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Sample records for bacterial antigen detection

  1. Antigen detection systems

    Science.gov (United States)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  2. Relationship between antigen concentration and bacterial load in Pacific salmon with bacterial kidney disease.

    Science.gov (United States)

    Hamel, Owen S; Anderson, James J

    2002-08-29

    Using data collected to test spawning female Pacific salmon (Oncorhynchus kisutch and O. tshawytscha for the presence and severity of bacterial kidney disease (BKD), a mathematical model of the relationship between bacterial load and antigen concentration in tissues and ovarian fluid is developed. Renibacterium salmoninarum, the causative agent of BKD, secretes large amounts of a 57 kDa protein ('p57'), its major soluble antigen, which eventually breaks down or is otherwise removed from free circulation. Bacterial load and soluble antigen concentration in tissues are strong indicators of fish health, while in ovarian fluid they are predictors of the success of offspring. Model results indicate either an exponentially increasing antigen removal rate or an exponentially decreasing per-bacterium antigen secretion rate with increasing antigen concentration. Possible mechanisms underlying the observed relationship include a nonlinear increasing autolytic rate of the 'p57' antigen and a bacterium-antigen interaction threshold which prevents bacterial antigen secretion. PMID:12363089

  3. 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)

    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 [125I]staphylococcal protein A ([125I]SpA) and optimal conditions for the assay defined by varying the amounts of antibodies, bacterial antigenic targets and [125I]SpA. In antibody excess, 100% of available [125I]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[125I]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.)

  4. Detection of Renibacterium salmoninarum antigen in migrating adult chum salmon (Oncorhynchus keta) in Japan.

    Science.gov (United States)

    Sakai, M; Atsuta, S; Kobayashi, M

    1992-01-01

    Renibacterium salmoninarum antigen was detected in the kidney of migrating chum salmon (Oncorhynchus keta) using the indirect dot blot assay and indirect fluorescent antibody test. The adult chum salmon had migrated into a bay in which cultured coho salmon infected with R. salmoninarum were present. Antigen was detected in 5% of the chum salmon although they did not have clinical signs of bacterial kidney disease (BKD). This report describes the first case of R. salmoninarum antigen detection among wild chum salmon populations in eastern Asia. PMID:1548789

  5. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866... Plasmodium species antigen detection assays. (a) Identification. A Plasmodium species antigen detection assay... malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum,...

  6. Detection of antigens in urine during acute toxoplasmosis.

    OpenAIRE

    Huskinson, J; Stepick-Biek, P; Remington, J S

    1989-01-01

    Toxoplasma antigens were detected in sera and urine of mice acutely infected with Toxoplasma gondii. The concentrations of antigens in the urine samples measured by enzyme-linked immunosorbent assay were similar to those detected in the sera of the corresponding mice. The major antigens were not dialyzable and were largely destroyed by treatment with trichloroacetic acid and heat (100 degrees C for 1 h). Toxoplasma antigens were demonstrable on Western blots (immunoblots) of the urine samples.

  7. 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. PMID:26984975

  8. 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.

  9. Detection of O antigens in Escherichia coli

    Science.gov (United States)

    Lipopolysaccharide on the surface of Escherichia coli constitute the O antigens, which are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in host-pathogen interactions. O antigens that are responsible for antigenic specificity of the ...

  10. Pneumocystis carinii antigen detection in rat serum and lung lavage.

    OpenAIRE

    McNabb, S J; Graves, D C; Kosanke, S.D.; Moyer, M J; Ivey, M H

    1988-01-01

    We developed a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that detected relatively low concentrations of known Pneumocystis carinii antigen added to buffer or rat sera. Artificial immunization-derived polyclonal rabbit anti-P. carinii antibody was used on the solid phase to capture the antigen. Infection-derived (after P. carinii pneumonia) polyclonal rat anti-P. carinii antibody or a mixture of five murine monoclonal antibodies was used as the antigen detecto...

  11. Antigen detection for human immunodeficiency virus.

    OpenAIRE

    Harry, D J; Jennings, M B; Yee, J.; Carlson, J. R.

    1989-01-01

    The recent development of enzyme immunoassay procedures for the direct determination of human immunodeficiency virus (HIV) antigens has been of significant benefit in both clinical and research applications. The historical development of HIV antigen assays as well as their current and future applications for use in the clinical microbiology laboratory are reviewed. A detailed description of selected commercially available assays is presented, and a comparison is made of various parameters, in...

  12. 'Nothing is permanent but change'- antigenic variation in persistent bacterial pathogens.

    Science.gov (United States)

    Palmer, Guy H; Bankhead, Troy; Lukehart, Sheila A

    2009-12-01

    Pathogens persist in immunocompetent mammalian hosts using various strategies, including evasion of immune effectors by antigenic variation. Among highly antigenically variant bacteria, gene conversion is used to generate novel expressed variants from otherwise silent donor sequences. Recombination using oligonucleotide segments from multiple donors is a combinatorial mechanism that tremendously expands the variant repertoire, allowing thousands of variants to be generated from a relatively small donor pool. Three bacterial pathogens, each encoded by a small genome (Treponema pallidum TprK and Anaplasma marginale Msp2 expression sites and donors are chromosomally encoded. Both T. pallidum and A. marginale generate antigenic variants in vivo in individual hosts and studies at the population level reveal marked strain diversity in the variant repertoire that may underlie pathogen strain structure and the capacity for re-infection and heterologous strain superinfection. Here, we review gene conversion in bacterial antigenic variation and discuss the short- and long-term selective pressures that shape the variant repertoire. PMID:19709057

  13. Analysis of protective antigen peptide binding motifs using bacterial display technology

    Science.gov (United States)

    Sarkes, Deborah A.; Dorsey, Brandi L.; Stratis-Cullum, Dimitra N.

    2015-05-01

    In today's fast-paced world, a new biological threat could emerge at any time, necessitating a prompt, reliable, inexpensive detection reagent in each case. Combined with magnetic-activated cell sorting (MACS), bacterial display technology makes it possible to isolate selective, high affinity peptide reagents in days to weeks. Utilizing the eCPX display scaffold is also a rapid way to screen potential peptide reagents. Peptide affinity reagents for protective antigen (PA) of the biothreat Bacillus anthracis were previously discovered using bacterial display. Bioinformatics analysis resulted in the consensus sequence WXCFTC. Additionally, we have discovered PA binding peptides with a WW motif, one of which, YGLHPWWKNAPIGQR, can pull down PA from 1% human serum. The strength of these two motifs combined, to obtain a WWCFTC consensus, is assessed here using Fluorescence Activated Cell Sorting (FACS). While monitoring binding to PA, overall expression of the display scaffold was assessed using the YPet Mona expression control tag (YPet), and specificity was assessed by binding to Streptavidin R-Phycoerythrin (SAPE). The importance of high YPet binding is highlighted as many of the peptides in one of the three replicate experiments fell below our 80% binding threshold. We demonstrate that it is preferable to discard this experiment, due to questionable expression of the peptide itself, than to try to normalize for relative expression. The peptides containing the WWCFTC consensus were of higher affinity and greater specificity than the peptides containing the WW consensus alone, validating further investigation to optimize known PA binders.

  14. 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.

  15. Detection of hepatitis A viral antigen by radioimmunoassay

    International Nuclear Information System (INIS)

    With coded samples, the effectiveness and specificity of a micro-SPIRA procedure for rapidly and quantitatively detecting type A hepatitis-associated antigen in large numbers of specimens from infected liver, stool, or serum has been demonstrated. Samples which were judged to be negative by IEM were found to contain significant levels of HAV antigen by this immunoradiometric technique. The detection of significant levels of HAV antigen in infected chimpanzees supports epidemiologic evidence of viremia during the acute stage of the disease. The results of this study suggest that the diagnosis of type A hepatitis by a convention serologic procedure may now be at hand. (auth)

  16. Autoradiographic detection of IgG and viral antigens

    International Nuclear Information System (INIS)

    Autoradiographic methods can be used as an alternative to indirect immunofluorescence to detect viral antigen expression or the presence of IgG in tissue sections. Iodinated protein A isolated from Staphylococcus aureus detects an influx of IgG into the central nervous system of mice inoculated with the coronavirus SD. Antispecies antibody that has been iodinated detects coronavirus antigen expression for 24 days post-inoculation while it is only detectable for 10 days by immunofluorescence. A direct comparison of indirect fluorescence and autoradiographic methods indicates that the autoradiographic techniques are considerably more sensitive. This increased sensitivity is sufficient to permit the detection of viral antigen in formalin fixed paraffin embedded tissue sections. (Auth.)

  17. DEVELOPMENT OF ANTIBODY TO RALSTONIA SOLANACEARUM AND ITS APPLICATION FOR DETECTION OF BACTERIAL WILT

    Directory of Open Access Journals (Sweden)

    YADI SURYADI

    2009-01-01

    Full Text Available The serological assay for the detection of bacterial wilt caused by Ralstonia solanacearum (RSwas able to provide information regarding the presence of the pathogen in plant materials. Theresearch is was aimed to develop polyclonal antibody (PAb for RS detection. Bacterial wholecells of RS isolates mixed with glutaraldehyde were used to immunize New Zealand femalewhite rabbit. The titre of antibody in culture supernatant was 1: 1024. The PAb developed froma ground nut RS isolates reacted with infected plant samples from various locations. It was ableto detect RS antigen of crude extract and pure cultures from tomato and potato plant samples4-5using dot blot ELISA; however, the minimum detectable concentration of RS antigen was 10cells/ml. The PAb obtained in this study is sensitive enough to detect RS isolates in routineserological assay

  18. Synthetic peptides with antigenic specificity for bacterial toxins.

    Science.gov (United States)

    Sela, M; Arnon, R; Jacob, C O

    1986-01-01

    The attachment of a diphtheria toxin-specific synthetic antigenic determinant and a synthetic adjuvant to a synthetic polymeric carrier led to production of a totally synthetic macromolecule which provoked protective antibodies against diphtheria when administered in aqueous solution. When peptides related to the B subunit of cholera toxin were synthesized and attached to tetanus toxoid, antibodies produced against the conjugate reacted in some but not all cases with intact cholera toxin and (especially with peptide CTP 3, residues 50-64) neutralized toxin reactivity, as tested by permeability in rabbit skin, fluid accumulation in ligated small intestinal loops and adenylate cyclase activation. Polymerization of the peptide without any external carrier, or conjugation with the dipalmityl lysine group, had as good an effect in enhancing the immune response as its attachment to tetanus toxoid. Prior exposure to the carrier suppressed the immune response to the epitope attached to it, whereas prior exposure to the synthetic peptide had a good priming effect when the intact toxin was given; when two different peptides were attached to the same carrier, both were expressed. Antisera against peptide CTP 3 were highly cross-reactive with the heat-labile toxin of Escherichia coli and neutralized it to the same extent as cholera toxin, which is not surprising in view of the great homology between the two proteins. A synthetic oligonucleotide coding for CTP 3 has been used to express the peptide in a form suitable for immunization. It led to a priming effect against the intact cholera toxin. PMID:2426052

  19. Sensitive, Rapid Detection of Bacterial Spores

    Science.gov (United States)

    Kern, Roger G.; Venkateswaran, Kasthuri; Chen, Fei; Pickett, Molly; Matsuyama, Asahi

    2009-01-01

    A method of sensitive detection of bacterial spores within delays of no more than a few hours has been developed to provide an alternative to a prior three-day NASA standard culture-based assay. A capability for relatively rapid detection of bacterial spores would be beneficial for many endeavors, a few examples being agriculture, medicine, public health, defense against biowarfare, water supply, sanitation, hygiene, and the food-packaging and medical-equipment industries. The method involves the use of a commercial rapid microbial detection system (RMDS) that utilizes a combination of membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and analysis of luminescence images detected by a charge-coupled-device camera. This RMDS has been demonstrated to be highly sensitive in enumerating microbes (it can detect as little as one colony-forming unit per sample) and has been found to yield data in excellent correlation with those of culture-based methods. What makes the present method necessary is that the specific RMDS and the original protocols for its use are not designed for discriminating between bacterial spores and other microbes. In this method, a heat-shock procedure is added prior to an incubation procedure that is specified in the original RMDS protocols. In this heat-shock procedure (which was also described in a prior NASA Tech Briefs article on enumerating sporeforming bacteria), a sample is exposed to a temperature of 80 C for 15 minutes. Spores can survive the heat shock, but nonspore- forming bacteria and spore-forming bacteria that are not in spore form cannot survive. Therefore, any colonies that grow during incubation after the heat shock are deemed to have originated as spores.

