oxidation current by approximately 16%, 52%, and 93%, respectively. The sensors exhibited linear trends with CEA concentration from 0 ng/mL to 10 ng/mL. The limit of detection and sensitivity of the electrode were estimated to be 0.25 ng/mL and 0.3827 μA/ng mL-1, respectively. Such sensors could be easily integrated into microfluidic platforms and could serve as a low-cost, rapid, point-of-care measurement method for CEA cancer biomarker.

  15. Comparison of Real Time Polymerase Chain Reaction with Microscopy and Antigen Detection Assay for the Diagnosis of Malaria

    International Nuclear Information System (INIS)

    Khan, S. A.; Ahmed, S.; Khan, F. A.; Shamshad, G. U.; Joyia, Z.; Mushahid, N.; Saeed, S.

    2013-01-01

    Objective: To determine the sensitivity of a real time polymerase chain reaction (PCR) for malaria diagnosis and to compare its accuracy with microscopy and an antigen based rapid diagnostic test (OptiMal). Study Design: Cross-sectional analytical study. Place and Duration of Study: Military Hospital, Armed Forces Institute of Transfusion and Armed Forces Institute of Pathology, Rawalpindi, from July to December 2011. Methodology: Venous blood samples of 300 clinically suspected patients of malaria were tested for malaria parasite by microscopy and OptiMal; and malaria parasite index was calculated for the positive samples. Plasmodium genus specific real time PCR was performed on all specimens, targeting small subunit rRNA gene. Diagnostic accuracy of three tests was compared and cost analysis was done. Results: Out of 300 patients, malaria parasite was detected in 110, 106 and 123 patients by microscopy, OptiMAL and PCR respectively. Real time PCR was 100% sensitive while microscopy and OptiMal had sensitivity of 89.4% and 86.2% respectively. All methods were 100% specific. The cost per test was calculated to be 0.2, 2.75 and 3.30 US dollar by microscopy, OptiMal and PCR respectively, excluding the once capital cost on PCR equipment. Conclusion: Genus specific real time PCR for the diagnosis of malaria was successfully established as a highly sensitive and affordable technology that should be incorporated in the diagnostic algorithm in this country. (author)

  16. [Use of Rapid Antigen Detection Test (RADT) among general practitioner teachers at the Paris Descartes University: 2005-2007].

    Science.gov (United States)

    Cornaglia, C; Robinet, J; Partouche, H

    2009-06-01

    The distribution of the Rapid Antigen Detection Test (RADT) and the National Health Insurance's information campaign should efficiently reduce the unjustified use antibiotic. However, a preliminary survey among GP trainers at the Paris Descartes University indicated that the RADT was seldom used. This study had for aim to describe the RADT use trend among trainers since 2005 and the main obstacles to its widespread use, and to assess the Mac Isaac score use and antibiotic prescriptions. Between February and May 2007, a survey was carried out among 66 GPs who were required to report their first ten patients over three years of age presenting with pharyngitis. RADT use and antibiotic prescriptions were compared with those of the 2005 survey. RADT use had decreased (52.5% [48.2-56.8] versus 57.5% [52.1-68.8], p<0.05). GPs did not use the RADT because they considered it "useless in decision making". Clinical findings were sufficient in most cases. The Mac Isaac score was not widely used by GPs (28.3%) and antibiotic prescription had increased except for macrolides which had decreased (10% vs 15%). Among patients with a negative RADT, 11.9% (vs 10.5% en 2005, p<0.001) were prescribed antibiotics. The RADT use decreased in two years among GP trainers. GPs still prescribe treatment according to clinical findings, most without using diagnostic tools.

  17. Chemical polyglycosylation and nanolitre detection enables single-molecule recapitulation of bacterial sugar export

    Science.gov (United States)

    Kong, Lingbing; Almond, Andrew; Bayley, Hagan; Davis, Benjamin G.

    2016-05-01

    The outermost protective layer of both Gram-positive and Gram-negative bacteria is composed of bacterial capsular polysaccharides. Insights into the interactions between the capsular polysaccharide and its transporter and the mechanism of sugar export would not only increase our understanding of this key process, but would also help in the design of novel therapeutics to block capsular polysaccharide export. Here, we report a nanolitre detection system that makes use of the bilayer interface between two droplets, and we use this system to study single-molecule recapitulation of sugar export. A synthetic strategy of polyglycosylation based on tetrasaccharide monomers enables ready synthetic access to extended fragments of K30 oligosaccharides and polysaccharides. Examination of the interactions between the Escherichia coli sugar transporter Wza and very small amounts of fragments of the K30 capsular polysaccharide substrate reveal the translocation of smaller but not larger fragments. We also observe capture events that occur only on the intracellular side of Wza, which would complement coordinated feeding by adjunct biosynthetic machinery.

  18. Development of Simple Bacterial Biosensor for Phenol Detection in Water at Medium Concentration using Glass Microelectrode

    Directory of Open Access Journals (Sweden)

    Setyawan Purnomo Sakti

    2016-01-01

    Full Text Available Water is one of the most fundamental natural resources in earth. The availability of clean water becomes a global interest. Many human activities result in water pollution. One from many pollution substances in water is phenol. Phenol is a very common residual compound in industrial activity. Extensive use of phenol in industry degrades water quality. Regulation has been set in many countries to prevent further damage to the water resource caused by phenol and limiting phenol concentration in water before released into the environment. Therefor it is importance to develop a sensor which can detect phenol concentration in water to be used as a wastewater quality control system. This paper presents a development of bacterial biosensor using Pseudomonas putida and Pseudomonas fluorescens as a biological sensitive material. The sensor was made from glass micro electrode using Ag/AgCl electrode as reference electrode, silver electrode and cellulose ester. The Pseudomonas putida was entrapped inside the nutrient solution and separated by cellulose ester membrane from water containing phenol. It was found that the Pseudomonas putida in used must be growth in 10 hours to reach its optimum growth condition. Linear relationship between biosensor output voltages to phenol concentration was measured for phenol concentration below 200 ppm. The sensitivity of the developed biosensor was 72mV/ppm for Pseudomonas putida and 68.8 mV/ppm for Pseudomonas fluorescens.

  19. Horseradish peroxidase-labeled silver/reduced graphene oxide thin film-modified screen-printed electrode for detection of carcinoembryonic antigen.

    Science.gov (United States)

    Lee, S X; Lim, H N; Ibrahim, I; Jamil, A; Pandikumar, A; Huang, N M

    2017-03-15

    In this study, a disposable and simple electrochemical immunosensor was fabricated for the detection of carcinoembryonic antigen. In this method, silver nanoparticles (AgNPs) were mixed with reduced graphene oxide (rGO) to modify the surface of screen-printed carbon electrode (SPE). Initially, AgNPs-rGO modified-SPEs were fabricated by using simple electrochemical deposition method. Then the carcinoembryonic antigen (CEA) was immobilized between the primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody onto AgNPs-rGO modified-SPEs to fabricate a sandwich-type electrochemical immunosensor. The proposed method could detect the CEA with a linear range of 0.05-0.50µgmL -1 and a detection limit down to 0.035µgmL -1 as compared to its non-sandwich counterpart, which yielded a linear range of 0.05-0.40µgmL -1 , with a detection limit of 0.042µgmL -1 . The immunosensor showed good performance in the detection of carcinoembryonic antigen, exhibiting a simple, rapid and low-cost. The immunosensor showed a higher sensitivity than an enzymeless sensor. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

    Science.gov (United States)

    Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

    2017-06-01

    For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Comparison of survival outcomes after recurrence detected by cancer antigen 125 elevation versus imaging study in epithelial ovarian cancer.

    Science.gov (United States)

    Paik, E Sun; Kim, Tae Joong; Lee, Yoo Young; Choi, Chel Hun; Lee, Jeong Won; Kim, Byoung Gie; Bae, Duk Soo

    2016-09-01

    The aim of this study was to compare survival outcomes in two groups of patients with recurrent epithelial ovarian cancer (EOC) with initial recurrence detection by cancer antigen 125 (CA-125) elevation or imaging, and underwent secondary cytoreductive surgery (SCS). A retrospective review of the medical records was performed on 99 recurrent EOC patients who underwent SCS at the Samsung Medical Center between January 2002 and December 2013. For follow-up after primary treatment, patients were routinely assessed by CA-125 levels every 3 months and computed tomography (CT) scan (or magnetic resonance imaging [MRI]) every 6 months for first 3 years, and by CA-125 every 6 months and CT scan (or MRI) every 12 months thereafter. The first recurrence was initially identified by either CA-125 elevation (n=41, 41.4%) or by imaging study (n=58, 58.6%). None of the patients showed the symptoms as initial sign of recurrence. There were higher percentages of extra-pelvic recurrence (87.8%) and multiple recurrences (78.0%) in the group diagnosed by CA-125 elevation. The proportion of no residual disease after SCS was comparably lower in the CA-125 group (22.0% vs. 72.4%). There were 19 cancer-associated deaths (19.2%) within a median follow-up period of 67 months. The group diagnosed by imaging had better overall survival from initial diagnosis (OS1), overall survival after SCS (OS2), progression-free survival after the initial treatment (PFS1) and progression-free survival after SCS compared to those of the CA-125 group (PFS2). EOC patients with recurrence initially detected by imaging study showed better survival outcomes than patients diagnosed by CA-125 elevation.