  20. Detection of gonococcal antigens in urine by radioimmunoassay

    International Nuclear Information System (INIS)

    A method of detecting gonococcal antigens by solid-phase radioimmunoassay with radioactively labelled antibody is described. A specificity test has been developed that enables this method to be used to detect gonococcal antigens in urine sediments. When sediments from samples of urine from male patients with gonorrhoea were tested, 31 (74%) of 42 gave positive results, clearly distinguishing them from sediments from urine samples from men with non-specific urethritis, none of which was positive. Ten of 14 urine sediments from urine samples from women with gonorrhoea gave positive results, as did 3 of 18 sediments from urine samples from women patients without gonorrhoea.These experiments demonstrate that gonococcal antigens can be detected in urine by radioimmunoassay; the method could be useful in diagnosis if, after refinement, its sensitivity and specificity were to be increased. (author)

  1. 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. PMID:26386068

  2. Bacteriophages for detection of bacterial pathogens

    International Nuclear Information System (INIS)

    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

  3. The detection of two antigenic groups among Renibacterium salmoninarum isolates.

    Science.gov (United States)

    Bandín, I; Santos, Y; Magariños, B; Barja, J L; Toranzo, A E

    1992-07-01

    The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected. PMID:1521757

  4. A microbial detection array (MDA for viral and bacterial detection

    Directory of Open Access Journals (Sweden)

    McLoughlin Kevin S

    2010-11-01

    Full Text Available Abstract Background Identifying the bacteria and viruses present in a complex sample is useful in disease diagnostics, product safety, environmental characterization, and research. Array-based methods have proven utility to detect in a single assay at a reasonable cost any microbe from the thousands that have been sequenced. Methods We designed a pan-Microbial Detection Array (MDA to detect all known viruses (including phages, bacteria and plasmids and developed a novel statistical analysis method to identify mixtures of organisms from complex samples hybridized to the array. The array has broader coverage of bacterial and viral targets and is based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms, and to have no significant matches to the human genome sequence. Results In blinded testing on spiked samples with single or multiple viruses, the MDA was able to correctly identify species or strains. In clinical fecal, serum, and respiratory samples, the MDA was able to detect and characterize multiple viruses, phage, and bacteria in a sample to the family and species level, as confirmed by PCR. Conclusions The MDA can be used to identify the suite of viruses and bacteria present in complex samples.

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

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    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.

  6. Seasonal Evaluation of Antigenic Bacterial Infections Among Working Class in the Inner City of Houston

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    Ebere C. Anyanwu

    2004-01-01

    Full Text Available This paper evaluates the monthly, quarterly, and seasonal variation of antigenic bacterial infections among the working class in the inner city of Houston using the Wellcogen Rapid Test methods. One of the aims was to demonstrate how this method could be used effectively in screening patients at risk and preventing the spread of antigenic bacteria such as Streptococcus pneumoniae, Haemophilus influenzae b, Streptococcus (Strep b, and Neisseria meningitidis (mainly group c and b. A total of 2,837 patients were screened for bacterial infections; 908 (32% were male and 1,929 (68% were female. The age range was between 2 and 70 years. Of the total group, 356 (12.5% patients were positive; 203 (57% were female while 153 (43% were male (male/female ratio of 1:1.3. Medically underserved and immune suppressed populations are the most affected by these bacterial infections. Blacks are the most affected (48% compared to Native Americans (1%, but children under 10 years of age have the highest incidence. This research showed, in addition, that the Wellcogen Rapid Tests are effective (356 cases identified for a rapid screening of infectious bacteria. Explanation for these results was probably due to poor living conditions, poor hygiene, and viral immune suppression in adults and immature immune systems in neonates and children under 10 years of age.

  7. Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

    Science.gov (United States)

    Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

    2008-06-22

    Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats. PMID:18182256

  8. Self-Adjuvanting Bacterial Vectors Expressing Pre-Erythrocytic Antigens Induce Sterile Protection against Malaria

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    Elke eBergmann-Leitner

    2013-07-01

    Full Text Available Genetically inactivated, Gram-negative bacteria that express malaria vaccine candidates represent a promising novel self-adjuvanting vaccine approach. Antigens expressed on particulate bacterial carriers not only target directly to antigen-presenting cells but also provide a strong danger signal thus circumventing the requirement for potent extraneous adjuvants. E. coli expressing malarial antigens resulted in the induction of either Th1 or Th2 biased responses that were dependent on both antigen and sub-cellular localization. Some of these constructs induced higher quality humoral responses compared to recombinant protein and most importantly they were able to induce sterile protection against sporozoite challenge in a murine model of malaria. In light of these encouraging results, two major Plasmodium falciparum pre-erythrocytic malaria vaccine targets, the Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS fused to the Maltose-binding protein in the periplasmic space and the Circumsporozoite Protein (CSP fused to the Outer membrane protein A in the outer membrane were expressed in a clinically relevant, attenuated Shigella strain (Shigella flexneri 2a. This type of live attenuated vector has previously undergone clinical investigations as a vaccine against shigellosis. Using this novel delivery platform for malaria, we find that vaccination with the whole organism represents an effective vaccination alternative that induces protective efficacy against sporozoite challenge. Shigella GeMI-Vax expressing malaria targets warrant further evaluation to determine their full potential as a dual disease, multivalent, self-adjuvanting vaccine system, against both shigellosis and malaria.

  9. 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.

  10. Direct Testing of Blood Cultures for Detection of Streptococcal Antigens

    OpenAIRE

    Wetkowski, Maryellen A.; Peterson, Ellena M.; de la Maza, Luis M.

    1982-01-01

    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 str...

  11. Enzyme immunoassay for the detection of group A streptococcal antigen.

    OpenAIRE

    Knigge, K M; Babb, J L; Firca, J R; Ancell, K; Bloomster, T G; Marchlewicz, B A

    1984-01-01

    A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 10(4) or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated w...

  12. Linkage of bacterial protein synthesis and presentation of MHC class I-restricted Listeria monocytogenes-derived antigenic peptides.

    Directory of Open Access Journals (Sweden)

    Silke Grauling-Halama

    Full Text Available The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217-225 and p60 449-457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.

  13. Detecting polysaccharide antigen of Neisseria meningitidis group C in cerebrospinal fluid by dot-ELISA assay.

    Science.gov (United States)

    Correia Barbosa, S F; Alkmin, M G; Landgraf, I M

    2000-06-01

    Cerebrospinal fluid (CSF) samples from 210 patients (200 with clinical evidence of bacterial meningitis, 10 with other clinical neurologic disease) were tested by a Dot-ELISA assay for detection of polysaccharide antigen of N. meningitidis group C. CSF samples were treated with EDTA 0.1 M, at pH 7.5 and heated to 90>C for 10 min. Polyclonal antiserum was purified by use of ethanol fractionation. The results were compared to those using bacterial culture (BC), latex agglutination (LA), counterimmunoelectrophoresis (CIE), and direct microscopy (DM) methods. Test results showed a correlation of 93.3%, 94.3%, 91.0% and 69.5% respectively, and sensitivity of 0.947 and specificity of 0.930. This study suggests that the dot-ELISA assay of CSF is a useful alternative technique for the diagnosis of group C meningitis. PMID:10934498

  14. Antigen

    Science.gov (United States)

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  15. Transplant immuno-diagnostics: crossmatch and antigen detection.

    Science.gov (United States)

    South, Andrew M; Grimm, Paul C

    2016-06-01

    Identifying and monitoring donor-directed anti-human leukocyte antigen antibodies are a rapidly evolving area of solid organ transplantation. Donor-specific antibodies dictate pre-transplant donor choice and donor-recipient matching and underlie much acute and chronic allograft rejection and loss. The evolution of available technology has driven this progress. Early, labor-intensive, whole-cell assays based on complement-dependent cytotoxicity suffered from poor sensitivity and specificity, technical challenges and lack of precision. Sequential improvement in assay performance included anti-human immunoglobulin-enhanced, complement-dependent cytotoxicity techniques followed by cell-based flow cytometry. However, variable specificity and sensitivity inherent in cell-based testing continued to limit flow cytometry. The introduction of solid-phase assays led to a second revolution in histocompatibility testing with the use of purified antigens bound to artificial surfaces rather than whole cells. These techniques augmented sensitivity and specificity to detect even low-titer antibodies to previously undetected antigens. Identification of complement-activating antibodies is being introduced, but current technology is in the developmental stage. While the detection of alloantibodies has improved dramatically, our comprehension of their importance remains imperfect. Variability in methodology and a lack of standardization limits the clinical application of these tests. In spite of the hurdles that remain, antibody-mediated rejection has become a key target to improve graft survival. PMID:26139577

  16. Detection of bacterial concentration variations based on dielectric magnetic flux.

    Science.gov (United States)

    Chen, Jingyao; Cai, Jie; Huang, Xingjian; Yi, Tian; Wang, Kexing; Pan, Siyi

    2016-02-01

    This study is based on the development of bacterial count quantification generated by medium dielectric variations and consequent polarization material release. The proposed approach is an action method for the fast detection of bacterial concentration in orange juice. The sensing method relies on bacterial attachment up to biofilm formation. Furthermore, different media provide more oxygen group content to enhance capacitance and self-discharge. The test took only 30 min and it provided the means for rapid bacterial detection in the juice industry. PMID:26304394

  17. Automated Image Analysis for Determination of Antibody Titers Against Occupational Bacterial Antigens Using Indirect Immunofluorescence.

    Science.gov (United States)

    Brauner, Paul; Jäckel, Udo

    2016-06-01

    Employees who are exposed to high concentrations of microorganisms in bioaerosols frequently suffer from respiratory disorders. However, etiology and in particular potential roles of microorganisms in pathogenesis still need to be elucidated. Thus, determination of employees' antibody titers against specific occupational microbial antigens may lead to identification of potentially harmful species. Since indirect immunofluorescence (IIF) is easy to implement, we used this technique to analyze immunoreactions in human sera. In order to address disadvantageous inter-observer variations as well as the absence of quantifiable fluorescence data in conventional titer determination by eye, we specifically developed a software tool for automated image analysis. The 'Fluorolyzer' software is able to reliably quantify fluorescence intensities of antibody-bound bacterial cells on digital images. Subsequently, fluorescence values of single cells have been used to calculate non-discrete IgG titers. We tested this approach on multiple bacterial workplace isolates and determined titers in sera from 20 volunteers. Furthermore, we compared image-based results with the conventional manual readout and found significant correlation as well as statistically confirmed reproducibility. In conclusion, we successfully employed 'Fluorolyzer' for determination of titers against various bacterial species and demonstrated its applicability as a useful tool for reliable and efficient analysis of immune response toward occupational exposure to bioaerosols. PMID:27026659

  18. Detection of filarial antigen in urine of humans with Wuchereria bancrofti infection by immunoradiometric assay

    International Nuclear Information System (INIS)

    Human urine samples of different groups were analyzed for the presence of filarial antigen by immuno-radiometric assay (IRMA) using 125I-Rabbit IgG antibodies to B. malayi antigen. Six out of ten microfilaraemia, one out of five clinical filariasis had detectable antigen, while none of the endemic and non-endemic normals (n=12) were positive. The effect of heat-acid treatment on the detectability of antigen in the urine and serum was explored. Heat-acid treatment in general did not reduce the level of filarial antigen in the urine samples while there was a measurable reduction in the antigen levels in the sera. (author)

  19. Optimisation of immuno-gold nanoparticle complexes for antigen detection

    OpenAIRE

    Van Der Heide, Susan; RUSSELL, David

    2016-01-01

    The aim of this investigation was to define the optimum method of binding antibodies to the surface of gold nanoparticles (AuNPs) and then to apply the optimised antibody-functionalised AuNPs for the detection of a target antigen. A detailed investigation of three different techniques for the functionalisation of AuNPs with anti-cocaine antibody and methods for the subsequent characterisation of the antibody-functionalised AuNP are reported. The addition of anti-cocaine antibody onto the AuNP...

  20. 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

  1. Optimisation of immuno-gold nanoparticle complexes for antigen detection.

    Science.gov (United States)

    van der Heide, Susan; Russell, David A

    2016-06-01

    The aim of this investigation was to define the optimum method of binding antibodies to the surface of gold nanoparticles (AuNPs) and then to apply the optimised antibody-functionalised AuNPs for the detection of a target antigen. A detailed investigation of three different techniques for the functionalisation of AuNPs with anti-cocaine antibody and methods for the subsequent characterisation of the antibody-functionalised AuNP are reported. The addition of anti-cocaine antibody onto the AuNP surface was facilitated by either: a polyethylene glycol (PEG) linker with a COOH terminal functional group; an aminated PEG ligand; or an N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-Protein A/G intermediate. Characterisation of the functionalised particles was performed using transmission electron microscopy, UV-Visible spectrophotometry and by agarose gel electrophoresis. In addition, the cocaine binding efficacy of the resultant AuNPs and their cocaine-binding capacity was determined using a cocaine-horseradish peroxidase conjugate, and by the application of a microtiter plate-based immunoassay. The results showed that the number of antibody per particle was the highest when the AuNP were functionalised with the Protein A/G intermediate. As compared to free antibody, the cocaine binding efficacy was significantly enhanced using the AuNP-Protein A/G-antibody complex. This optimal antibody-antigen binding efficacy is thought to be the result of the large number of antibody per particle and the oriented binding of the antibody to the Protein A/G on the AuNP surface. These results highlight the ideal immuno-gold nanoparticle characteristics for the detection of target antigens such as cocaine. PMID:26994353

  2. Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations

    International Nuclear Information System (INIS)

    We have previously reported the identification of the cyclic enterobacterial common antigen (ECACYC) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol.185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N-acetyl signals of ECACYC have been observed in 15N-1H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECACYC in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate

  3. 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.

  4. 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.

  5. An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.

    Science.gov (United States)

    Jahnmatz, Peter; Bengtsson, Theresa; Zuber, Bartek; Färnert, Anna; Ahlborg, Niklas

    2016-06-01

    The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs. PMID:26930550

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

    Directory of Open Access Journals (Sweden)

    Shin-Ichiro Nishimura

    2008-07-01

    Full Text Available 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.

  7. Detection of Mycobacterial Antigens in Leprosy Serum Immune Complex

    OpenAIRE

    1986-01-01

    The antigens from immune complexes of sera from patients with mycobacterial diseases were released by sodium dodecyl sulfate. The antigenic activity of the released proteins was tested by agar gel diffusion and immunoelectrophoresis. This simple method provided direct evidence for the presence of mycobacterial antigens in the immune complexes of sera from patients with leprosy and tuberculosis.

  8. [Detection of T-antigen in colorectal adenocarcinoma and polyps].

    Science.gov (United States)

    Xu, S; Lu, Y; Wang, Q

    1995-10-01

    Galactose oxidase method was employed to detect the beta-D-Gal (1-->3) -D-Gal NAc residue of T-antigen present in the large intestinal mucus of 156 subjects. The positive rates of the test were 84.4%, 29.1%, and 7.2% in the mucus samples obtained from 32 patients with colorectal adenocarcinomas, 55 with polyps and 69 controls respectively. Chi-square test demonstrated that there were significant differences between the group of carcinoma and control (P < 0.001) as well as between also polyp and control (P < 0.01). The test had a high sensitivity (84.4%) and specificity (92.8%) in the diagnosis of colorectal cancer and may be used as a practical mass screening test for colorectal neoplasms. PMID:8731834

  9. 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.

  10. Bacteriophage Amplification-Coupled Detection and Identification of Bacterial Pathogens

    Science.gov (United States)

    Cox, Christopher R.; Voorhees, Kent J.

    Current methods of species-specific bacterial detection and identification are complex, time-consuming, and often require expensive specialized equipment and highly trained personnel. Numerous biochemical and genotypic identification methods have been applied to bacterial characterization, but all rely on tedious microbiological culturing practices and/or costly sequencing protocols which render them impractical for deployment as rapid, cost-effective point-of-care or field detection and identification methods. With a view towards addressing these shortcomings, we have exploited the evolutionarily conserved interactions between a bacteriophage (phage) and its bacterial host to develop species-specific detection methods. Phage amplification-coupled matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was utilized to rapidly detect phage propagation resulting from species-specific in vitro bacterial infection. This novel signal amplification method allowed for bacterial detection and identification in as little as 2 h, and when combined with disulfide bond reduction methods developed in our laboratory to enhance MALDI-TOF-MS resolution, was observed to lower the limit of detection by several orders of magnitude over conventional spectroscopy and phage typing methods. Phage amplification has been combined with lateral flow immunochromatography (LFI) to develop rapid, easy-to-operate, portable, species-specific point-of-care (POC) detection devices. Prototype LFI detectors have been developed and characterized for Yersinia pestis and Bacillus anthracis, the etiologic agents of plague and anthrax, respectively. Comparable sensitivity and rapidity was observed when phage amplification was adapted to a species-specific handheld LFI detector, thus allowing for rapid, simple, POC bacterial detection and identification while eliminating the need for bacterial culturing or DNA isolation and amplification techniques.

  11. 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. PMID:27379844

  12. Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

    OpenAIRE

    Pramanik, J; Lodam, A. N.; Badole, C.M.; Reddy, M. V. R.; Patond, K. R.; Harinath, B. C.

    2000-01-01

    Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity ...

  13. Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations.

    Science.gov (United States)

    Canales, Mario; Labruna, Marcelo B; Soares, João F; Prudencio, Carlos R; de la Fuente, José

    2009-12-01

    The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. PMID:19835826

  14. Label-free detection of antigens using implantable SERS nanosensors

    Science.gov (United States)

    Li, Honggang; Baum, Caitlin E.; Cullum, Brian M.

    2005-11-01

    Monitoring the presence, production and transport of proteins inside individual living cells can provide vital information about cellular signaling pathways and the overall biological response of an organism. For example, cellular response to external stimuli, such as biological warfare (BW) agents, can be monitored by measuring interleukin-II (IL-2) expression inside T-cells as well as other chemical species associated with T-cell activation. By monitoring such species, pre-symptomatic detection of exposure to BW agents can be achieved, leading to significantly increased post-exposure survival rates. To accomplish such monitoring, we have developed and optimized implantable nanosphere-based nanosensors for the intracellular analysis of specific proteins in a label-free fashion. These sensors consist of 300-520 nm diameter silica spheres that have been coated with silver and antibodies to allow for trace protein detection via surface enhanced Raman spectroscopy (SERS). They have been optimized for SERS response by evaluating the size of the nanospheres best suited to 632.8 nm laser excitation, as well as the various nanosensor fabrication steps (i.e., silver deposition process, antibody binding, etc.). During usage, the presence of the specific protein of interest is monitored by either directly measuring SERS signals associated with the protein and/or changes in the SERS spectrum of the antibodies resulting from conformational changes after antigen binding. In this work, human insulin was used as a model compound for initial studies into the sensitivity of these optimized nanosensors.

  15. 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.

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

    OpenAIRE

    2015-01-01

    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 appli...

  17. CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles

    OpenAIRE

    Bergman, Molly A.; Cummings, Lisa A.; Barrett, Sara L. Rassoulian; Smith, Kelly D.; Lara, J. Cano; Aderem, Alan; Cookson, Brad T.

    2005-01-01

    A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysacc...

  18. Aromatic-dependent salmonella as anti-bacterial vaccines and as presenters of heterologous antigens or of DNA encoding them.

    Science.gov (United States)

    Stocker, B A

    2000-09-29

    The development of live bacterial vaccines is reviewed, in particular aromatic-dependent Salmonella, either for protection against the corresponding infections (including typhoid fever) or as carrier-presenter of antigens of unrelated pathogens or of DNA specifying them. Aromatic-dependent Salmonella live vaccines are also compared with BCG and Ty21a and the recent records of exceptional situations are discussed in which aroA (deletion) strains of Salmonella typhimurium cause progressive disease in mice. PMID:11000459

  19. [Detection of dengue virus antigen in post-mortem tissues].

    Science.gov (United States)

    Rivera, Jorge; Neira, Marcela; Parra, Edgar; Méndez, Jairo; Sarmiento, Ladys; Caldas, María Leonor

    2014-01-01

    The epidemiological situation of dengue has worsened over the last decade. The difficulties in preventing its transmission and the absence of a vaccine or specific treatment have made dengue a serious risk to public health, health centers and research systems at different levels. Currently, most studies on the pathogenesis of dengue infection focus on the T-cell immune response almost exclusively in secondary infections and are aimed at identifying the mechanisms involved in the development of vascular permeability and bleeding events that accompany the infection. This report describes the case of a baby girl less than 45 days of age with clinical signs of severe dengue, whose diagnosis was confirmed by reverse transcription polymerase chain reaction in post-mortem tissue samples and by the ancillary diagnostic use of immunohistochemistry, which detected viral antigens in all organs obtained at autopsy. This case highlights the importance of studying primary infections associated with severe dengue, particularly in children, who are more likely to develop the severe form of the disease without previous infection, and it further stresses the importance of a diagnosis that should not be based solely on the examination of liver tissue samples when studying the pathogenesis of the viral infection. PMID:25504239

  20. Control of tick infestations in cattle vaccinated with bacterial membranes containing surface-exposed tick protective antigens.

    Science.gov (United States)

    Almazán, Consuelo; Moreno-Cantú, Orlando; Moreno-Cid, Juan A; Galindo, Ruth C; Canales, Mario; Villar, Margarita; de la Fuente, José

    2012-01-01

    Vaccines containing the Rhipicephalus (Boophilus) microplus BM86 and BM95 antigens protect cattle against tick infestations. Tick subolesin (SUB), elongation factor 1a (EF1a) and ubiquitin (UBQ) are new candidate protective antigens for the control of cattle tick infestations. Previous studies showed that R. microplus BM95 immunogenic peptides fused to the Anaplasma marginale major surface protein (MSP) 1a N-terminal region (BM95-MSP1a) for presentation on the Escherichia coli membrane were protective against R. microplus infestations in rabbits. In this study, we extended these results by expressing SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins on the E. coli membrane using this system and demonstrating that bacterial membranes containing the chimeric proteins BM95-MSP1a and SUB-MSP1a were protective (>60% vaccine efficacy) against experimental R. microplus and Rhipicephalus annulatus infestations in cattle. This system provides a novel, simple and cost-effective approach for the production of tick protective antigens by surface display of antigenic protein chimera on the E. coli membrane and demonstrates the possibility of using recombinant bacterial membrane fractions in vaccine preparations to protect cattle against tick infestations. PMID:22085549

  1. Microfluidic immunomagnetic separation for enhanced bacterial detection

    DEFF Research Database (Denmark)

    Hoyland, James; Kunstmann-Olsen, Casper; Ahmed, Shakil;

    A combined lab-on-a-chip approach combining immunomagnetic separation (IMS) and flow cytometry was developed for the enrichment and detection of salmonella contamination in food samples. Immunomagnetic beads were immobilized in chips consisting of long fractal meanders while contaminated samples ...... to obtain maximum capturing efficiency. The effects of channel volume, path length and number of bends of microfluidic chip on IMS efficiency were also determined....

  2. Improved method for detection of glycosidases in bacterial colonies.

    OpenAIRE

    Paoni, N F; Arroyo, R L

    1984-01-01

    An assay has been developed to detect bacterial glycosidases in colonies grown on the surface of agar plates. Advantages of this technique over previously described methods include elimination of the need for replica plating, better visualization of chromagenic reaction products, and a simple permeabilization step to enable better penetration by chromagenic substrates.

  3. Molecular detection, monitoring and modulation of antigen specific immune responses.

    OpenAIRE

    Aubert, G

    2004-01-01

    Stem cell transplantati.on (SCT) represents the only curative treatment option for leukaemia. The bone marrow or peripheral blood stem cells transferred in the transplant procedure restore immune functions, allowing the targeting of infected cells or cells expressing tumour antigens. Cytotoxic T lymphocytes (CTL), key mediators of antigen specific killing, were investigated in the context of cytomegalovirus (CMV) infection or chronic myeloid leukaemia (CML). CMV infection after SCT in the abs...

  4. 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.

  5. Detection of polysaccharide antigens of Candida albicans interfering with specific antibodies in human sera

    International Nuclear Information System (INIS)

    A double antibody sandwich radioimmunoassay was developed for the detection of circulating polysaccharide antigens of Candida albicans. The sensitivity of the assay for polysaccharides was 1 ng/mL. The in-vitro interference of specific polysaccharide antibodies, even from sera with low antibody levels, could be demonstrated. The sensitivity of the antigen detection decreased proportionally to the amount of polysaccharide antibodies in the sera. The sensitivity of the assay was almost completely restored by heating the sera. This procedure destroyed antibodies and the released polysaccharide antigens were detectable in the test system by using radiolabelled anti-polysaccharide antibodies. (author)

  6. Solid phase radioimmunoassay for detection of antibodies to extractable nuclear antigens

    Energy Technology Data Exchange (ETDEWEB)

    Whittingham, S. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1983-06-24

    A solid phase radioimmunoassay is described for the detection of autoantibodies to the saline-soluble extractable nuclear antigens, ribonucleoprotein (RNP) and SS-B (or La). This assay depends on enrichment of antigens from a crude, commercially available (Pel Freez, USA) extract of rabbit thymus by absorption to the F(ab)/sub 2/ fraction of specific high titre antibody attached to a microtitre plate. Serum antibody reactive with this antigen is then detected by /sup 125/I-labelled Protein A. The assay is simple and is more sensitive than the gel diffusion assays in general use for detecting such antibodies.

  7. A solid phase radioimmunoassay for detection of antibodies to extractable nuclear antigens

    International Nuclear Information System (INIS)

    A solid phase radioimmunoassay is described for the detection of autoantibodies to the saline-soluble extractable nuclear antigens, ribonucleoprotein (RNP) and SS-B (or La). This assay depends on enrichment of antigens from a crude, commercially available (Pel Freez, USA) extract of rabbit thymus by absorption to the F(ab)2 fraction of specific high titre antibody attached to a microtitre plate. Serum antibody reactive with this antigen is then detected by 125I-labelled Protein A. The assay is simple and is more sensitive than the gel diffusion assays in general use for detecting such antibodies. (Auth.)

  8. 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

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene...... against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the...... polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use....

  9. Detection of Brucella antigen in the aborted ovine fetal stomach contents using a modified ELISA test

    Directory of Open Access Journals (Sweden)

    Th. M. Al-Nima

    2010-01-01

    Full Text Available This study was conducted on two flocks of sheep suffering from abortion in Mosul city, Iraq. The clinical findings in ewes were abortion during the 3-4 months of gestation period in the both flocks. The total percentage of abortion was 11.7 %, whereas stillbirth percentage was 4 %. Brucella spp. was isolated from four (33.3 % of the 12 samples (stomach contents of the aborted fetuses. All culture – positive samples had also positive with direct smears. By a modified enzyme-linked immunosorbent assay (ELISA, Brucella antigens were detected in the fetal stomach contents of 5 samples. The sensitivity and specificity of the modified ELISA were 100 % and 87.5 % respectively. The test had a good negative predictive value but only a moderate positive predictive value. Therefore, the test would be useful for confirming the existence of suspect disease. Comparison of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92. Of the 12 serum samples from aborted ewes, eight samples were positive with Rose Bengle test (66.7 %, more than 10 samples (83.3 % were detected by indirect ELISA test.