  2. Long-term in vitro reactivity for human leukocyte antigen antibodies and comparison of detection using serum versus plasma.

    Science.gov (United States)

    Norris, Philip J; Lee, Jar-How; Carrick, Danielle M; Gottschall, Jerome L; Lebedeva, Mila; de Castro, B R; Kleinman, Steven H; Busch, Michael P

    2009-02-01

    Human leukocyte antigen (HLA) antibodies are a possible cause of transfusion-related acute lung injury (TRALI), and fluorescent bead assays are often used for antibody detection. Serum is the manufacturer's recommended sample, but plasma may be easier to obtain for studies of HLA antibody prevalence and TRALI case investigations. Specimens were obtained from 44 multiparous females positive for the presence of HLA antibodies by lymphocytotoxicity testing at least 13 years prior and from 1000 contemporary blood donors. Screening tests were performed using a multiplex bead-based assay. In addition to comparing results obtained with paired plasma and serum samples, the effects of storage at 4 degrees C for 1 week and of multiple freeze-thaw cycles were evaluated. Of 42 evaluable subjects with HLA antibodies documented more than 13 years earlier, only 1 showed loss of detectable antibodies, with 39 (93%) positive in the screening assay for HLA Class I and 24 (57%) positive in the screening assay for HLA Class II antibodies. In 968 evaluable contemporary donors, 291 screened positive for the presence of HLA Class I and 206 for HLA Class II antibodies using a low assay cutoff. Screening test concordance using paired plasma and serum samples was high, particularly for subjects with higher-level antibodies. Refrigeration of samples for 1 week did not significantly affect assay results, while repeated freeze-thaw cycles caused a decrement in signal level. Serum and plasma samples gave concordant results in the majority of cases, particularly for specimens with higher-level antibodies. High-level HLA antibodies were present in most individuals for more than 13 years.

  3. Development of an FPW Biosensor with Low Insertion Loss and High Fabrication Yield for Detection of Carcinoembryonic Antigen.

    Science.gov (United States)

    Lan, Je-Wei; Huang, I-Yu; Lin, Yu-Cheng; Lin, Chang-Yu; Chen, Jian-Lin; Hsieh, Chia-Hsu

    2016-11-08

    In the last two decades, various flexural plate-wave (FPW)-based biosensors with low phase velocity, low operation frequency, high sensitivity, and short response time, have been developed. However, conventional FPW transducers have low fabrication yield because controlling the thickness of silicon/isolation/metal/piezoelectric multilayer floating thin-plate is difficult. Additionally, conventional FPW devices usually have high insertion loss because of wave energy dissipation to the silicon substrate or outside area of the output interdigital transducers (IDTs). These two disadvantages hinder the application of FPW devices. To reduce the high insertion loss of FPW devices, we designed two focus-type IDTs (fan-shaped and circular, respectively) that can effectively confine the launched wave energy, and adopted a focus-type silicon-grooved reflective grating structure (RGS) that can reduce the wave propagation loss. To accurately control the thickness of the silicon thin-plate and substantially improve the fabrication yield of FPW transducers, a 60 °C/27 °C two-step anisotropic wet etching process was developed. Compared with conventional FPW devices (with parallel-type IDTs and without RGS), the proposed FPW devices have lower insertion loss (36.04 dB) and higher fabrication yield (63.88%). Furthermore, by using cystamine-based self-assembled monolayer (SAM) nanotechnology, we used the improved FPW device to develop a novel FPW-based carcinoembryonic antigen (CEA) biosensor for detection of colorectal cancer, and this FPW-CEA biosensor has a low detection limit (5 ng/mL), short response time (<10 min), high sensitivity (60.16-70.06 cm²/g), and high sensing linearity (R-square = 0.859-0.980).

  4. A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen.

    Science.gov (United States)

    van Dam, Govert J; de Dood, Claudia J; Lewis, Melanie; Deelder, André M; van Lieshout, Lisette; Tanke, Hans J; van Rooyen, Louis H; Corstjens, Paul L A M

    2013-10-01

    An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA-ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP-LF test indicates a better performance than the ELISA. The UCP-LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings. The dry format UCP-LF assay was shown to provide a robust and easy to use format for rapid testing of CAA antigen in serum. It performed at least as good as the ELISA with respect to sensitivity and specificity, and was found to be superior with respect to speed and simplicity of use. Worldwide shipping at ambient temperature of the assay reagents, and the availability of small scanners to analyze the CAA UCP-LF strip, are two major steps towards point-of-care (POC) applications in remote and resource poor environments to accurately identify low (30 pg CAA/mL serum; equivalent to about 10 worm pairs) to heavy Schistosoma

  5. Nanolitre real-time PCR detection of bacterial, parasitic, and viral agents from patients with diarrhoea in Nunavut, Canada

    Directory of Open Access Journals (Sweden)

    David M. Goldfarb

    2013-04-01

    Full Text Available Background. Little is known about the microbiology of diarrhoeal disease in Canada's Arctic regions. There are a number of limitations of conventional microbiology testing techniques for diarrhoeal pathogens, and these may be further compromised in the Arctic, given the often long distances for specimen transport. Objective. To develop a novel multiple-target nanolitre real-time reverse transcriptase (RT-PCR platform to simultaneously test diarrhoeal specimens collected from residents of the Qikiqtani (Baffin Island Region of Nunavut, Canada, for a wide range of bacterial, parasitic and viral agents. Study design/methods. Diarrhoeal stool samples submitted for bacterial culture to Qikiqtani General Hospital in Nunavut over an 18-month period were tested with a multiple-target nanolitre real-time PCR panel for major diarrhoeal pathogens including 8 bacterial, 6 viral and 2 parasitic targets. Results. Among 86 stool specimens tested by PCR, a total of 50 pathogens were detected with 1 or more pathogens found in 40 (46.5% stool specimens. The organisms detected comprised 17 Cryptosporidium spp., 5 Clostridium difficile with toxin B, 6 Campylobacter spp., 6 Salmonella spp., 4 astroviruses, 3 noroviruses, 1 rotavirus, 1 Shigella spp. and 1 Giardia spp. The frequency of detection by PCR and bacterial culture was similar for Salmonella spp., but discrepant for Campylobacter spp., as Campylobacter was detected by culture from only 1/86 specimens. Similarly, Cryptosporidium spp. was detected in multiple samples by PCR but was not detected by microscopy or enzyme immunoassay. Conclusions. Cryptosporidium spp., Campylobacter spp. and Clostridium difficile may be relatively common but possibly under-recognised pathogens in this region. Further study is needed to determine the regional epidemiology and clinical significance of these organisms. This method appears to be a useful tool for gastrointestinal pathogen research and may also be helpful for clinical

  6. 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

  7. Differential in vivo expression of mycobacterial antigens in Mycobacterium tuberculosis infected lungs and lymph node tissues.

    Science.gov (United States)

    Mustafa, Tehmina; Leversen, Nils Anders; Sviland, Lisbet; Wiker, Harald Gotten

    2014-10-03

    The clinical course of tuberculosis (TB) infection, bacterial load and the morphology of lesions vary between pulmonary and extrapulmonary TB. Antigens expressed in abundance during infection could represent relevant antigens in the development of diagnostic tools, but little is known about the in vivo expression of various M. tuberculosis antigens in different clinical manifestations. The aim of this study was to study the differences in the presence of major secreted as well as somatic mycobacterial antigens in host tissues during advanced rapidly progressing and fatal pulmonary disease with mainly pneumonic infiltrates and high bacterial load, and to compare this to the presence of the same antigens in TB lymphadenitis cases, which is mainly chronic and self-limiting disease with organised granulomas and lower bacterial load. Human pulmonary (n = 3) and lymph node (n = 17) TB biopsies, and non-TB controls (n = 12) were studied. Ziehl-Neelsen stain, nested PCR 1S6110 and immunohistochemistry were performed. Major secreted (MPT32, MPT44, MPT46, MPT51, MPT53, MPT59, MPT63, and MPT64) and somatic mycobacterial antigens (Mce1A, Hsp65, and MPT57) were detected by using rabbit polyclonal antibodies. Plenty of bacilli were detectable with Ziehl-Neelsen stain in the lung biopsies while no bacilli were detected in the lymph node biopsies. All the cases were shown to be positive by PCR. Both secretory and somatic antigens were expressed in abundance in pulmonary infiltrates, while primarily somatic antigens were detected in the lymphadenitis cases. Of the secreted antigens, only MPT64 was consistently detected in both cases, indicating a preferential accumulation of this antigen within the inflammatory cells, even if the cells of the granuloma can efficiently restrict bacterial growth and clear away the secreted antigens. This study shows that major secreted mycobacterial antigens were found in high amounts in advanced pulmonary lesions without proper granuloma

  8. Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection.