  10. Bacterial antigen expression is an important component in inducing an immune response to orally administered Salmonella-delivered DNA vaccines.

    Directory of Open Access Journals (Sweden)

    Michelle E Gahan

    Full Text Available BACKGROUND: The use of Salmonella to deliver heterologous antigens from DNA vaccines is a well-accepted extension of the success of oral Salmonella vaccines in animal models. Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology. An important aspect of the basic biology of the Salmonella/DNA vaccine platform is the relative contributions of prokaryotic and eukaryotic expression in production of the vaccine antigen. Gene expression in DNA vaccines is commonly under the control of the eukaryotic cytomegalovirus (CMV promoter. The aim of this study was to identify and disable putative bacterial promoters within the CMV promoter and evaluate the immunogenicity of the resulting DNA vaccine delivered orally by S. typhimurium. METHODOLOGY/PRINCIPAL FINDINGS: The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter. These promoters have homology to the bacterial consensus sequence and functional activity. To disable prokaryotic expression from the CMV promoter a series of genetic manipulations were performed to remove the two major bacterial promoters and add a bacteria transcription terminator downstream of the CMV promoter. S. typhimurium was used to immunise BALB/c mice orally with a DNA vaccine encoding the C-fragment of tetanus toxin (TT under control of the original or the modified CMV promoter. Although both promoters functioned equally well in eukaryotic cells, as indicated by equivalent immune responses following intramuscular delivery, only the original CMV promoter was able to induce an anti-TT specific response following oral delivery by S. typhimurium. CONCLUSIONS: These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful

  11. 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.

  12. 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.

  13. Detection of Rabies Virus Antigen in Dog Saliva Using a Latex Agglutination Test

    OpenAIRE

    Kasempimolporn, S.; Saengseesom, W.; Lumlertdacha, B.; Sitprija, V.

    2000-01-01

    Dog bites are responsible for more than 90% of human rabies deaths in Asia. We developed a simple and inexpensive test based on latex agglutination (LA) for rabies virus antigen detection in dog saliva. Rabies virus antigen could be detected by agglutination on a glass slide using latex particles coated with gamma globulin. By evaluation of paired saliva-brain specimens from 238 dogs, the LA test using saliva was 99% specific and 95% sensitive compared to the fluorescent antibody test (FAT) o...

  14. Post immunization Hib antigen detection in the CSF of a patient with meningococcal meningitis.

    Science.gov (United States)

    Samaha, A N; Araj, G F; Mroueh, S M

    1997-01-01

    We report a case of meningococcal meningitis where the cerebrospinal fluid was negative for Neisseria meningitidis but positive for Haemophilus influenzae type b by rapid antigen detection test. We believe that this was due to prior immunization with Haemophilus influenzae type b vaccine. We recommend caution in interpretation of the rapid antigen detection tests especially in patients who had been vaccinated against organisms screened by these tests. PMID:9421943

  15. Enzyme immunoassay for direct detection of influenza type A and adenovirus antigens in clinical specimens.

    OpenAIRE

    Harmon, M W; Pawlik, K M

    1982-01-01

    Detection of viral antigens in specimens without prior cultivation in cell culture provides the most rapid method for specific viral diagnosis. A solid-phase, double-antibody enzyme immunoassay was developed for this purpose and tested with clinical specimens containing influenza type A and adenovirus. Polystyrene microtiter wells were the solid phase and were coated with virus-specific guinea pig immunoglobulins. Specimens were added, and bound viral antigens were detected by addition of vir...

  16. Cocktail of Theileria equi antigens for detecting infection in equines

    Institute of Scientific and Technical Information of China (English)

    Shimaa; Abd; El-Salam; El-Sayed; Mohamed; Abdo; Rizk; Mohamed; Alaa; Terkawi; Ahmed; Mousa; El; Said; El; Shirbini; El; Said; Gehad; Elsayed; Mohamed; Fouda; Naoaki; Yokoyama; Ikuo; Igarashi

    2015-01-01

    Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain,Theileria equi(T.equi)protein 82(Te 82)and T.equi 104 k Da microneme-rhoptry antigen precursor(Te 43),to diagnose T.equi infection in horses as compared with equi merozoite antigen-2(EMA-2).Methods:In the current study,we applied a cocktail-ELISA containing two antigens(EMA-2+Te 82)to diagnose T.equi infection either in experimentally infected horses or in field infection.Results:Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T.equi infection in horses as compared with Te 82 or Te 43 alone.Conclusions:The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

  17. Cocktail of Theileria equi antigens for detecting infection in equines

    Institute of Scientific and Technical Information of China (English)

    Shimaa Abd El-Salam El-Sayed; Mohamed Abdo Rizk; Mohamed Alaa Terkawi; Ahmed Mousa; El Said El Shirbini El Said; Gehad Elsayed; Mohamed Fouda; Naoaki Yokoyama; Ikuo Igarashi

    2015-01-01

    Objective: To use two diagnostic antigens belonging to the frequently associated in Theileria domain, Theileria equi (T. equi) protein 82 (Te 82) and T. equi 104 kDa microneme-rhoptry antigen precursor (Te 43), to diagnose T. equi infection in horses as compared with equi merozoite antigen-2 (EMA-2). Methods: In the current study, we applied a cocktail-ELISA containing two antigens (EMA-2+Te 82) to diagnose T. equi infection either in experimentally infected horses or in field infection. Results: Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T. equi infection in horses as compared with Te 82 or Te 43 alone. Conclusions: The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T. equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

  18. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent for salmonid bacterial kidney disease

    Science.gov (United States)

    Pascho, R.J.; Mulcahy, D.

    1987-01-01

    A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

  19. Bacterial Flora of Osteoradionecrosis Detected by Molecular techniques

    OpenAIRE

    2006-01-01

    The following is a report of a study undertaken to identify the bacterial species in bone samples from patients suffering from osteoradionecrosis. This was done using polymerase chain reaction (PCR), cloning and sequencing techniques, where 16S rRNA was the gene used for analysis. The results from two patient samples will be presented. As far as we know, this is the first study that includes molecular genetic techniques to detect bacteria associated with osteoradionecrosis.

  20. Molecular Detection of Common Bacterial Pathogens Causing Meningitis

    Directory of Open Access Journals (Sweden)

    H Sadighian

    2009-03-01

    Full Text Available "nBackground: The clinical diagnosis of meningitis is crucial, particularly in children. The early diagnosis and empiric an­tibi­otic treatments have led to a reduction in morbidity and mortality rates. PCR and the enzymatic digestion of 16SrDNA frag­ment which is produced by universal primers led up fast and sensitive determination. The purpose of this study was to investi­gate a rapid method for detection of common bacterial pathogens causing meningitis."nMethods: According to the gene encoding 16SrDNA found in all bacteria, a pair of primers was designed. Then the univer­sal PCR was performed for bacterial agents of meningitis (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influ­enzae, etc. by employing broad- range DNA extraction method. The ob­tained uni­versal PCR products were digested with restriction enzymes (HaeIII, AluI and MnlI to identify bacterial species. "nResults: By the enzymatic digestion of the universal products of each standard strain of the above bacteria, specific patterns were achieved. These specific patterns may be used for comparison in CSF examination. The analytical sensitivity of the as­say was approximately 1.5´102 CFU/ml of CSF even in samples with high amount of proteins. Conclusion: The universal PCR coupled with enzymatic digestion can be used to detect and identify bacterial pathogens in clini­cal specimens rapidly and accurately. Molecular diagnostic of bacterial meningitis, though expensive and labor-inten­sive, but is valuable and critical in patient management.

  1. Evaluation of Diagnos Malaria Stix test (antigen detection assay) for diagnosis of malaria.

    Science.gov (United States)

    Khan, Haris M; Shujatullah, Fatima; Shahid, M; Raza, Adil; Malik, Ritu

    2010-06-01

    Malaria is one of the most common parasitic infection in India. The diagnosis largely depends on peripheral blood smear examination. Newer diagnostic methods like various antigen detection assays are now in use for prompt diagnosis and treatment. This study was done to determine the effectiveness of Diagnos Malaria Stix (antigen detection) assay in diagnosis of malaria. This involves detection of PfHRP-2 antigen and P.V. specific pLDH antigen. 162 patients with signs and symptoms of malaria included in the study. Leishman stained blood smear examination was done for all patients. Commercially available Diagnos Malaria Stix assay was used. Diagnos Malaria Stix showed sensitivity, specificity positive and negative predictive values of 100% each while Sensitivity, specificity, positive and negative predictive values of Leishman stained blood smear examination were 45.45%, 100%, 100% and 92% respectively. PMID:22471175

  2. Variable detection of myeloid antigens in childhood acute lymphoblastic leukaemia.

    OpenAIRE

    Howard, M R; Thomas, L; Reid, M. M.

    1994-01-01

    AIMS--To determine whether the use of different sources of anti-CD13 and anti-CD33 monoclonal antibodies leads to discrepant results in childhood acute lymphoblastic leukaemia (ALL), which might contribute to the wide variation in the reported incidence of myeloid antigen expressing ALL in childhood. METHODS--Stored leukaemic cells from 10 children with previously defined myeloid positive ALL were examined. A range of commercially available anti-CD13 and anti-CD33 monoclonal antibodies, direc...

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

    Science.gov (United States)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-11-15

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene 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 °C 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 against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use. PMID:25076184

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

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; Souza, Wayner Vieira de; Silva, Edmilson Domingos; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2016-01-01

    The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies. PMID:27517281

  5. 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; Wredstrøm, K.; Pedersen, B.; Andresen, Lars Ole; Heegaard, Peter M. H.; Jakobsen, M.H.

    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......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...... microtiter 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...

  6. 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.

  7. Changes in the antigenic and molecular structure of γ-irradiated bacterial lipopolysaccharide (LPS)

    International Nuclear Information System (INIS)

    Ionization radiation is known to alter the biological properties of LPS. The author treated a highly purified LPS from E. coli in aqueous medium with 60Co-radiation. The changes in the antigenic and molecular structure of LPS were studied in double immunodiffusion/immunoelectrophoresis (rabbit antiserum) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Untreated LPS revealed two major antigenic components and, due to varying lengths of the O-polysaccharide side chain, a series of homopolymers (SDS-PAGE). High doses of γ-radiation destroyed all antigenic reactivities and all stainable bands on SDS-PAGE. However, lower doses of radiation were selective. Disappearance of the more radiation-sensitive, electrophoretically fast-migrating antigenic component paralleled elimination of the long O-side chain containing molecules. The relatively radiation-resistant, less anodic second antigenic component cross-reacted with LPS of another E. coli strain and corresponded to LPS molecules composed of R-core and lipid A (SDS-PAGE). These findings explain the in vivo loss of antibody protection from shock before non-specific resistance with γ-irradiation of LPS

  8. Usefulness of an enzyme-linked immunosorbent assay for detection of Giardia antigen in feces.

    OpenAIRE

    Nash, T E; Herrington, D A; Levine, M M

    1987-01-01

    The usefulness of a recently developed enzyme-linked immunosorbent assay which detects Giardia lamblia antigen in feces was determined in experimentally infected humans. Giardia antigen was determined in serially collected fecal specimens from humans inoculated with two Giardia isolates, GS/M and Isr. A total of 277 stools from 18 volunteers were tested, 74 from Isr-inoculated volunteers and 203 from GS/M-inoculated volunteers. None of the five Isr-inoculated volunteers became infected, and n...

  9. Failure of a novel, rapid antigen and antibody combination test to detect antigen-positive HIV infection in African adults with early HIV infection.

    Directory of Open Access Journals (Sweden)

    William Kilembe

    Full Text Available BACKGROUND: Acute HIV infection (prior to antibody seroconversion represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent. METHODS: Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1 Antigen positive, antibody negative (acute infection; 2 Antigen positive, antibody positive; 3 Antigen negative, antibody positive; 4 Antigen negative, antibody negative; and 5 Antigen false positive, antibody negative (HIV uninfected. A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both. RESULTS: Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106, 1 (2.9% was detected by the Combo antigen component, 7 (20.6% others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18. Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9. One false positive Combo antibody result (1/30, 3.3% was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL. CONCLUSIONS: Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal.

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

    International Nuclear Information System (INIS)

    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)

  11. A novel inhibition ELISA for the detection and monitoring of Penicillium marneffei antigen in human serum.

    Science.gov (United States)

    Prakit, K; Nosanchuk, J D; Pruksaphon, K; Vanittanakom, N; Youngchim, S

    2016-04-01

    The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment. PMID:26838686

  12. Selective detection of bacterial layers with terahertz plasmonic antennas

    CERN Document Server

    Berrier, Audrey; Nonglaton, Guillaume; Bergquist, Jonas; Rivas, Jaime Gómez

    2012-01-01

    Current detection and identification of micro-organisms is based on either rather unspecific rapid microscopy or on more accurate complex, time-consuming procedures. In a medical context, the determination of the bacteria Gram type is of significant interest. The diagnostic of microbial infection often requires the identification of the microbiological agent responsible for the infection, or at least the identification of its family (Gram type), in a matter of minutes. In this work, we propose to use terahertz frequency range antennas for the enhanced selective detection of bacteria types. Several microorganisms are investigated by terahertz time-domain spectroscopy: a fast, contactless and damage-free investigation method to gain information on the presence and the nature of the microorganisms. We demonstrate that plasmonic antennas enhance the detection sensitivity for bacterial layers and allow the selective recognition of the Gram type of the bacteria.