    Science.gov (United States)

    Cram, Erik D; Simmons, Ryan S; Palmer, Amy L; Hildebrand, William H; Rockey, Daniel D; Dolan, Brian P

    2016-02-01

    The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8(+) cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8(+) T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8(+) killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Immunodiagnosis of paramphistomosis using monoclonal antibody-based sandwich ELISA for detection of Paramphistomum gracile circulating 16 kDa antigen.

    Science.gov (United States)

    Anuracpreeda, Panat; Tepsupornkul, Kullanid; Chawengkirttikul, Runglawan

    2017-06-01

    In this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected with P. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL-1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected with P. gracile, Fasciola gigantica, Moniezia benedeni, Trichuris sp., Strongyloides sp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

  10. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    reduce or delay bacterial biofilm formation of a range of urinary tract infectious E.coli and Klebsiella isolates. Several other proteinaceous coatings were also found to display anti-adhesive properties, possibly providing a measure for controlling the colonization of implant materials. Several other...... components. These substances may both mediate and stabilize the bacterial biofilm. Finally, several adhesive structures were examined, and a novel physiological biofilm phenotype in E.coli biofilms was characterized, namely cell chain formation. The autotransporter protein, antigen 43, was implicated...

  11. Performance evaluation of point-of-care test for detection of Cryptosporidium stool antigen in children and HIV infected adults.

    Science.gov (United States)

    Shimelis, Techalew; Tadesse, Endale

    2014-05-16

    Gastro-enteritis is associated with significant morbidity and mortality in patients with HIV/AIDS and children, and Cryptosporidium is the most important parasite implicated. To date, several commercial companies have developed simple and rapid point-of-care tests for the detection of Cryptosporidium infection; however, information is scarce regarding their diagnostic significance in Ethiopia. This study aimed at evaluating the performance of a rapid diagnostic test (RDT) for the detection of Cryptosporidium stool antigen. A hospital-based cross-sectional study was conducted in Hawassa University Hospital, southern Ethiopia from May to November 2013. Faecal samples were collected from a total of 100 children and 250 HIV infected individuals with diarrhea or CD4 T-cell count lower than 200 cells/μl. Specimens were processed using direct, formol-ether concentration and modified Ziehl-Neelsen techniques for diagnosis of Cryptosporidium and other parasites. One hundred faecal samples (50 positives for Cryptosporidium, 35 positives for other parasites and 15 negatives for any intestinal parasites) were tested using the CoproStrip™Cryptosporidium kit (Savyon Diagnostics Ltd, Israel). Test parameters were calculated using microscopy of the modified Ziehl-Neelsen stained stool smear as reference method. The performance of the RDT was first compared to routine microscopic analysis (examination ≤10 min). The CoproStrip™Cryptosporidium RDT correctly detected 31 of 42 positive samples and 49 of 50 negative samples (i.e., 11 false negatives and 1 false positive). Sensitivity, specificity, PPV, NPV and accuracy were calculated to be 74, 98, 97, 84 and 88%, respectively. Upon thorough microscopic analysis (examination >10 min), 8 more samples with very low oocyst density were found. However, these were missed by the kit and lower the sensitivity and NPV to 62 and 72%, respectively. No cross-reactivity was observed with any of the helminthic or other protozoan parasites

  12. Simultaneous Detection of Three Bacterial Seed-Borne Diseases in Rice Using Multiplex Polymerase Chain Reaction

    OpenAIRE

    Kang, In Jeong; Kang, Mi-Hyung; Noh, Tae-Hwan; Shim, Hyeong Kwon; Shin, Dong Bum; Heu, Suggi

    2016-01-01

    Burkholderia glumae (bacterial grain rot), Xanthomonas oryzae pv. oryzae (bacterial leaf blight), and Acidovorax avenae subsp. avenae (bacterial brown stripe) are major seedborne pathogens of rice. Based on the 16S and 23S rDNA sequences for A. avenae subsp. avenae and B. glumae, and transposase A gene sequence for X. oryzae pv. oryzae, three sets of primers had been designed to produce 402 bp for B. glumae, 490 bp for X. oryzae, and 290 bp for A. avenae subsp. avenae with the 63°C as an opti...

  13. Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy

    DEFF Research Database (Denmark)

    Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H.

    2017-01-01

    cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8...... day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues...

  14. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Kingsley, Mark T.

    2001-03-13

    The threat to American interests from terrorists is not limited to attacks against humans. Terrorists might seek to inflict damage to the U.S. economy by attacking our agricultural sector. Infection of commodity crops by bacterial or fungal crop pathogens could adversely impact U.S. agriculture, either directly from damage to crops or indirectly from damage to our ability to export crops suspected of contamination. Recognizing a terrorist attack against U.S. agriculture, to be able to prosecute the terrorists, is among the responsibilities of the members of Hazardous Material Response Unit (HMRU) of the Federal Bureau of Investigation (FBI). Nucleic acid analysis of plant pathogen strains by the use of polymerase chain reaction (PCR) amplification techniques is a powerful method for determining the exact identity of pathogens, as well as their possible region of origin. This type of analysis, however, requires that PCR assays be developed specific to each particular pathogen strain, and analysis protocols developed that are specific to the particular instrument used for detection. The objectives of the work described here were threefold: 1) to assess the potential terrorist threat to U.S. agricultural crops, 2) to determine whether suitable assays exist to monitor that threat, and 3) where assays are needed for priority plant pathogen threats, to modify or develop those assays for use by specialists at the HMRU. The assessment of potential threat to U.S. commodity crops and the availability of assays for those threats were described in detail in the Technical Requirements Document (9) and will be summarized in this report. This report addresses development of specific assays identified in the Technical Requirements Document, and offers recommendations for future development to ensure that HMRU specialists will be prepared with the PCR assays they need to protect against the threat of economic terrorism.

  15. The Effect of Antibiotic Exposure and Specimen Volume on the Detection of Bacterial Pathogens in Children With Pneumonia.

    Science.gov (United States)

    Driscoll, Amanda J; Deloria Knoll, Maria; Hammitt, Laura L; Baggett, Henry C; Brooks, W Abdullah; Feikin, Daniel R; Kotloff, Karen L; Levine, Orin S; Madhi, Shabir A; O'Brien, Katherine L; Scott, J Anthony G; Thea, Donald M; Howie, Stephen R C; Adrian, Peter V; Ahmed, Dilruba; DeLuca, Andrea N; Ebruke, Bernard E; Gitahi, Caroline; Higdon, Melissa M; Kaewpan, Anek; Karani, Angela; Karron, Ruth A; Mazumder, Razib; McLellan, Jessica; Moore, David P; Mwananyanda, Lawrence; Park, Daniel E; Prosperi, Christine; Rhodes, Julia; Saifullah, Md; Seidenberg, Phil; Sow, Samba O; Tamboura, Boubou; Zeger, Scott L; Murdoch, David R

    2017-06-15

    Antibiotic exposure and specimen volume are known to affect pathogen detection by culture. Here we assess their effects on bacterial pathogen detection by both culture and polymerase chain reaction (PCR) in children. PERCH (Pneumonia Etiology Research for Child Health) is a case-control study of pneumonia in children aged 1-59 months investigating pathogens in blood, nasopharyngeal/oropharyngeal (NP/OP) swabs, and induced sputum by culture and PCR. Antibiotic exposure was ascertained by serum bioassay, and for cases, by a record of antibiotic treatment prior to specimen collection. Inoculated blood culture bottles were weighed to estimate volume. Antibiotic exposure ranged by specimen type from 43.5% to 81.7% in 4223 cases and was detected in 2.3% of 4863 controls. Antibiotics were associated with a 45% reduction in blood culture yield and approximately 20% reduction in yield from induced sputum culture. Reduction in yield of Streptococcus pneumoniae from NP culture was approximately 30% in cases and approximately 32% in controls. Several bacteria had significant but marginal reductions (by 5%-7%) in detection by PCR in NP/OP swabs from both cases and controls, with the exception of S. pneumoniae in exposed controls, which was detected 25% less frequently compared to nonexposed controls. Bacterial detection in induced sputum by PCR decreased 7% for exposed compared to nonexposed cases. For every additional 1 mL of blood culture specimen collected, microbial yield increased 0.51% (95% confidence interval, 0.47%-0.54%), from 2% when volume was ≤1 mL to approximately 6% for ≥3 mL. Antibiotic exposure and blood culture volume affect detection of bacterial pathogens in children with pneumonia and should be accounted for in studies of etiology and in clinical management. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

  16. An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.

    Directory of Open Access Journals (Sweden)

    Jin-Ju Zhang

    Full Text Available Real-time polymerase chain reaction (RT-PCR enables effective and sensitive screening for infectious risk in the field of blood safety. However, when using RT-PCR to detect bacterial contamination, several intractable points must be considered, one of which is the lack of appropriate quality control. In this study, we developed a simplified RT-PCR assay in which the same primer set and two distinct probes were used to detect both, an internal reference control and the target in a reaction. The copy number of the internal reference control represents the positive detection limit of the assay; therefore, when the threshold-cycle value of the target is less than or equal to that of the internal reference control, the result obtained for the target can be considered to be a true positive. When human gDNA was spiked with Escherichia coli gDNA and the detection limit for the internal reference control was set to five copies, the measured detection limit for E. coli gDNA was two copies. The internal reference control duplex RT-PCR assay showed high efficiency (0.91-1.02, high linearity (R2 > 0.99, and good reproducibility in intra- and inter-assay comparisons. Lastly, when human platelet-rich plasma samples were spiked with E. coli or other bacterial species, all species were detected efficiently, and the results of a two-sample pooled t test showed that the limit of detection for E. coli was 1 cfu/mL. Here, we present a synthetic internal reference control molecule and a new statistical method for improving the reliability of RT-PCR assays when screening for bacterial contamination in blood products.