  13. Bacterial histo-blood group antigens contributing to genotype-dependent removal of human noroviruses with a microfiltration membrane.

    Science.gov (United States)

    Amarasiri, Mohan; Hashiba, Satoshi; Miura, Takayuki; Nakagomi, Toyoko; Nakagomi, Osamu; Ishii, Satoshi; Okabe, Satoshi; Sano, Daisuke

    2016-05-15

    We demonstrated the genotype-dependent removal of human norovirus particles with a microfiltration (MF) membrane in the presence of bacteria bearing histo-blood group antigens (HBGAs). Three genotypes (GII.3, GII.4, and GII.6) of norovirus-like particles (NoVLPs) were mixed with three bacterial strains (Enterobacter sp. SENG-6, Escherichia coli O86:K61:B7, and Staphylococcus epidermidis), respectively, and the mixture was filtered with an MF membrane having a nominal pore size of 0.45 μm. All NoVLP genotypes were rejected by the MF membrane in the presence of Enterobacter sp. SENG-6, which excreted HBGAs as extracellular polymeric substances (EPS). This MF membrane removal of NoVLPs was not significant when EPS was removed from cells of Enterobacter sp. SENG-6. GII.6 NoVLP was not rejected with the MF membrane in the presence of E. coli O86:K61:B7, but the removal of EPS of E. coli O86:K61:B7 increased the removal efficiency due to the interaction of NoVLPs with the exposed B-antigen in lipopolysaccharide (LPS) of E. coli O86:K61:B7. No MF membrane removal of all three genotypes was observed when S. epidermidis, an HBGA-negative strain, was mixed with NoVLPs. These results demonstrate that the location of HBGAs on bacterial cells is an important factor in determining the genotype-dependent removal efficiency of norovirus particles with the MF membrane. The presence of HBGAs in mixed liquor suspended solids from a membrane bioreactor (MBR) pilot plant was confirmed by immune-transmission electron microscopy, which implies that bacterial HBGAs can contribute to the genotype-dependent removal of human noroviruses with MBR using MF membrane. PMID:27095709

  14. 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.

  15. Use of in vivo-induced antigen technology (IVIAT for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

    Directory of Open Access Journals (Sweden)

    Lu Chengping

    2009-09-01

    Full Text Available Abstract Background Streptococcus suis serotype 2 (SS2 is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, in vivo-induced antigen technology (IVIAT, an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated in vivo during SS2 infection. Results Convalescent-phase sera from pigs infected with SS2 were pooled, adsorbed against in vitro antigens, and used to screen SS2 genomic expression libraries. Upon analysis of the identified proteins, we were able to assign a putative function to 40 of the 48 proteins. These included proteins implicated in cell envelope structure, regulation, molecule synthesis, substance and energy metabolism, transport, translation, and those with unknown functions. The in vivo-induced changes in the expression of 10 of these 40 genes were measured using real-time reverse transcription (RT-PCR, revealing that the expression of 6 of the 10 genes was upregulated in the in vivo condition. The strain distribution of these 10 genes was analyzed by PCR, and they were found in the most virulent SS2 strains. In addition, protein sequence alignments of the newly identified proteins demonstrate that three are putative virulence-associated proteins. Conclusion Collectively, our results suggest that these in vivo-induced or upregulated genes may contribute to SS2 disease development. We hypothesize that the identification of factors specifically induced or upregulated during SS2 infection will aid in our understanding of SS2 pathogenesis and may contribute to the control SS2 outbreaks. In addition, the proteins identified

  16. Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels.

    Science.gov (United States)

    Sjöling, Åsa; Sadeghipoorjahromi, Leila; Novak, Daniel; Tobias, Joshua

    2015-03-01

    Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples. PMID:25542594

  17. 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.

  18. Development of an antigen-capture ELISA for the detection of avian leukosis virus p27 antigen.

    Science.gov (United States)

    Yun, Bingling; Li, Delong; Zhu, Haibo; Liu, Wen; Qin, Liting; Liu, Zaisi; Wu, Guan; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei; Gao, Yulong

    2013-02-01

    An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs. PMID:23201286

  19. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

    Science.gov (United States)

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Ling, Shigan; Zhang, Heqiu

    2011-01-01

    A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies. PMID:21029749

  20. Functionalized nanowire-based antigen detection using frequency-based signals.

    Science.gov (United States)

    Nguyen, Thanh Cong; Qiu, Wanzhi Z; Skafidas, Efstratios

    2012-01-01

    As part of clinical diagnosis, a clinician is required to detect disease causing antigens, bacteria, or viruses in serum, saliva, or other biological samples. Usually, this requires the sample to be sent to a pathology laboratory for analysis. Silicon nanowires can be made into sensitive molecular sensors. When being functionalized with antibodies, they are capable of detecting femto molar concentrations of antigens in real time. Biological molecules at a pH different from their isoelectric point exhibit a net charge. When an antigen attaches to the antibody on the nanowire, the net charged on the antigen displaces free carriers in the nanowire changing its conductance. To date, detection methods have been based upon directly measuring the change in dc conductance. This is difficult and requires sensitive low-noise amplifiers and high-resolution analog-to-digital converters. This is not ideal for low-cost and highly integrated systems. In this paper, it is demonstrated that nanowires exhibit an ac-transfer function that resembles that of a high-pass filter. To the authors' knowledge, this is the first time this effect has been reported. Furthermore, it is illustrated that as molecules with a higher net charge attach to the nanowire and displace more charge carriers within the nanowire channel, the filter's corner frequency decreases. This property of silicon nanowires is exploited to build a low-cost real-time antigen detection system. PMID:21968710

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

    International Nuclear Information System (INIS)

    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 105 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)

  2. Detection of HIV-1 antigen based on magnetic tunnel junction sensor and magnetic nanoparticles

    CERN Document Server

    Li, L; Zhou, Y; Pong, P W T

    2016-01-01

    In recent years, it is evidenced that the individuals newly infected HIV are transmitting the virus prior to knowing their HIV status. Identifying individuals that are early in infection with HIV antibody negative (window period) remains problematic. In the newly infected individuals, HIV antigen p24 is usually present in their serum or plasma 7-10 days before the HIV antibody. After antibody production initiates, the p24 antigen is bound into immune complexes. That means the detectable p24 antigens in serum/plasma are short-lived, and their amount is in the pg/ml range. Thus, a rapid quantitative bio-detection system with high-sensitivity is required to achieve early disease diagnosis. Magnetoresistive (MR) biosensor with ultra-high sensitivity possesses great potential in this area. In this study, a p24 detection assay using MgO-based magnetic tunnel junction (MTJ) sensor and 20-nm magnetic nanoparticles is reported.

  3. Detection of Intracellular Bacterial Communities in Human Urinary Tract Infection

    OpenAIRE

    Opal, Steven M

    2007-01-01

    Steven Opal reviews the phenomenon of bacterial communities and discusses the role played by bacterial communication and cooperation in host-pathogen interactions, particularly in urinary tract infection.

  4. Rapid detection of antibodies against cytomegalovirus induced immediate early and early antigens by an enzyme linked immunosorbent assay.

    OpenAIRE

    M. Musiani; Carpi, C; Zerbini, M

    1984-01-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibodies against cytomegalovirus induced immediate early antigens and early antigens was developed using purified nuclear antigens and was compared with the indirect immunofluorescence test. The tests were comparable in their ability to detect positive and negative sera, and antibody titres determined by both assays were similar. The use of ELISA for the detection of antibodies against cytomegalovirus induced immediate early and ear...

  5. Radioimmunoassay for the detection of hepatitis e antigen (HBeAG) and antibody (ANTI-HBe)

    International Nuclear Information System (INIS)

    A solid phase micro-immunoradiometric assay (micro-SPIRA) for the detection of hepatitis e antigen (HBeAg) and antibody has been developed. Chimpanzee anti-HBe/2 was developed by repeated immunizations with purified antigen containing HBeAg/1 and HBeAg/2. An anti-HBe/2 titer of 1:4 was determined by immunodiffusion (ID) analysis. Anti-HBe/1 was not detected. The anti-HBe IgG used in the assay was purified from plasma by a combination of DEAE-cellulose and affinity chromatography. The sensitivity of the micro-SPIRA for antigen and antibody was 193 ng/ml and 65 ng/ml, respectively. By comparing relative endpoint titers obtained by ID to micro-SPIRA, it was determined that micro-SPIRA for antigen and antibody is 320 and greater than 1300 times more sensitive, respectively, than ID. The specificity of the assay was ascertained by the examination of various non-B specimens. The application of the assay to a panel of 50 hepatitis B surface antigen (HBsAg)-positive specimens resulted in an increase in positivity of 18% for antigen and 22% for antibody

  6. 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

  7. Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

    OpenAIRE

    Anderson, L J; Tsou, C; Parker, R. A.; Chorba, T L; Wulff, H; Tattersall, P; Mortimer, P P

    1986-01-01

    Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscop...

  8. Detection of bacterial pathogens in environmental samples using DNA microarrays.

    Science.gov (United States)

    Call, Douglas R; Borucki, Monica K; Loge, Frank J

    2003-05-01

    Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes. PMID:12654494

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

    OpenAIRE

    Kazuki Morioka; Katsuhiko Fukai; Kazuo Yoshida; Rie Kitano; Reiko Yamazoe; Manabu Yamada; Tatsuya Nishi; Toru Kanno

    2015-01-01

    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 det...

  10. Radioimmunoassay in the detection of the hepatitis Be antigen/antibody system in asymptomatic carriers of hepatitis B surface antigen

    International Nuclear Information System (INIS)

    A radioimmunoassay for hepatitis e antigen (HBeAg) and antibody to e (anti-HBe) was developed and sera of 71 asymptomatic chronic carriers of hepatitis B surface antigen (HBsAg), in 44 of whom liver biopsy was obtained, were tested. In addition, testing for Dane particle associated DNA polymerase activity was performed in all sera. HBeAg was detected in 14 subjects (19.7%) and anti-HBe in 46 (64.8%). The highest proportion of HBeAg positivity (40%) was found among carriers with histological evidence of chronic hepatitis, whereas anti-HBe was present in 80% of carriers with normal liver histology, in 58% of carriers with non-specific reactive hepatitis and in 60% of carriers with chronic liver lesions. DNA polymerase activity was present in 92.8% of sera positive for HBeAg, in 13% of sera positive for anti HBe, and in 9% of sera negative for both markers. Our results demonstrate that not all HBsAg carriers reactive to HBeAg show evidence of chronic hepatitis nor, conversely, that anti-HBe is invariably associated with the healthy carrier state of HBsAg. Finally, circulating Dane particles, as revealed by the presence of serum specific DNA polymerase activity, may also be present in anti-HBe positive sera other than those of some HBsAg carriers lacking both HBeAg and anti-HBe. (orig.)

  11. 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

    -induced release of sVEGF and sVEGFR1 from whole blood in vitro. MATERIAL AND METHODS: Sixty-one patients with abdominal diseases undergoing five different surgical procedures were included in the study. Blood samples were drawn from patients before and after the operation. White blood cells and platelets were......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...... counted, and plasma sVEGF and sVEGFR1 were determined. Whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lipopolysaccharides or protein A) and sVEGF and sVEGFR1 levels were subsequently determined in the supernatants. RESULTS: Neither sVEGF nor sVEGFR1...

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

    International Nuclear Information System (INIS)

    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.)

  13. Subdominant antigens in bacterial vaccines: Am779 is subdominant in the anaplasma marginale outer membrane vaccine but does not associate with protective immunity

    Science.gov (United States)

    Identification of specific antigens responsible for the ability of complex immunogens to induce protection is a major goal in development of bacterial vaccines. Much of the investigation has focused on highly abundant and highly immunodominant outer membrane proteins. Recently however, genomic and p...

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

    OpenAIRE

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

    2004-01-01

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

  15. 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.

  16. Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata

    Science.gov (United States)

    Bakırcı, Serkan; Tait, Andrew; Kinnaird, Jane; Eren, Hasan

    2016-01-01

    Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals. PMID:27270235

  17. Resonant mass biosensor for ultrasensitive detection of bacterial cells

    Science.gov (United States)

    Gupta, Amit; Akin, Demir; Bashir, Rashid

    2003-01-01

    This paper describes a surface micromachined cantilever beam based oscillator detector for biological applications. This study used a novel microfabrication technique of merged epitaxial lateral overgrowth (MELO) and chemical mechanical polishing (CMP) to fabricate thin, low stress, single-crystal silicon cantilever beams. Vibration spectra of the cantilever beams, excited by thermal and ambient noise, was measured in air using a Digital Instrument Dimension 3100 Series scanning probe microscope (SPM). The cantilever beams were calibrated by obtaining the spring constant using the added-mass method. The sensors were used to detect the presence of Listeria innocua bacteria by applying increasing concentration of bacteria suspension on the same cantilever beam and measuring the resonant frequency changes in air. Cantilever beams were also used to detect the mass of the adsorbed antibodies and used to show selective capture of bacterial cells. The results indicate that the developed biosensor is capable of rapid and ultra-sensitive detection of bacteria and promises significant potential for enhancement of microbiological research and diagnostics.

  18. Detection of an Antigenic Group 2 Coronavirus in an Adult Alpaca with Enteritis▿

    OpenAIRE

    Genova, Suzanne G.; Streeter, Robert N.; Simpson, Katharine M.; Kapil, Sanjay

    2008-01-01

    Antigenic group 2 coronavirus was detected in a fecal sample of an adult alpaca by reverse transcription-PCR. The presence of alpaca coronavirus (ApCoV) in the small intestine was demonstrated by immune histochemistry with an antinucleocapsid monoclonal antibody that reacts with group 2 coronaviruses. Other common causes of diarrhea in adult camelids were not detected. We conclude that nutritional stress may have predisposed the alpaca to severe ApCoV infection.

  19. Prostate-specific antigen in the early detection of prostate cancer

    OpenAIRE

    Thompson, Ian M; Ankerst, Donna P.

    2007-01-01

    Throughout Canada, the United States and much of Europe, prostate-specific antigen (PSA) screening for prostate cancer has proliferated over the past 2 decades, leading to dramatic increases in detection rates of prostate cancer. Although it has unquestionably led to increased detection of cancer and a migration to lower-stage and -volume tumours, it is still unknown whether PSA screening significantly reduces mortality from prostate cancer. Often thought to be dichotomous (i.e., either norma...

  20. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

    OpenAIRE

    Bezeaud, A; Rosenswajg, M; Guillin, M C

    1989-01-01

    Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar,...

  1. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits.

    OpenAIRE

    Haque, R; Neville, L. M.; Hahn, P.; Petri, W A

    1995-01-01

    Humans are infected by two morphologically identical species of Entamoeba: Entamoeba histolytica causes amebic colitis and liver abscess, and Entamoeba dispar is noninvasive. Several weeks of culture and isoenzyme (zymodeme) analysis are required to differentiate E. histolytica from E. dispar. Here we report a field trial of commercial antigen detection kits designed to rapidly detect and differentiate E. histolytica from E. dispar in stool specimens. Stool specimens from 202 patients with di...

  2. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens.

    OpenAIRE

    Ungar, B L

    1990-01-01

    Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with crypto...

  3. Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

    Science.gov (United States)

    Pascho, R.J.; Goodrich, T.D.; McKibben, C.L.

    1997-01-01

    Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

  4. 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.

  5. Pneumolysin in urine: A rapid antigen detection method to diagnose pneumococcal pneumonia in children

    Directory of Open Access Journals (Sweden)

    Rajalakshmi B

    2002-01-01

    Full Text Available PURPOSE: Etiological diagnosis of pneumococcal pneumonia is difficult in small children in whom blood culture cannot be done or who have already been started on antibiotics. A simple technique which can be applied at the bedside or in the outpatient department may help in obviating this problem. Detection of pneumolysin, a product of invasive pneumococci is being exploited as a diagnostic tool. METHODS: An attempt was made to detect this protein in urine of seventy children, clinically suspected and radiologically diagnosed cases of pneumonia. Seventy age and sex matched controls were included in the study. Purified pneumolysin was prepared from clinical isolates of invasive pneumococcal infections. This was used to raise polyclonal antisera in rabbits. The antisera was used to sensitise Cowan 1 Staphylococcus aureus (CoA. A slide agglutination was performed with 25 µL urine and equal quantity of the reagent. RESULTS: Results were compared with CoA reagent sensitised with antisera raised against a genetically derived pneumolysoid and capsular polysaccharide for antigen detection in the urine. Pneumolysin could be detected in 42.9% (30/70 urine samples from cases with pneumonia by the genetically derived antigen and in 37.1% samples by the in house prepared antigen, in contrast to 2.1% in healthy controls and 4.2% in children with infections other than pneumonia. The result was statistically significant. Detection of pneumolysin was slightly better than detection of capsular polysaccharide antigen in urine although the result was not statistically significant. Blood culture proved to be positive in only 29.5% cases. CONCLUSIONS: Pneumolysin detection in urine showed promising results and was found to be simple and rapid. It will help in quickening the diagnosis of pneumococcal pneumonia.

  6. [Immunotherapy by polyvalent bacterial antigen (Broncasma Berna) in the prevention of pneumonia in the elderly].

    Science.gov (United States)

    Suzuki, K; Yamamoto, K; Adachi, S; Yamamoto, T

    1989-03-01

    Pneumonia in the elderly often occurs repeatedly, and the mortality rate from pneumonia continues to remain high today despite the usual use of antibacterial chemotherapy. Therefore, we conducted immunotherapy using a polyvalent bacterial vaccine (broncasma Berna). We treated 54 elderly patients with Broncasma Berna, containing chief bacterial pathogens responsible for pneumonia in the elderly. Clinical results obtained during 2 years were compared with those of 18 subjects not treated with Broncasma Berna. The survival rate was 64.8% for the group treated with Broncasma Berna and 50% for the group not treated. The frequency of contraction of pneumonia decreased significantly in the group treated. Clinical efficacy was obtained in 63% of the group treated to prevent pneumonia. The death rate from pneumonia was 17.6% for the group treated and 44.4% for the group not treated. Immunologically, reinforcement in humoral and cellular immunities was indicated by immunoglobulin values, positive tuberculin skin tests, and an increase in lymphocyte stimulation index values for Broncasma Berna. Significant pathogens in sputum disappeared or decreased in 6 (54.6%) out of 11 patients. Side effects such as pain or redness at the site of injection were observed in 6 patients. From the above results, it may be concluded that Broncasma Berna can be considered to be effective as a long-term immunoprophylactic agent in the prevention of pneumonia in the elderly. PMID:2504831

  7. Antigens of Trypanosoma cruzi detected by different classes and subclasses of antibodies.

    Science.gov (United States)

    Araujo, F G; Heilman, B; Tighe, L

    1984-01-01

    The kinetics of the appearance of specific IgM and of subclasses of IgG antibodies following infection of mice with Trypanosoma cruzi and the antigens of amastigotes and epimastigotes recognized by these antibodies were investigated by using the indirect immunofluorescent antibody test and the protein transfer technique. IgM and IgG2 antibodies were detected almost at the same time and peaked on day 30 and 40 of infection respectively. On day 150 of infection IgM antibodies were barely detectable whereas IgG2 antibodies were still at a high titre. IgG3 and IgG1 antibodies were first detected on days 20 and 30, peaked on days 30 and 50 respectively, and were still detected at low titres on day 150 of infection. The immunofluorescent test with each antibody revealed differences in the patterns of the fluorescent staining of the organisms, particularly with amastigotes. These differences were most striking with IgG3 antibodies. Fluorescent staining with IgM or IgG1 was localized mostly on one or two poles of the amastigotes; with IgG2 it was over the entire body of either amastigotes or epimastigotes; and with IgG3 it was in the form of very small spots over the entire body of organisms of both stages. The Western blots revealed that each antibody apparently recognized the same antigens in both the epimastigote and amastigote antigen preparations. The 90 Kd MW antigen of epimastigotes as well as two antigens of MW 92 Kd and 90 Kd of amastigotes were recognized by each of the antibodies examined. PMID:6438837

  8. Performance evaluation of four rapid antigen tests for the detection of Respiratory syncytial virus.

    Science.gov (United States)

    Jung, Bo Kyeung; Choi, Sung Hyuk; Lee, Jong Han; Lee, JungHwa; Lim, Chae Seung

    2016-10-01

    Rapid identification of Respiratory syncytial virus (RSV) is important in the management of infected patients. Rapid diagnostic tests (RDT) are widely used for this purpose. This study aimed to evaluate the clinical performance of four RSV antigen tests including the BinaxNow RSV Card test, SD Bioline RSV test, BD Veritor RSV test, and Humasis RSV antigen test in comparison with real-time RT-PCR as the reference method. Nasopharyngeal swabs were collected from 280 patients with symptoms of lower respiratory tract infection and stored at -80°C. All swabs were tested for RSV using four rapid antigen tests and real time RT-PCR. The sensitivity of the BinaxNow RSV Card test, SD Bioline RSV test, BD Veritor RSV test, and Humasis RSV Antigen tests were 62.5%, 61.3%, 65.0%, and 67.5% for RSV A, and 61.3%, 65.0%, 61.3%, and 67.5% for RSV B compared to real time RT-PCR, respectively. The specificity of BD Veritor RSV test was 95.8% and those of the other three RDTs was 100%. Commercial RSV antigen detection assays are useful tools for the rapid diagnosis of RSV infection. However, confirmatory testing is always recommended. J. Med. Virol. 88:1720-1724, 2016. © 2016 Wiley Periodicals, Inc. PMID:26990654

  9. 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

    -dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8......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...

  10. Detection of avian influenza antigens in proximity fiber, droplet, and optical waveguide microfluidics

    Science.gov (United States)

    Yoon, Jeong-Yeol; Heinze, Brian C.; Gamboa, Jessica; You, David J.

    2009-05-01

    Virus antigens of avian influenza subtype H3N2 were detected on two different microfluidic platforms: microchannel and droplet. Latex immunoagglutination assays were performed using 920-nm highly carboxylated polystyrene beads that are conjugated with antibody to avian influenza virus. The bead suspension was merged with the solutions of avian influenza virus antigens in a Y-junction of a microchannel made by polydimethylsiloxane soft lithography. The resulting latex immunoagglutinations were measured with two optical fibers in proximity setup to detect 45° forward light scattering. Alternatively, 10 μL droplets of a bead suspension and an antigen solution were merged on a superhydrophobic surface (water contact angle = 155°), whose movement was guided by a metal wire, and 180° back light scattering is measured with a backscattering optical probe. Detection limits were 0.1 pg mL-1 for both microchannel with proximity fibers and droplet microfluidics, thanks to the use of micro-positioning stages to help generate reproducible optical signals. Additionally, optical waveguide was tested by constructing optical waveguide channels (filled with mineral oil) within a microfluidic device to detect the same light scattering. Detection limit was 0.1 ng mL-1 for an optical waveguide device, with a strong potential of improvement in the near future. The use of optical waveguide enabled smaller device setup, easier operation, smaller standard deviations and broader linear range of assay than proximity fiber microchannel and droplet microfluidics. Total assay time was less than 10 min.

  11. Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

    Science.gov (United States)

    Kang, Yu; Gohlke, Ulrich; Engström, Olof; Hamark, Christoffer; Scheidt, Tom; Kunstmann, Sonja; Heinemann, Udo; Widmalm, Göran; Santer, Mark; Barbirz, Stefanie

    2016-07-27

    Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system. PMID:27045683

  12. Natural Killer Cells and Helicobacter pylori Infection: Bacterial Antigens and Interleukin-12 Act Synergistically To Induce Gamma Interferon Production

    Science.gov (United States)

    Yun, Cheol H.; Lundgren, Anna; Azem, Josef; Sjöling, Åsa; Holmgren, Jan; Svennerholm, Ann-Mari; Lundin, B. Samuel

    2005-01-01

    Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-γ in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-γ), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-γ by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-γ by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor β2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-γ when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection. PMID:15731046

  13. 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. PMID:16517916

  14. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System.

    Science.gov (United States)

    van Coevorden-Hameete, Marleen H; Titulaer, Maarten J; Schreurs, Marco W J; de Graaff, Esther; Sillevis Smitt, Peter A E; Hoogenraad, Casper C

    2016-01-01

    Autoimmune encephalitis (AIE) is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients' serum or CSF therefore has serious consequences for the patients' treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: (1) Immunohistochemistry (IHC) and immunofluorescence on rat/primate brain sections; (2) Immunocytochemistry (ICC) of living cultured hippocampal neurons; and (3) Cell Based Assay (CBA). In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs. PMID:27303263

  15. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Marleen eVan Coevorden-Hameete

    2016-05-01

    Full Text Available Autoimmune encephalitis (AIE is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients’ serum or CSF therefore has serious consequences for the patients’ treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: 1 Immunohistochemistry and immunofluorescence on rat/ primate brain sections, 2 Immunocytochemistry of living cultured hippocampal neurons, 3 Cell Based Assay (CBA. In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs.

  16. Comparative efficacy of antigen and antibody detection tests for human trichinellosis

    International Nuclear Information System (INIS)

    Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation

  17. 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.

  18. Detection of HBs antigen in routine paraffin embedded liver tissue by enzyme-labelled antibody technique

    Directory of Open Access Journals (Sweden)

    Tsuji,Takao

    1976-02-01

    Full Text Available HB surface antigen (HBs Ag was detected using the enzyme-labelled antibody technique on routinely processed liver biopsy material fixed in Bouin's fixative and embedded in paraffin. Of 85 examined specimens, 45 cases were HBs Ag positive by both the immunofluorescent test and the enzyme labelled antibody technique. The remaining 40 cases were negative by both techniques. The specificity of HBs Ag detected by the enzyme-labelled antibody technique was confirmed by the blocking test using guinea pig specific HBs antibody. The results indicate that the enzyme-labelled antibody technique may be useful for detecting HBs Ag on routine paraffin sections.