  17. 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.

  18. Evaluation of a point-of-care immunoassay test kit 'StrongStep' for cryptococcal antigen detection.

    Directory of Open Access Journals (Sweden)

    Edward Mpoza

    Full Text Available HIV-associated cryptococcal meningitis is the leading cause of adult meningitis in Sub-Saharan Africa, accounting for 15%-20% of AIDS-attributable mortality. The development of point-of-care assays has greatly improved the screening and diagnosis of cryptococcal disease. We evaluated a point-of-care immunoassay, StrongStep (Liming Bio, Nanjing, Jiangsu, China lateral flow assay (LFA, for cryptococcal antigen (CrAg detection in cerebrospinal fluid (CSF and plasma.We retrospectively tested 143 CSF and 77 plasma samples collected from HIV-seropositive individuals with suspected meningitis from 2012-2016 in Uganda. We prospectively tested 90 plasma samples collected from HIV-seropositive individuals with CD4 cell count <100 cells/μL from 2016-2017 as part of a cryptococcal antigenemia screening program. The StrongStep CrAg was tested against a composite reference standard of positive Immy CrAg LFA (Immy, Norman, OK, USA or CSF culture with statistical comparison by McNemar's test.StrongStep CrAg had a 98% (54/55 sensitivity and 90% (101/112 specificity in plasma (P = 0.009, versus reference standard. In CSF, the StrongStep CrAg had 100% (101/101 sensitivity and 98% (41/42 specificity (P = 0.99. Adjusting for the cryptococcal antigenemia prevalence of 9% in Uganda and average cryptococcal meningitis prevalence of 37% in Sub-Saharan Africa, the positive predictive value of the StrongStep CrAg was 50% in plasma and 96% in CSF.We found the StrongStep CrAg LFA to be a sensitive assay, which unfortunately lacked specificity in plasma. In lower prevalence settings, a majority of positive results from blood would be expected to be false positives.

  19. Detection of GAD-Ab index in diabetic patients using 35S labeled recombinant human GAD65 antigen

    International Nuclear Information System (INIS)

    Huang Gan; Zhao Zhiguang; Peng Jian; Yan Xiang; Zhu Xuping; Yang Lin; Li Xia; Wang Jianping; Jiang Tiejian

    2003-01-01

    Objective: To establish a novel method for measuring glutamic acid decarboxylase autoanti-bodies(GAD-Ab). Methods: Recombinant human GAD 65 was used as the antigen, in vitro transcribed and translated 35 S-GAD 65 as the tracer, a self-designed rotating incubation apparatus as the incubator, protein-A sepharose as the precipitator, and the liquid scintillation counter was used to measure radioactive count value to detect GAD-Ab. The positive cut-off point of GAD-Ab index was determined as > 0.05 by the 99.5% percentile in 109 healthy individuals. GAD-Ab levels were determined in 43 type 1 and 226 type 2 diabetic patients. Results: The optimized working conditions included SJ1515 35 S-methionine for in vitro transcription and translation, 20-30 r/min setup of rotating incubation apparatus, test temperature 4-25 degree C, freshly prepared buffer of pH 7.2-7.4, and horizontal rotor centrifuge. The new method was better than original one, with intra-assay CV of 4.9%-8.3% and inter-assay CV of 7.1%-10.8 %, specificity of 98.2%. The results were comparable with the figures issued by an international standardized laboratory (concordance was 98.3%, Kappa value 0.971). The positive rate of GAD-Ab was 58.1% (25 of 43) in type 1 and 10.2%(23 of 226) in type 2 diabetes patients, but only 1.8% (2 of 109) in healthy individuals. Conclusion: The new assay for GAD-Ab is a highly sensitive, accurate, specific and reproducible method for clinical use

  20. Hepatocellular carcinoma. A study of 50 autopsy cases with detection of hepatitis B surface antigen in fixed tissues.

    Science.gov (United States)

    Perez-Barrios, A; Colina-Ruizdelgado, F; Gallego, I; Martinez-Tello, F J

    1983-03-01

    Fifty patients who died of hepatocellular carcinoma (HCC) were autopsied at the Ciudad Sanitaria "1 degree de Octubre" and the Hospital de la Cruz Roja (Madrid) from 1974 to 1980. Formalin fixed paraffin-embedded autopsy tissue of liver and tumor from the 50 HCC and liver tissue from 50 liver cirrhosis (LC) and from 50 autopsy of non cirrhotic control cases were examined for the presence of cytoplasmic hepatitis B surface antigen (HBsAg). The study was carried out using orcein staining, immunoperoxidase technique (IP) and indirect immunofluorescence (IF). In livers with HCC the HBsAg was detected in the cytoplasm of the hepatocytes in 10 cases (20%) with the orcein staining and in 11 (22%) with the IP and IF techniques. In one case (2%) HBsAg was found in the cytoplasm of tumor cells with the three methods--In four cases (8%) of LC and 2 (4%) control cases cytoplasmic positive cells were found. In 41 patients with HCC HBsAg was studied in the serum by radio-immunoassay (RIA) (13 cases) and immunodiffussion (28 cases). 5 patients (12,1%) were positive and 36 (72%) were negative. In the 5 serum positive HBsAg HCC the staining methods for cytoplasmic HBsAg were positive (100%). In 36 serum negative HBsAg HCC the staining method were positive in 2 cases. The results let us to conclude that HBV is a probable important etiologic factor of HCC in our milieu. 54% of the patients with HCC had a previous history of alcohol abuse; however, histologic features compatible with an alcoholic etiology were found in only 5 cases. Nevertheless we consider that the described histopathologic findings do not exclude excess alcohol consumption as a possible etiologic factor for HCC in our series.

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

    International Nuclear Information System (INIS)

    Mbwambo, H.A.

    1997-01-01

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

  2. Detection of bacterial contaminants and hybrid sequences in the genome of the kelp Saccharina japonica using Taxoblast

    Directory of Open Access Journals (Sweden)

    Simon M. Dittami

    2017-11-01

    Full Text Available Modern genome sequencing strategies are highly sensitive to contamination making the detection of foreign DNA sequences an important part of analysis pipelines. Here we use Taxoblast, a simple pipeline with a graphical user interface, for the post-assembly detection of contaminating sequences in the published genome of the kelp Saccharina japonica. Analyses were based on multiple blastn searches with short sequence fragments. They revealed a number of probable bacterial contaminations as well as hybrid scaffolds that contain both bacterial and algal sequences. This or similar types of analysis, in combination with manual curation, may thus constitute a useful complement to standard bioinformatics analyses prior to submission of genomic data to public repositories. Our analysis pipeline is open-source and freely available at http://sdittami.altervista.org/taxoblast and via SourceForge (https://sourceforge.net/projects/taxoblast.

  3. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. DIAGNOSTIC ROLE OF FLUORINE-18 (18F) FLUORODEOXYGLUCOSE POSITRON EMISSION TOMOGRAPHY COMPUTED TOMOGRAPHY IN DETECTING RECURRENT DISEASE IN PATIENTS WITH COLORECTAL CANCER AND ELEVATED CARCINOEMBRYONIC ANTIGEN.

    Science.gov (United States)

    Matovina, Emil; Mihailović, Jasna; Nikoletić, Katarina; Srbovan, Dolores

    2015-01-01

    Early detection of recurrence is an important factor for long term survival of patients with colorectal cancer. Measurement of serum levels of carcinoembryonic antigen has been commonly used in the postoperative surveillance of colorectal cancer. The purpose of this study was to evaluate the ability of positron emission tomography-computed tomography to detect pathological substrate of elevated serum carcinoembryonic antigen in patients with colorectal cancer. The patients with colorectal cancer who underwent curative surgical resection and/ or chemotherapy, who were found in our database, were analyzed retrospectively. Forty-eight 18F-fluorodeoxyglucose positron emission tomography-computed tomography studies including 45 patients (14 women, 31 men; mean age: 62.93 years) with elevated serum, carcinoembryonic antigen levels, which had been performed between January 2011 and January 2014, were evaluated. Serum levels of carcinoembryonic antigen were measured within 3 months after positron emission tomography-computed tomography examination. Final diagnosis of recurrence was made by histopathological findings, radiology studies or clinical follow-up. Recurrences were diagnosed in 37 patients, the prevalence being 77.1%. Liver metastases were found in 18 patients, abdominal, pelvic and/or mediastinal lymph nodes were positive in 19 patients, 11 patients had loco regional recurrences and 4 patients had pulmonary metastasis, and bone metastases were found in one patient. One patient was diagnosed with metastasis in scar tissue. The overall sensitivity and specificity of positron emission tomography-computed tomography was 90.24% and 71.42%, respectively. The positive and negative predictive values were 94.87% and 55.56%, respectively. 18F-fluorodeoxyglucose positron emission tomography-computed tomography is a powerful tool that could be used in determining colorectal cancer recurrence in patients with elevated carcinoembryonic antigen levels and could have an

  5. Detection of irradiated chicken and fish meats by the determination of Gram negative bacterial count and bacterial endotoxins

    International Nuclear Information System (INIS)

    Badr, H.M.