  19. Procalcitonin for detecting community-acquired bacterial pneumonia

    OpenAIRE

    Devi Gusmaiyanto; Finny Fitry Yani; Efrida Efrida; Rizanda Machmud

    2016-01-01

    Background Pneumonia is a major cause of morbidity andmortality in children under five years of age. Pneumonia can be ofbacterial or viral origin. It is difficult to distinguish between thesetwo agents based on clinical manifestations, as well as radiologicaland laboratory examinations. Furthermore, bacterial cultures taketime to incubate and positive results may only be found in 10-30%of bacterial pneumonia cases. Procalcitonin has been used as amarker to distinguish etiologies, as bacterial...

  20. The detection of canine autoantibodies to thyroid antigens by enzyme-linked immunosorbent assay, hemagglutination and indirect immunofluorescence.

    OpenAIRE

    Haines, D M; Lording, P M; Penhale, W. J.

    1984-01-01

    Autoantibodies to thyroid antigens in serums from 34 clinically hypothyroid dogs were detected by various methods. Antibodies to thyroglobulin were detectable by enzyme-linked immunosorbent assay (ELISA) in 59%, by chromic chloride hemagglutination in 53% and by indirect immunofluorescence on formalin-fixed, paraffin-embedded, trypsin-digested thyroid tissue in 73% of samples. Antibody to thyroid microsomal antigen was detectable by ELISA in 29% of serums. Indirect immunofluorescence showed c...

  1. Biomimetic/Optical Sensors for Detecting Bacterial Species

    Science.gov (United States)

    Homer, Margie; Ksendzov, Alexander; Yen, Shiao-Pin; Ryan, Margaret; Lazazzera, Beth

    2006-01-01

    Biomimetic/optical sensors have been proposed as means of real-time detection of bacteria in liquid samples through real-time detection of compounds secreted by the bacteria. Bacterial species of interest would be identified through detection of signaling compounds unique to those species. The best-characterized examples of quorum-signaling compounds are acyl-homoserine lactones and peptides. Each compound, secreted by each bacterium of an affected species, serves as a signal to other bacteria of the same species to engage in a collective behavior when the population density of that species reaches a threshold level analogous to a quorum. A sensor according to the proposal would include a specially formulated biomimetic film, made of a molecularly imprinted polymer (MIP), that would respond optically to the signaling compound of interest. The MIP film would be integrated directly onto an opticalwaveguide- based ring resonator for optical readout. Optically, the sensor would resemble the one described in Chemical Sensors Based on Optical Ring Resonators (NPO-40601), NASA Tech Briefs, Vol. 29, No. 10 (October 2005), page 32. MIPs have been used before as molecular- recognition compounds, though not in the manner of the present proposal. Molecular imprinting is an approach to making molecularly selective cavities in a polymer matrix. These cavities function much as enzyme receptor sites: the chemical functionality and shape of a cavity in the polymer matrix cause the cavity to bind to specific molecules. An MIP matrix is made by polymerizing monomers in the presence of the compound of interest (template molecule). The polymer forms around the template. After the polymer solidifies, the template molecules are removed from the polymer matrix by decomplexing them from their binding sites and then dissolving them, leaving cavities that are matched to the template molecules in size, shape, and chemical functionality. The cavities thus become molecular-recognition sites

  2. TO EVALUATE THE ROLE OF NS1 ANTIGEN FOR EARLY DETECTION OF DENGUE FEVER

    OpenAIRE

    Chithambaram; Kushal D

    2014-01-01

    In India, Dengue epidemics are becoming more frequent and are straining the limited resources of the public health system. Diagnosing dengue early is challenging because the initial symptoms of dengue infection are often non-specific. It is for this reason diagnosing dengue infection early becomes important. AIM: To evaluate the role of NS1 antigen for early detection of dengue fever. SETTING AND DESIGN: Prospective Study done in a tertiary care hospital between January 20...

  3. Response of human lymphocytes to PHA and tumour-associated antigens as detected by fluorescence polarization.

    OpenAIRE

    Balding, P.; Light, P A; Preece, A. W.

    1980-01-01

    Fluorescence polarization measurement during the progress of fluorochromasia has been used to study the response of human lymphocytes to phytohaemagglutinin (PHA) and to tumour-associated antigens, as a basis for the detection of malignant disease. Polarization (P) values of both stimulated and unstimulated lymphocytes decreased with increasing intracellular fluorescence intensity, and with the duration of the fluorochromatic reaction. When these effects were taken into account, there was no ...

  4. An enzyme-linked immunosorbent assay for the detection of Entamoeba histolytica antigens in faecal material.

    Science.gov (United States)

    Grundy, M S; Voller, A; Warhurst, D

    1987-01-01

    This paper describes a method for the detection of Entamoeba histolytica antigens in stool samples using a multi-layer ELISA. The method is sensitive and specific, showing no interference with other intestinal parasites, e.g. E. coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii, Hymenolepis nana, Giardia lamblia, Trichomonas and Ascaris. The method provides a rapid and simple screening assay for E. histolytica infections and should assist in diagnosis and epidemiological studies of the disease. PMID:2895514

  5. A COMPARATIVE STUDY OF BLOOD SMEAR, QBC AND ANTIGEN DETECTION FOR DIAGNOSIS OF MALARIA

    OpenAIRE

    Suthar, Dr. Mitesh N.; Mevada, Dr. Amita K.; Pandya, Dr. Neha H.; Desai, Dr. Kinnar S.; Patel, Dr. Vaishali; Goswami, Dr. Toral

    2013-01-01

    Background & objectives:Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. Microscopy has been the Gold standard for malaria diagnosis for decades. We made an attempt to compare blood smear, Quantitative Buffy Coat (QBC) and rapid antigen detection methods for the rapid diagnosis of malaria.Methods & materials:A Cross sectional prospective study was conducted for 6 months in G.G.Hospital, Jamnagar. A total number of 90 hospitalized...

  6. Detection of Chikungunya Virus Antigen by a Novel Rapid Immunochromatographic Test

    OpenAIRE

    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

    2014-01-01

    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 fro...

  7. 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.

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

    OpenAIRE

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

    2014-01-01

    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 pep...

  9. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System

    OpenAIRE

    Marleen eVan Coevorden-Hameete; Maarten eTitulaer; Marco eSchreurs; Esther ede Graaff; Peter eSillevis Smitt; Casper eHoogenraad

    2016-01-01

    Autoimmune encephalitis (AIE) is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients’ serum or CSF therefore has serious consequences for the patients’ treatment and follow-up and requires the availability of sensitive and specific diagnostic tests...

  10. Validity of an enzyme immunoassay for detection of Neisseria gonorrhoeae antigens.

    OpenAIRE

    Papasian, C J; Bartholomew, W R; Amsterdam, D

    1984-01-01

    An enzyme immunoassay (EIA; Gonozyme, Abbott Laboratories) for the antigenic detection of Neisseria gonorrhoeae in endocervical or urethral specimens was evaluated. EIA results were compared with results of conventional culture tests for N. gonorrhoeae. Specimens from 208 males (113 culture positive) and 252 females (72 culture positive) were tested. The sensitivity and specificity of EIA for specimens from males were 97.3 and 95.8%, respectively. The sensitivity and specificity of EIA for sp...

  11. Detection of Neisseria gonorrhoeae antigen by a solid-phase enzyme immunoassay.

    OpenAIRE

    Aardoom, H A; hoop, D', B.B.; Iserief, C O; Michel, M F; Stolz, E

    1982-01-01

    The Gonozyme test (Abbott Laboratories) is a new enzyme immunoassay for detecting Neisseria gonorrhoeae antigens in specimens from the urethra in men and the endocervix in women. To evaluate the usefulness of the assay 249 patients were investigated. The results obtained with the immunoassay were compared with those of culture and microscopy of Gram-stained smears. The sensitivity and specificity of the test were high in men with urethritis and acceptable in different groups of women. As the ...

  12. Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

    OpenAIRE

    Weare, J A; Robertson, E F; Madsen, G; Hu, R; Decker, R H

    1991-01-01

    Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a...

  13. 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; Herbert B. Tanowitz

    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.

  14. Detection of Histoplasma capsulatum Antigen in Panamanian Patients with Disseminated Histoplasmosis and AIDS▿

    OpenAIRE

    Gutierrez, Maria Eugenia; Canton, Alfredo; Connolly, Patricia; Zarnowski, Robert; Wheat, L. Joseph

    2008-01-01

    Histoplasmosis is a common endemic mycosis in the Americas, often causing severe disease in patients with AIDS. Antigen detection has become an important method for rapid diagnosis of histoplasmosis in the United States but not in Central or South America. Isolates from patients in the United States are predominantly found to be class 2 isolates when typed using the nuclear gene YPS3, while isolates from Latin America are predominantly typed as class 5 or class 6. Whether infection with these...

  15. Evaluation of a Candida Antigen Detection Method (Cand-Tec): Experience from a University Teaching Hospital

    OpenAIRE

    Anderson, Todd J.; Bryant, Heather E; Deirdre L Church

    1992-01-01

    The usefulness of a rapid latex agglutination method for the detection of Candida antigen (Cand-Tec; Ramco Laboratories. Texas) was retrospectively assessed in a university teaching hospital over a one year period. Patients were enrolled when the managing physician requested Cand-Tec testing for confirmation of possible invasive candidal infection. The majority of patients were critically ill; 56% were in the intensive care unit, and 30% subsequently died. Analyses were available from 79 pati...

  16. Antigen detection of rabies virus in brain smear using direct Rapid Immunohistochemistry Test

    OpenAIRE

    Damayanti R; Rahmadani I; Fitria Y

    2014-01-01

    Rabies is zoonotic disease caused by a fatal, neurotropic virus. Rabies virus is classified into the Genus of Lyssavirus under the yang family of Rhabdoviridae. Rabies affecting hot- blooded animals, as well as human. Dogs, cats, monkeys are the vectors or reservoirs for rabies and the virus was transmitted through the saliva after infected animal’s bites. The aim of this study was to conduct rapid diagnosis to detect rabies viral antigen in brain smear using immunohistochemical (IHC) method ...

  17. Development of Monoclonal Antibodies Suitable for Rabies Virus Antibody and Antigen Detection

    OpenAIRE

    Chander, Vishal; Singh, R.P.; Verma, P. C.

    2012-01-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the pro...

  18. Detection of cytomegalovirus in shell vial cultures by using a DNA probe and early nuclear antigen monoclonal antibody.

    OpenAIRE

    Scott, A A; K. A. Walker; Hennigar, L M; Williams, C H; Manos, J P; Gansler, T

    1988-01-01

    An in situ biotinylated DNA probe assay was evaluated as an adjunct to anti-cytomegalovirus early nuclear antigen indirect immunofluorescence and cytopathic effect on cytomegalovirus-infected monolayers in shell vial cultures. Viral infection was detected by early nuclear antigen indirect immunofluorescence at 24 h and by DNA probe assay and shell vial cytopathic effect at 5 days.

  19. Aerosol delivered radiolabeled antibodies to ectopic lung carcinoma antigens in scintigraphic tumour detection

    International Nuclear Information System (INIS)

    Biologically specific antibodies offer increased specificity of inhalation scintigraphy in pulmonary malignancy at present limited to detection of airways obstruction. Polypeptide ectopic antigens produced by epithelial lung tumours provide a targeting focus for radiolabeled antibodies. Image resolution using the intravenous route is poor due to the small proportion of the dose targeting to the tumor site and ineffective clearance of the background. The inhalation route minimizes non-specific distribution and utilizes mucociliary clearance as a contrast enhancement mechanism. Mucociliary deposition is cleared more effectively than alveolar deposition. Submicronic aerosol droplets produced by airlet nebulisers ensure peripheral airways penetration and deposition. Affinity purified polyclonal goat antibodies against calcitonin labeled with 150 MBq of Tc-99m are delivered to the lungs to produce dynamic images over 24 hours. Pure synthetic human calcitonin ensures production of monospecific antibodies that can be affinity purified. Labeling with Tc-99m by stannous chloride reduction is effected on the solid phase Sepharose beads. An antibody-antigen concentration gradient over a small distance can be provided with radiolabeled antibodies from aerosol deposition. Histologically neoplastic cells are typically separated from the mucous layer by bronchial mucosa and a thin uninvolved lamina. A localised high concentration of labile antigen is present in extra-cellular spaces at the tumour site. A study is progressing to determine if specific antibody-antigen agglutination contributes towards localised impairment of mucociliary clearance at tumour sites creating contrast

  20. Detection of circulating polysaccharide antigen of Schistosoma mansoni in hamster sera by crossed immunoelectrophoresis

    Directory of Open Access Journals (Sweden)

    Margareth Fernandes

    1988-04-01

    Full Text Available Crossed immunoelectrophoresis (IEC was used for detection of free and complexed circulating polisaccharide anodic antigen (AgCA of Schistosoma mansoni in sera of infected hamsters. An attempt was also done to detect AgCA in human sera from patients infected with S. mansoni. The conditions for isolation and detection of complexed AgCA were established. The sensitivity of IEC was increased by incorporation of 2% polyethylene glicol (PEG to the agarose and by maintaining the system at 4°C during the electrophoretic run. Free AgCA was detected in 12 and the complexed in 30 of the 37 hamsters sera analysed. Correlation between AgCA (free and complexed and the parasite load was observed. AgCA was not detected, under the experimental conditions used, in human sera from 7 patients in the acute and 23 in the chronic phase of infection.

  1. 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.