    2010-01-01

    The aim of this investigation was to study the possibility of detecting irradiated chicken and fish meats by the determination of Gram negative bacteria combined with the determination of endotoxin concentrations. Samples of chicken breast with skin, skinless chicken breast and eviscerated Bolti fish (Tilabia nilotica) were irradiated at room temperature at doses of 0, 1.5 and 3 kGy followed by storage at refrigeration temperature (4 ± 1 degree C) for 12 days or frozen storage at -18 degree C for 60 days. Furthermore, other samples of chicken and Bolti fish were irradiated in the frozen sate at doses of 0, 3, and 7 kGy followed by frozen storage at - 18 degree C for 60 days. Then the enumeration of Gram negative bacteria in conjunction with the determination of endotoxin concentrations were carried out for both irradiated and non-irradiated samples post treatments and during storage in addition to the discovery of Pseudomonas spp. The obtained results showed that chicken and fish samples irradiated at dose of 1.5 kGy could be identified during refrigerated storage for 6 and 9 days, respectively, while all samples irradiated at dose of 3 kGy were identifiable during 12 days of refrigerated storage. Moreover, all irradiated and frozen stored samples were identifiable during their frozen storage (- 18 degree C). The absence of Pseudomonads in all irradiated samples may aid in the differentiation of irradiated and non-irradiated samples especially during refrigerated storage. This method can be applied as a general screening method to predict the possible treatment of chicken and fish meats by ionizing radiation

  6. Detection of a pathogen shift among the pectolytic bacterial pathogens of potato in Washington State

    Science.gov (United States)

    Bacterial tuber soft rot, aerial stem rot and blackleg are significant diseases of potatoes in Washington State. These diseases are caused by Pectobacterium carotovorum subsp. carotovorum, Pectobacterium atrosepticum, and Dickeya chrysanthemi, all characterized by the ability to produce pectolytic ...

  7. Clinical usefulness and accuracy of polymerase chain reaction in the detection of bacterial meningitis agents in pediatric cerebrospinal fluid.

    Science.gov (United States)

    Nour, M; Alaidarous, A

    2018-02-16

    Bacterial meningitis poses enormous healthcare challenges due to a high mortality, morbidity and sequelae. Neisseria (N.) meningitidis, Haemophilus (H.) influenzae, Streptococcus (S.) pneumoniae and S. agalactiae remain among the most prevalent infectious agents that cause bacterial meningitis in children. The objective of this study was the simultaneous detection of these pathogens in suspected cerebrospinal fluid (CSF) by using multiplex polymerase chain reaction (mPCR) and compare PCR results with standard diagnostics currently used in clinical practice. CSF specimens were obtained from 515 children (<5 years) clinically suspected of having acute bacterial meningitis. Based on bacterial culture, four isolates of salmonella sp and one Citrobacter freundii isolate were identified. The remaining 510 CSF specimens, having negative culture, were subjected to mPCR. Twenty-three (4.51%) CSF samples yielded a PCR positive signal. The pathogens identified were: S. pneumoniae (n=13), H. influenzae (n=7) and N. meningitidis (n=3). S. agalactiae was not detected. Using sequential multiplex PCR, serogrouping of S. pneumoniae revealed 3 different serotypes: serotype 19A (n=6), 19F (n=4) and serotype 23F (n=3). Only the serotype A was identified for the 3N. meningitidis isolates. Despite vaccination, S. pneumoniae remains a leading cause of pediatric invasive disease. Detecting causative organism remains the most critical aspect for management of children with suspected meningitis. PCR method is more sensitive and rapid than culture for detecting the infectious agents. Institution of PCR diagnostics is recommended for early and appropriate therapy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  8. Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies.

    Science.gov (United States)

    Amakishi, Etsuko; Irie, Yoriko; Nishizawa, Kanae; Kamada, Hiromi; Nakajima, Fumiaki; Matsuyama, Nobuki; Ishii, Hiroyuki; Matsukura, Harumichi; Yasui, Kazuta; Hirayama, Fumiya

    2015-04-01

    Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

  9. Development of a serogroup-specific multiplex PCR assay to detect a set of Escherichia coli serogroups based on the identification of their O-antigen gene clusters.

    Science.gov (United States)

    Wang, Quan; Ruan, Xiaojuan; Wei, Dongmei; Hu, Zhidong; Wu, Lixia; Yu, Ting; Feng, Lu; Wang, Lei

    2010-10-01

    The Escherichia coli serogroups O115, O126, O137, O158, O165, and O173 are pathogenic strains associated with diarrhea. Molecular approaches such as PCR have been proven to be rapid, inexpensive, and accurate. The sequences of the O-antigen-processing genes wzx and wzy are specific for different O antigens and are generally used as the target genes for the detection and identification of E. coli strains belonging to different O serogroups. In this report, the O-antigen gene clusters of these 6 O serogroups were sequenced, and genes were identified on the basis of homology. By screening these sequences against all 186 E. coli and Shigella strains, we found that the sequences of the wzx and wzy genes were serogroup-specific, and 2 specific primer pairs for each serogroup were screened out. A multiplex PCR assay targeting all 6 serogroups was developed. Twenty-nine strains were used to validate the specificity of the assay. The detection sensitivity was 1ng genomic DNA. As the assay was shown to be accurate and sensitive, it can be used for the identification and detection of strains belonging to these serogroups in stool and other environmental samples after being isolated by culture. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  10. Adsorption according to the Langmuir-Freundlich model is the detection mechanism of the antigen p53 for early diagnosis of cancer.

    Science.gov (United States)

    Soares, Juliana Coatrini; Soares, Andrey Coatrini; Pereira, Paulo Augusto Raymundo; Rodrigues, Valquiria da Cruz; Shimizu, Flavio Makoto; Melendez, Matias Eliseo; Scapulatempo Neto, Cristovam; Carvalho, André Lopes; Leite, Fábio L; Machado, Sergio A S; Oliveira, Osvaldo N

    2016-03-28

    Biosensors for early detection of cancer biomarkers normally depend on specific interactions between such biomarkers and immobilized biomolecules in the sensing units. Though these interactions are expected to yield specific, irreversible adsorption, the underlying mechanism appears not to have been studied in detail. In this paper, we show that adsorption explained with the Langmuir-Freundlich model is responsible for detection of the antigen p53 associated with various types of cancers. Irreversible adsorption was proven between anti-p53 antibodies immobilized on the biosensors and the antigen p53, with the adequacy of the Langmuir-Freundlich model being confirmed with three independent experimental methods, viz. polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), nanogravimetry using a quartz crystal microbalance and electrochemical impedance spectroscopy. The method based on this irreversible adsorption was sufficiently sensitive (limit of detection of 1.4 pg mL(-1)) for early diagnosis of Hodgkin lymphoma, pancreatic and colon carcinomas, and bladder, ovarian and lung cancers, and could distinguish between MCF7 cells containing the antigen p53 from Saos-2 cells that do not contain it.

  11. Detecting the dormant: a review of recent advances in molecular techniques for assessing the viability of bacterial endospores.

    Science.gov (United States)

    Mohapatra, Bidyut R; La Duc, Myron T

    2013-09-01

    Due to their contribution to gastrointestinal and pulmonary disease, their ability to produce various deadly exotoxins, and their resistance to extreme temperature, pressure, radiation, and common chemical disinfecting agents, bacterial endospores of the Firmicutes phylum are a major concern for public and environmental health. In addition, the hardy and dormant nature of endospores renders them a particularly significant threat to the integrity of robotic extraterrestrial life-detection investigations. To prevent the contamination of critical surfaces with seemingly ubiquitous bacterial endospores, clean rooms maintained at exceedingly stringent cleanliness levels (i.e., fewer than 100,000 airborne particles per ft(3)) are used for surgical procedures, pharmaceutical processing and packaging, and fabrication and assembly of medical devices and spacecraft components. However, numerous spore-forming bacterial species have been reported to withstand typical clean room bioreduction strategies (e.g., UV lights, maintained humidity, paucity of available nutrients), which highlights the need for rapid and reliable molecular methods for detecting, enumerating, and monitoring the incidence of viable endospores. Robust means of evaluating and tracking spore burden not only provide much needed information pertaining to endospore ecophysiology in different environmental niches but also empower decontamination and bioreduction strategies aimed at sustaining the reliability and integrity of clean room environments. An overview of recent molecular advances in detecting and enumerating viable endospores, as well as the expanding phylogenetic diversity of pathogenic and clean room-associated spore-forming bacteria, ensues.

  12. The bladder tumor antigen (BTA) test compared to voided urine cytology in the detection of bladder neoplasms.

    Science.gov (United States)

    Murphy, W M; Rivera-Ramirez, I; Medina, C A; Wright, N J; Wajsman, Z

    1997-12-01

    Tests to detect recurrent bladder neoplasms are limited and none is consistently accurate. Recent studies suggest that the bladder tumor antigen (BTA) test, an agglutination reaction for basement membrane complexes, is superior to voided urine cytology in clinical practice. We compared BTA and voided urine cytology to bladder washings and cystoscopy, emphasizing diagnostic yield among patients with causes of basement membrane complexes other than bladder cancer. Random voided urine specimens from 67 patients with a history of bladder neoplasms were collected before cystoscopy and bladder washing. Urine also was obtained from 34 patients with inflammatory bladder conditions including 5 with a history of prostate cancer. Each urine was tested for BTA according to a commercial kit. Positive results were indicated by yellow on a test pad. Blinded to all other results, each urine and each bladder washing were examined microscopically, and a positive test had malignant/suspicious cells. Bladder biopsies were performed when endoscopic lesions were seen. Specimens were grouped into 4 categories: group 1--biopsy proved bladder neoplasm, group 2--history of bladder cancer but not biopsy proved, group 3--history of prostate cancer and group 4--no history of urological cancer. Voided urine cytology was positive in 54% of specimens from patients with biopsy proved bladder neoplasms compared to 29% for BTA. Relative yield for voided urine cytology versus BTA was not changed if all group 2 cases having a positive bladder washing and positive cystoscopy were assumed to have bladder cancer, nor was relative yield altered by subsequent short-term followup. Of voided urine specimens 14% from group 1 patients and 41% from group 2 patients had scant cells. Overall diagnostic yield was superior for bladder washing. False-positive BTA occurred in 7 of 34 patients with no history of urological or prostate cancer. There were no false-positive voided urine cytology interpretations in these

  13. Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections.

    Science.gov (United States)

    Olesen, Cathrine Holm; Brahimi, Karima; Vandahl, Brian; Lousada-Dietrich, Susana; Jogdand, Prajakta S; Vestergaard, Lasse S; Dodoo, Daniel; Højrup, Peter; Christiansen, Michael; Larsen, Severin Olesen; Singh, Subhash; Theisen, Michael

    2010-10-26

    In endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies. Naturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa) or low molecular weight (immunity against malaria.

  14. Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.

    Science.gov (United States)

    Skaik, Younis; Battermann, Anja; Hiller, Oliver; Meyer, Oliver; Figueiredo, Constanca; Salama, Abdulgabar; Blasczyk, Rainer

    2013-05-31

    Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin.

    Science.gov (United States)

    Kong, Minsuk; Shin, Joong Ho; Heu, Sunggi; Park, Je-Kyun; Ryu, Sangryeol

    2017-10-15

    The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, Bacillus cereus (B. cereus). To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in Escherichia coli (E. coli) and incorporated in our assays. The sensitivity of the strip to detect B. cereus was 1×10 4 CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Novel, sensitive and low-cost diagnostic tests for 'river blindness'--detection of specific antigens in tears, urine and dermal fluid.

    Science.gov (United States)

    Ngu, J L; Nkenfou, C; Capuli, E; McMoli, T E; Perler, F; Mbwagbor, J; Tume, C; Nlatte, O B; Donfack, J; Asonganyi, T

    1998-05-01

    Sensitive, specific and low-cost diagnostic tests for onchocerciasis are indispensable for monitoring the efficacy of control programs, as well as for preventing blindness (when the tests are combined with efficacious chemotherapy. Three new tests to detect Onchocerca-specific antigens in tears, dermal fluid and urine employ antibodies to O. volvulus-specific recombinant proteins, Oncho-C27 and OvD3B, encoded by genes within the immunodominant Onchocerca OV 33-3 gene family, and expressed in yeast and in E. coli, respectively. In these assays, Onchocerca-specific antigens in test samples are bound onto a solid surface and revealed using appropriate enzyme-labelled antibodies. Proteins in the samples are first transferred to Hybond-N + membrane disks or nitrocellulose paper using either a transblot or a dotblot machine, and then reacted with specific O. volvulus antibodies. Bound antibodies are revealed with species-specific peroxidase-labelled antibodies and peroxidase substrate. Positive tests give a brown colour. In one of the two assays developed to detect Onchocerca antigens in tears, the sensitivity was enhanced by first adsorbing the specific antibodies onto the membrane surface in order to immobilize and concentrate the Onchocerca-specific antigen molecules on the membrane. The specificity of the recombinant proteins for Onchocerca volvulus had been verified by ELISA, classical Western blot and modified DSIA. The tests are a dipstick immunobinding assay for ocular microfilariae (DSIA), a transblot immunobinding assay for the detection of skin microfilariae (TADA) and a dot-blot immunobinding assay for detecting urinary microfilariae and their antigens (DIA). Their specificity and sensitivity were evaluated in the field on 110 subjects with proven ocular microfilariae, 130 subjects with clinical and parasitological evidence of onchocerciasis, 25 subjects infected with other helminths and 120 normal controls. The minimal detection limits of Oncho-C27 protein

  17. Development of Nested-PCR Assay to Detect Acidovorax citrulli, a Causal Agent of Bacterial Fruit Blotch at Cucurbitaceae

    Directory of Open Access Journals (Sweden)

    Young-Tak Kim

    2015-06-01

    Full Text Available The specific and sensitive nested-PCR method to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch on cucurbitaceae, was developed. PCR primers were designed from the draft genome sequence which was obtained with the Next Generation Sequencing of A. citrulli KACC10651, and the nested-PCR primer set (Ac-ORF 21F/Ac-ORF 21R were selected by checking of specificity to A. citrulli with PCR assays. The selected nested-PCR primer amplified the 140 bp DNA only from A. citrulli strains, and detection sensitivity of the nested PCR increased 10,000 times of 1st PCR detection limit (10 ng genomic DNA/PCR. The nested PCR detected A. citrulli from the all samples of seed surface wash (external seed detection of the artificially inoculated watermelon seeds with 101 cfu/ml and above population of A. citrulli while the nested PCR could not detected A. citrulli from the mashed seed suspension (internal seed detection of the all artificially inoculated watermelon seeds. When the naturally infested watermelon seeds (10% seed infested rate with grow-out test used, the nested PCR detected A. citrulli from 2 seed samples out of 10 replication samples externally and 5 seed samples out of 10 replication samples internally. We believe that the nested-PCR developed in this study will be useful method to detect A. citrulli from the Cucurbitaceae seeds.

  18. Microfluidic immunosensor with integrated liquid core waveguides for sensitive Mie scattering detection of avian influenza antigens in a real biological matrix.

    Science.gov (United States)

    Heinze, Brian C; Gamboa, Jessica R; Kim, Keesung; Song, Jae-Young; Yoon, Jeong-Yeol

    2010-11-01

    This work presents the use of integrated, liquid core, optical waveguides for measuring immunoagglutination-induced light scattering in a microfluidic device, towards rapid and sensitive detection of avian influenza (AI) viral antigens in a real biological matrix (chicken feces). Mie scattering simulations were performed and tested to optimize the scattering efficiency of the device through proper scatter angle waveguide geometry. The detection limit is demonstrated to be 1 pg mL(-1) in both clean buffer and real biological matrix. This low detection limit is made possible through on-chip diffusional mixing of AI target antigens and high acid content microparticle assay reagents, coupled with real-time monitoring of immunoagglutination-induced forward Mie scattering via high refractive index liquid core optical waveguides in close proximity (100 μm) to the sample chamber. The detection time for the assay is <2 min. This device could easily be modified to detect trace levels of any biological molecules that antibodies are available for, moving towards a robust platform for point-of-care disease diagnostics.

  19. Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

    Science.gov (United States)

    Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

    2015-01-01

    The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

  20. Predictive value of serum prostate specific antigen in detecting bone metastasis in prostate cancer patients using bone scintigraphy

    International Nuclear Information System (INIS)

    Kamaleshwaran, Koramadai Karuppusamy; Mittal, Bhagwant Rai; Harisankar, Chidambaram Natrajan Balasubramanian; Bhattacharya, Anish; Singh, Shrawan Kumar; Mandal, Arup K

    2012-01-01

    Radionuclide bone scan (BS) used to be the investigation of choice for detecting osseous metastases in prostate cancer. Now, with the availability serum prostate specific antigen (PSA) testing, clinicians do have a timely, cost-effective method to determine those patients who are highly unlikely to have osseous metastases. We determine the utility of PSA for predicting the presence of skeletal metastasis on BSs in prostate cancer patients. Retrospective analysis of medical records of 322 consecutive prostate cancers patients subjected to BS during the last 3 years was done. 52 cases were excluded due to following reasons: Serum PSA not available, hormonal or other therapy given prior to serum PSA measurement, and/or BS, and symptomatic for bone metastasis. In remaining 270 cases, PSA value and BS were evaluated. BS was performed with Tc99m methylene diphosphonate (MDP) as per the standard protocol. BS was found to be positive in 153/270 (56%) and negative in 117 (46%) patients. Of the 153 positive cases, 108 (70%) had serum PSA > 100 ng/ml, 42 (28%) had PSA of 20-100 ng/ml and only 3 (2%) had PSA < 20 ng/ml. All the patients with PSA > 100 ng/ml had multiple skeletal metastasis. Of the 117 negative cases, 110 (94%) had a PSA < 20 ng/ml, 5 had between 20 and 100 ng/ml and only 2 (1.8%) had PSA > 100 ng/ml. Of the 113 patients with serum PSA < 20 ng/ml, 110 (97.4%) did not show any bony metastasis. 150/157 (95.5%) patients with PSA > 20 ng/ml had bone metastasis. Using this criterion, 110 (40.7%) scans would have been omitted. Serum PSA < 20 ng/ml have high predictive value in ruling out skeletal metastasis. Our data are in corroboration with results from previous studies that BS should be performed only if PSA > 20 ng/ml. Using this cut-off, unnecessary investigation can be avoided. Avoiding BS in this group of patients would translate into a significant cost-saving and reduction in their psychological and physical burden

  1. Fluorescent detection of dipicolinic acid as a biomarker of bacterial spores using lanthanide-chelated gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Donmez, Mert [Department of Chemistry, Faculty of Art and Sciences, Duzce University, Duzce 81620 (Turkey); Yilmaz, M. Deniz, E-mail: deniz.yilmaz@gidatarim.edu.tr [Department of Bioengineering, Faculty of Engineering and Architecture, Konya Food and Agriculture University, Konya 42080 (Turkey); Kilbas, Benan, E-mail: benankilbas@duzce.edu.tr [Department of Chemistry, Faculty of Art and Sciences, Duzce University, Duzce 81620 (Turkey)

    2017-02-15

    Highlights: • The nanosensors based on gold nanoparticles functionalized with lanthanide complexes were synthesized. • The nanosensors selectively and sensitively detected DPA, a biomarker of bacterial spores. • Ratiometric sensing of DPA by a ternary complex was achieved by ligand displacement strategy. - Abstract: Gold nanoparticles (GNPs) functionalized with ethylenediamine-lanthanide complexes (Eu-GNPs and Tb-GNPs) were used for the selective fluorescent detection of dipicolinic acid (DPA), a unique biomarker of bacterial spores, in water. Particles were characterized by transmission electron microscopy and zeta potential measurements. The coordination of DPA to the lanthanides resulted in the enhancement of the fluorescence. A selective response to DPA was observed over the nonselective binding of aromatic ligands. The ligand displacement strategy were also employed for the ratiometric fluorescent detection of DPA. 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedion (TFNB) was chosen as an antenna to synthesize ternary complexes. The addition of DPA on EuGNP:TFNB ternary complex quenched the initial emission of the complex at 615 nm and increased the TFNB emission at 450 nm when excited at 350 nm. The results demonstrated that the ratiometric fluorescent detection of DPA was achieved by ligand displacement strategy.

  2. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    Science.gov (United States)

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

  3. Development of a panel of recombinase polymerase amplification assays for detection of common bacterial urinary tract infection pathogens.

    Science.gov (United States)

    Raja, B; Goux, H J; Marapadaga, A; Rajagopalan, S; Kourentzi, K; Willson, R C

    2017-08-01

    To develop and evaluate the performance of a panel of isothermal real-time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis. All five RPAs required reaction times of under 12 min to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross-react with high concentrations of nontarget bacterial genomic DNA. In a 50-sample retrospective clinical study, the five-RPA assay panel was found to have a specificity of 100% (95% CI, 78-100%) and a sensitivity of 89% (95% CI, 75-96%) for UTI detection. The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Rapid identification of the causative pathogens of UTIs can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture's 48-72-h turnaround time. The routine and widespread use of RPA to supplement or replace culture-based methods could profoundly impact UTI management and the emergence of multidrug-resistant pathogens. © 2017 The Society for Applied Microbiology.

  4. Detection and validation of QTL affecting bacterial cold water disease resistance in rainbow trout using restriction-site associated DNA sequencing

    Science.gov (United States)

    Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellites genome scan we have previously detected significant and suggestive QTL with major effects on the phenotypic variation of survival following challenge with Flavobacterium psychrophilum...

  5. Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens

    Directory of Open Access Journals (Sweden)

    Harvinder Talwar

    2015-04-01

    Full Text Available Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB. No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

  6. Detection of Immune-Complex Dissociated Nonstructural-1 (NS-1) Antigen in Patients with Acute Dengue Virus Infections

    NARCIS (Netherlands)

    P. Koraka (Penelope); C.P. Burghoorn-Maas; A. Falconar; T.E. Setiati (Tatty); K. Djamiatun; J. Groen (Jan); A.D.M.E. Osterhaus (Albert)

    2003-01-01

    textabstractAccurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot

  7. Sponge-specific unknown bacterial groups detected in marine sponges collected from Korea through barcoded pyrosequencing.

    Science.gov (United States)

    Jeong, Jong-Bin; Kim, Kyoung-Ho; Park, Jin-Sook

    2015-01-01

    The bacterial diversity of 10 marine sponges belonging to the species Cliona celata, an unidentified Cliona species, Haliclona cinerea, Halichondria okadai, Hymeniacidon sinapium, Lissodendoryx isodictyalis, Penares incrustans, Spirastrella abata, and Spirastrella panis collected from Jeju Island and Chuja Island was investigated using amplicon pyrosequencing of the 16S rRNA genes. The microbial diversity of these sponges has as of yet rarely or never been investigated. All sponges, except Cliona celata, Lissodendoryx isodictyalis, and Penares incrustans, showed simple bacterial diversity, in which one or two bacterial OTUs occupied more than 50% of the pyrosequencing reads and their OTU rank abundance curves saturated quickly. Most of the predominant OTUs belonged to Alpha-, Beta-, or Gammaproteobacteria. Some of the OTUs from the sponges with low diversity were distantly (88%~89%) or moderately (93%~97%) related to known sequences in the GenBank nucleotide database. Phylogenetic analysis showed that many of the representative sequences of the OTUs were related to the sequences originating from sponges and corals, and formed sponge-specific or -related clades. The marine sponges investigated herein harbored unexplored bacterial diversity, and further studies should be done to understand the microbes present in sponges.

  8. System automation for a bacterial colony detection and identification instrument via forward scattering

    International Nuclear Information System (INIS)

    Bae, Euiwon; Hirleman, E Daniel; Aroonnual, Amornrat; Bhunia, Arun K; Robinson, J Paul

    2009-01-01

    A system design and automation of a microbiological instrument that locates bacterial colonies and captures the forward-scattering signatures are presented. The proposed instrument integrates three major components: a colony locator, a forward scatterometer and a motion controller. The colony locator utilizes an off-axis light source to illuminate a Petri dish and an IEEE1394 camera to capture the diffusively scattered light to provide the number of bacterial colonies and two-dimensional coordinate information of the bacterial colonies with the help of a segmentation algorithm with region-growing. Then the Petri dish is automatically aligned with the respective centroid coordinate with a trajectory optimization method, such as the Traveling Salesman Algorithm. The forward scatterometer automatically computes the scattered laser beam from a monochromatic image sensor via quadrant intensity balancing and quantitatively determines the centeredness of the forward-scattering pattern. The final scattering signatures are stored to be analyzed to provide rapid identification and classification of the bacterial samples

  9. An iron detection system determines bacterial swarming initiation and biofilm formation

    NARCIS (Netherlands)

    Lin, Chuan-Sheng; Tsai, Yu-Huan; Chang, Chih-Jung; Tseng, Shun-Fu; Wu, Tsung-Ru; Lu, Chia-Chen; Wu, Ting-Shu; Lu, Jang-Jih; Horng, Jim-Tong; Martel, Jan; Ojcius, David M.; Lai, Hsin-Chih; Young, John D.; Andrews, S. C.; Robinson, A. K.; Rodriguez-Quinones, F.; Touati, D.; Yeom, J.; Imlay, J. A.; Park, W.; Marx, J. J.; Braun, V.; Hantke, K.; Cornelis, P.; Wei, Q.; Vinckx, T.; Troxell, B.; Hassan, H. M.; Verstraeten, N.; Lewis, K.; Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P.; Kearns, D. B.; Losick, R.; Butler, M. T.; Wang, Q.; Harshey, R. M.; Lai, S.; Tremblay, J.; Deziel, E.; Overhage, J.; Bains, M.; Brazas, M. D.; Hancock, R. E.; Partridge, J. D.; Kim, W.; Surette, M. G.; Givskov, M.; Rather, P. N.; Houdt, R. Van; Michiels, C. W.; Mukherjee, S.; Inoue, T.; Frye, J. G.; McClelland, M.; McCarter, L.; Silverman, M.; Matilla, M. A.; Wu, Y.; Outten, F. W.; Singh, P. K.; Parsek, M. R.; Greenberg, E. P.; Welsh, M. J.; Banin, E.; Vasil, M. L.; Wosten, M. M.; Kox, L. F.; Chamnongpol, S.; Soncini, F. C.; Groisman, E. A.; Laub, M. T.; Goulian, M.; Krell, T.; Lai, H. C.; Lin, C. S.; Soo, P. C.; Tsai, Y. H.; Wei, J. R.; Wyckoff, E. E.; Mey, A. R.; Leimbach, A.; Fisher, C. F.; Payne, S. M.; Livak, K. J.; Schmittgen, T. D.; Clarke, M. B.; Hughes, D. T.; Zhu, C.; Boedeker, E. C.; Sperandio, V.; Stintzi, A.; Clarke-Pearson, M. F.; Brady, S. F.; Drake, E. J.; Gulick, A. M.; Qaisar, U.; Rowland, M. A.; Deeds, E. J.; Garcia, C. A.; Alcaraz, E. S.; Franco, M. A.; Rossi, B. N. Passerini de; Mehi, O.; Skaar, E. P.; Visaggio, D.; Nishino, K.; Dietz, P.; Gerlach, G.; Beier, D.; Bustin, S. A.; Schwyn, B.; Neilands, J. B.

    2016-01-01

    Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising

  10. Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

    Science.gov (United States)

    Tekewe, Alemu; Fan, Yuanyuan; Tan, Emilyn; Middelberg, Anton P J; Lua, Linda H L

    2017-02-01

    A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. 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

  12. Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with neurocryptococcosis.

    Science.gov (United States)

    Rocha, Katya C; Pinhal, Cinthia; Cavalcanti, Sônia; Vidal, Monica S M; Toscano, Matheus; Moraes-Vasconcelos, Dewton; Duarte, Alberto J S; Fonseca, Fernando L A; de Abreu, Luiz Carlos; Valenti, Vitor E; Grumach, Anete S G

    2012-10-30

    Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis. Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of 3H-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05. The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis. Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

  13. Detection of prostate specific antigen (PSA) in human saliva using an ultra-sensitive nanocomposite of graphene nanoplatelets with diblock-co-polymers and Au electrodes.

    Science.gov (United States)

    Khan, M S; Dighe, K; Wang, Z; Srivastava, I; Daza, E; Schwartz-Dual, A S; Ghannam, J; Misra, S K; Pan, D

    2018-02-26

    Prostate-specific antigen (PSA) is a commonly used biomarker for the detection of prostate cancer (PCa) and there are numerous data available for its invasive detection in the serum and whole blood. In this work, an electrochemical sensing method was devised to detect traces of PSA in human saliva using a hybrid nanocomposite of graphene nanoplatelets with diblock co-polymers and Au electrodes (GRP-PS 67 -b-PAA 27 -Au). The pure graphitic composition on filter paper provides significantly high electrical and thermal conductivity while PS 67 -b-PAA 27 makes an amphiphilic bridge between GRP units. The sensor utilizes the binding of an anti-PSA antibody with an antigen-PSA to act as a resistor in a circuit providing an impedance change that in turn allows for the detection and quantification of PSA in saliva samples. A miniaturized electrical impedance analyzer was interfaced with a sensor chip and the data were recorded in real-time using a Bluetooth-enabled module. This fully integrated and optimized sensing device exhibited a wide PSA range of detection from 0.1 pg mL -1 to 100 ng mL -1 (R 2 = 0.963) with a lower limit of detection of 40 fg mL -1 . The performance of the biosensor chip was validated with an enzyme-linked immunosorbent assay technique with a regression coefficient as high as 0.940. The advantages of the newly developed saliva-PSA electrical biosensor over previously reported serum-PSA electrochemical biosensors include a faster response time (3-5 min) to achieve a stable electrical signal for PSA detection, high selectivity, improved sensitivity, no additional requirement of a redox electrolyte for electron exchange and excellent shelf life. The presented sensor is aimed for clinical commercialization to detect PSA in human saliva.

  14. Molecular Detection of Leptospira in Two Returned Travelers: Higher Bacterial Load in Cerebrospinal Fluid versus Serum or Plasma

    Science.gov (United States)

    Waggoner, Jesse J.; Soda, Elizabeth A.; Seibert, Ryan; Grant, Philip; Pinsky, Benjamin A.

    2015-01-01

    Leptospirosis is a potentially severe illness in returned travelers. Patients often present with fever, headache, and neck pain, which may lead to a workup for meningitis including the acquisition of cerebrospinal fluid (CSF). Although Leptospira DNA has been detected in CSF by polymerase chain reaction (PCR), little data exist regarding the utility of testing CSF in addition to serum or plasma obtained on presentation. In this report, we present two cases of leptospirosis in returned travelers presenting with fever and headache. Our first patient had neutrophilic meningitis, and Leptospira was detectable only in CSF obtained on admission. The second patient had a normal CSF profile, but Leptospira was detected in CSF at a bacterial load 5- to 10-fold higher than that in plasma. CSF is an important specimen for the diagnosis of Leptospira by molecular methods and may yield an actionable diagnosis in the absence of leptospiremia. PMID:26033024

  15. Counterimmunoelectrophoresis in the diagnosis of bacterial meningitis

    DEFF Research Database (Denmark)

    Colding, H; Lind, I

    1977-01-01

    was performed with antisera to Neisseria meningitidis (groups A, B and C), Streptococcus pneumoniae (omni-serum and pools A to 1), and Haemophilus influenzae type b. Antigen was detected in 57% (72/126) of specimens in which cultures revealed these three kinds of microorganisms in CSF and in 12% (17....... influenzae type b. Specific diagnosis was achieved in 60% (170/283) of the specimens studied and could be extablished within 1 h in 85% (145/170) by the combined results of microscopy and CIE. Ten specimens, nine of which showed a reaction with antiserum to N. meningitidis group A, were positive by CIE only......./139) of the culture-negative specimens. CSF specimens from 21 patients with bacterial meningitis caused by other species were all negative in CIE, except four, three of which contained Escherichia coli antigen reacting with antiserum to N. meningitidis group B and one E. coli antigen reacting with antiserum to H...

  16. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks.

    Science.gov (United States)

    Wei, B; Cha, S-Y; Kang, M; Park, I-J; Moon, O-K; Park, C-K; Jang, H-K

    2013-05-01

    Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples.

  17. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Viral and Bacterial Pathogens of Infectious Diarrhea

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2014-01-01

    Full Text Available Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1, and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2. The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20–200 copies for a single virus and 102-103 CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus.

  18. RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids

    Science.gov (United States)

    Abdullah, Zeinab; Schlee, Martin; Roth, Susanne; Mraheil, Mobarak Abu; Barchet, Winfried; Böttcher, Jan; Hain, Torsten; Geiger, Sergej; Hayakawa, Yoshihiro; Fritz, Jörg H; Civril, Filiz; Hopfner, Karl-Peter; Kurts, Christian; Ruland, Jürgen; Hartmann, Gunther; Chakraborty, Trinad; Knolle, Percy A

    2012-01-01

    Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria. PMID:23064150

  19. Bacterial Biofilm Infection Detected in Breast Implant-Associated Anaplastic Large-Cell Lymphoma.

    Science.gov (United States)

    Hu, Honghua; Johani, Khalid; Almatroudi, Ahmad; Vickery, Karen; Van Natta, Bruce; Kadin, Marshall E; Brody, Garry; Clemens, Mark; Cheah, Chan Yoon; Lade, Stephen; Joshi, Preeti Avinash; Prince, H Miles; Deva, Anand K

    2016-06-01

    A recent association between breast implants and the development of anaplastic large-cell lymphoma (ALCL) has been observed. The purpose of this study was to identify whether bacterial biofilm is present in breast implant-associated ALCL and, if so, to compare the bacterial microbiome to nontumor capsule samples from breast implants with contracture. Twenty-six breast implant-associated ALCL samples were analyzed for the presence of biofilm by real-time quantitative polymerase chain reaction, next-generation sequencing, fluorescent in situ hybridization, and scanning electron microscopy, and compared to 62 nontumor capsule specimens. Both the breast implant-associated ALCL and nontumor capsule samples yielded high mean numbers of bacteria (breast implant-associated ALCL, 4.7 × 10 cells/mg of tissue; capsule, 4.9 × 10 cells/mg of tissue). Analysis of the microbiome in breast implant-associated ALCL specimens showed significant differences with species identified in nontumor capsule specimens. There was a significantly greater proportion of Ralstonia spp. present in ALCL specimens compared with nontumor capsule specimens (p capsule specimens compared with breast implant-associated ALCL specimens (p < 0.001). Bacterial biofilm was visualized both on scanning electron microscopy and fluorescent in situ hybridization. This novel finding of bacterial biofilm and a distinct microbiome in breast implant-associated ALCL samples points to a possible infectious contributing cause. Breast implants are widely used in both reconstructive and aesthetic surgery, and strategies to reduce their contamination should be more widely studied and practiced. Risk, V.

  20. Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction

    Directory of Open Access Journals (Sweden)

    Consolandi Clarissa

    2002-09-01

    Full Text Available Abstract Background PCR amplification of bacterial