  2. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test

    Directory of Open Access Journals (Sweden)

    A.H.L. Bruning

    2016-05-01

    Full Text Available Clinically relevant diagnosis of human bocavirus 1 (HBoV1 is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days.

  3. Field Evaluation of an Antigen Detection Immunochromatographic Test for Diagnosis of Plasmodium falciparum Malaria in India

    OpenAIRE

    Yadav, R. S.; V. P. Sharma; Srivastava, H. C.

    1998-01-01

    An immunochromatographic test (ICT Malaria P. f.) based on the detection of Plasmodium falciparum HRP-II antigen was evaluated in Gujarat state, India. Whole blood of 148 clinically suspected malaria patients was tested blind by microscopy and ICT simultaneously. Compared with the examination of Giemsa stained thick blood films, ICT test was 99% specific and 92% efficient. It was 100% sensitive for detection of >5000 parasites/μL blood, 90% sensitive (95% CI 72-109) for 1001-5000 parasites/μL...

  4. Detection of Breda virus antigen and antibody in humans and animals by enzyme immunoassay.

    OpenAIRE

    Brown, D W; Beards, G M; Flewett, T. H.

    1987-01-01

    Enzyme immunoassays were developed for the detection of Breda virus antibody and antigen. Cattle sera collected in the United Kingdom were found to have a high prevalence of antibody (55%) to Breda virus when examined in a competitive enzyme-linked immunosorbent assay. A low prevalence of antibody was found in pigs (2.2%), and no antibody was found in sheep or goat sera. No antibody to either Breda virus or Berne virus was detected in human sera collected from veterinarians and farm workers. ...

  5. [Detections of hepatitis C virus RNA and NS3 antigen and their relation to liver histopathology].

    Science.gov (United States)

    Wang, F; Wang, S; Jin, L

    1995-11-01

    To detect the distribution of hepatitis C virus and investigate the pathogenesis mechanisms of the viral infection in the liver tissues of the patients with acute or chronic hepatitis C, we examined HCV antigen expression by using the murine monoclonal antibody against HCV C33c peptide in the paraffin-embedded liver tissues from 28 patients with acute or chronic hepatitis C. The NS3 antigen was detected in 85.7% (24/28) of all the biopsy specimens. The distribution and staining density of the antigen immunoreactive signal varied according to different types of patients and the regions in the liver sections, but they obviously had a topographical relationship with the inflammatory-necrosis areas such as fatty and ballooning degeneration and focal necrosis in the liver tissues of nearly all the patients. In addition, the localization of HCV RNA investigated by in situ hybridization assay in 20 liver tissues the above 28 biopsy HD in the Chinese. They also provide valuable data for HD molecular diagnosis, genetic counselling and genetic health. PMID:8697087

  6. Solid-phase RIA for the detection of subtype-specific hepatitis-B antigen determinants

    International Nuclear Information System (INIS)

    With sensitive methods like radioimmunoassay (RIA) and enzyme immunoassay (EIA), HBsAg can be detected already at a concentration of 2 ng/ml specific antigen protein. Until now, the HBsAg subtype determinants have usually been detected by migration electrophoresis with a detection limit of 1000 ng/ml and immunodiffusion (1D) with a detection limit of about 3000 ng/ml. As these methods are rather unsensitive, a great part of RIA positive sera could never be typified until now. The paper describes an RIA with labelled subtype-specific antibodies for a specific detection of the antigen determinants d and y even at only 10 ng/ml. In an investigation of 251 sera not typified so far, 11 sera had the subtype determinant y, and 65 sera could still not be typed. The amount of non-typfiable sera is thus reduced from 23.2 to 6.4%. The relative frequency of subtype adw against ayn in RIA-typified sera corresponded to the distribution obtained in the earlier method. In the RIA the adw /ayw ratio was 63:37, in the earlier method 60:40. (orig.)

  7. A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes

    International Nuclear Information System (INIS)

    Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favourably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants. (Auth.)

  8. 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.

  9. Procalcitonin for detecting community-acquired bacterial pneumonia

    Directory of Open Access Journals (Sweden)

    Devi Gusmaiyanto

    2015-03-01

    Full Text Available Background Pneumonia is a major cause of morbidity and mortality in children under five years of age. Pneumonia can be of bacterial or viral origin. It is difficult to distinguish between these two agents based on clinical manifestations, as well as radiological and laboratory examinations. Furthermore, bacterial cultures take time to incubate and positive results may only be found in 10-30% of bacterial pneumonia cases. Procalcitonin has been used as a marker to distinguish etiologies, as bacterial infections tend to increase serum procalcitonin levels. Objective To determine the sensitivity, specificity, positive predictive value and negative predictive value of procalcitonin in community-acquired bacterial pneumonia. Method This cross-sectional study was conducted in the Pediatric Health Department of Dr. M. Djamil Hospital, Padang. Subjects were selected by consecutive sampling. Procalcitonin measurements and PCR screening were performed on blood specimens from 32 pneumonia patients and compared. Results Of the 32 subjects, most were boys (56.25%, under 5 years of age (99%, and had poor nutritional status (68.75%. Using a cut-off point of 0.25 ng/mL, procalcitonin level had a sensitivity of 92%, specificity 50%, positive predictive value 88%, and negative predictive value 60% for diagnosing bacterial pneumonia. Using a cut-off point of 0.5 ng/mL, procalcitonin level had a specificity of 46%, specificity 83%, positive predictive value 91%, and negative predictive value 25%. Conclusion A cut-off point of 0.25 ng/mL of procalcitonin level may be more useful to screen for bacterial pneumonia than a cut-off point of 0.5 ng / mL. However, if the 0.25 ng/mL cut-off point is used, careful monitoring will be required for negative results, as up to 40% may actually have bacterial pneumonia. [Paediatr Indones. 2015;55:65-9.].

  10. Detection of bacterial endospores in soil by terbium fluorescence

    OpenAIRE

    Andrea Brandes Ammann; Linda Kölle; Helmut Brandl

    2011-01-01

    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 photoluminescenc...

  11. Procalcitonin for detecting community-acquired bacterial pneumonia

    Directory of Open Access Journals (Sweden)

    Devi Gusmaiyanto

    2016-06-01

    Full Text Available Background Pneumonia is a major cause of morbidity andmortality in children under five years of age. Pneumonia can be ofbacterial or viral origin. It is difficult to distinguish between thesetwo agents based on clinical manifestations, as well as radiologicaland laboratory examinations. Furthermore, bacterial cultures taketime to incubate and positive results may only be found in 10-30%of bacterial pneumonia cases. Procalcitonin has been used as amarker to distinguish etiologies, as bacterial infections tend toincrease serum procalcitonin levels.Objective To determine the sensitivity, specificity, positivepredictive value and negative predictive value of procalcitoninin community-acquired bacterial pneumonia.Method This cross-sectional study was conducted in thePediatric Health Department of Dr. M. Djamil Hospital, Padang.Subjects were selected by consecutive sampling. Procalcitoninmeasurements and PCR screening were performed on bloodspecimens from 32 pneumonia patients and compared.Results Of the 32 subjects, most were boys (56.25%, under 5years of age (99%, and had poor nutritional status (68.75%.Using a cut-off point of 0.25 ng/mL, procalcitonin level hada sensitivity of 92%, specificity 50%, positive predictive value 88%, and negative predictive value 60% for diagnosing bacterial pneumonia. Using a cut-off point of 0.5 ng/mL, procalcitonin level had a specificity of 46%, specificity 83%, positive predictive value 91%, and negative predictive value 25%.Conclusion A cut-off point of 0.25 ng/mL of procalcitonin level may be more useful to screen for bacterial pneumonia than a cutoff point of 0.5 ng / mL. However, if the 0.25 ng/mL cut-off point is used, careful monitoring will be required for negative results, as up to 40% may actually have bacterial pneumonia. [PaediatrIndones. 2015;55:65-9.].

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

    Directory of Open Access Journals (Sweden)

    Helena Lúcia Carneiro Santos

    2007-06-01

    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.

  13. Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

    Directory of Open Access Journals (Sweden)

    Hafez Heydari Zarnagh

    2015-04-01

    Full Text Available Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3 cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

  14. Pelacakan Secara Imunohistokimiawi Antigen Ekskretori-Sekretori pada Sapi Bali yang Terinfeksi Fasciola gigantica (IMMUNOHISTOCHEMICAL DETECTION OF EXCRET0RY-SECRETORY ANTIGENS IN BALI CATLLE INFECTED BY FASCIOLA GIGANTICA

    Directory of Open Access Journals (Sweden)

    Ida Bagus Oka Winaya

    2014-10-01

    Full Text Available In order to study the distribution of excretory-secretory (ES F. gigantica in liver tissue of infected balicattle a research was establisihed using monoclonal antibodies (mAbs againts ES antigens. Immortalmouse myeloma cells were fused with the lymphocytes derived from the spleen of mice that immunizedwith ES antigen. The mAbs were tested for their specificity by using enzyme linked immunosorbent assay(ELISA. Five specific mAbs againts ES antigens were isolated and two mAbs were used for immunodetectionof ES antigens in liver tissue of bali cattle. Immunohistochemical ES antigens were not detected in paraffinembeded tissue of negative confirmed fasciolosis samples. ES antigens was detected in hepatocytes andcytoplasm of bile duct epithelims in the bali cattle that infected with fasciolosis in moderate intensity.Therfore indicated that mAbs produced in this study are applicable for detecting ES antigens in bali cattleinfected by F. gigantica.

  15. 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.

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

    OpenAIRE

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

    2015-01-01

    Background 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...

  17. Limitations of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) for detection of filarial antigens in serum

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, R.G.; Alexander, E.; Adkinson, N.F. (Johns Hopkins Univ., Baltimore, MD (USA). School of Medicine); Hussain, R. (National Institutes of Health, Bethesda, MD (USA))

    1984-03-30

    The performance of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) was examined for quantitation of filarial antigens (Brugia malayi and Dirofilaria immitis) in serum from infected human and animal hosts and non-infected controls. Multiple PEG concentrations were employed to determine the level of non-specific binding (NSB) in non-exposed human sera (NEHS) containing no filarial antigen. The NSB observed when 3 different /sup 125/I-labelled IgG antibodies were added to 26 NEHS varied 3-fold and was correlated significantly with total serum IgM (r = 0.80, P < 0.005, n = 24) but not with serum IgA (r = 0.37) or IgG (r = 0.45). NSB levels were significantly reduced when a Fab'/sub 2/ fragment of the /sup 125/I-labelled antibody was used, but the correlation of NSB with total serum IgM remained significant (r = 0.57, P < 0.01). The presence of rheumatoid factor in NEHS sera also significantly increased NSB by an average of 3-fold. These effects eliminated the assay's ability to detect in sera from infected hosts filarial antigen the presence of which could be readily demonstrated by an immunoradiometric assay. The RIPEGA's precision (intra-assay coefficient of variation (CV) = 21% at 35% Bsub(max)) and reproducibility (inter-assay CV = 29% at 35% Bsub(max)) are less satisfactory than many alternative immunoassays.

  18. Anisakis antigens detected in fish muscle infested with Anisakis simplex L3.

    Science.gov (United States)

    Solas, M Teresa; García, Maria Luisa; Rodriguez-Mahillo, Ana I; Gonzalez-Munoz, Miguel; de las Heras, Cristina; Tejada, Margarita

    2008-06-01

    Anisakis simplex is a fish parasite that is a public health risk to those consuming raw or poorly cooked marine fish and cephalopods because of the possibility of becoming infested with live larvae. In humans, penetration of the larvae into the gastrointestinal track can cause acute and chronic symptoms and allergic anisakiasis. Excretion and secretion products released by the larvae are thought to play a role in migration through the tissues and induce an immunoglobulin E-mediated immune response. The aim of this preliminary study was to detect parasite antigens and allergens in fish tissues surrounding the migrating larvae. Hake and anchovy fillets were artificially parasitized with Anisakis larvae and stored in chilled conditions for 5 days. Larvae were evaluated for fluorescence, fish muscle tissue was examined with transmission electron microscopy, and immunohistochemical reactions of two rabbit polyclonal antisera against a parasite crude extract and the allergen Ani s 4 were recorded. Larvae immediately migrated into the fish muscle, and no emission of bluish fluorescence was observed. Fish muscle areas in contact with the parasite showed disruptions in the structure and inclusion of granules within sarcomeres. Both parasite antigens and the Ani s 4 allergen were located in areas close to the larvae and where sarcomere structure was preserved. These findings indicate that parasite antigens and allergens are dispersed into the muscle and might cause allergic symptoms such as dyspnea, vomiting, diarrhea, urticaria, angioedema, or anaphylaxis in some individuals sensitive to A. simplex. PMID:18592760

  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. Universal primer PCR with DGGE for rapid detection of bacterial pathogens.

    Science.gov (United States)

    Ji, Niannian; Peng, Bo; Wang, Guizhong; Wang, Sanying; Peng, Xuanxian

    2004-06-01

    A universal primer PCR (UPPCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens. The results show that this method is efficient at amplifying the conserved regions of bacterial 16S rRNA genes with universal primers and can detect causative bacterial pathogens rapidly. Six species of bacteria from fisheries (Pseudomonas fluorescens, Vibrio anguillarum, Aeromonas hydrophila, Vibrio fluvialis, Providencia rettgeri and Aeromonas sobria) were examined. Our results indicate that the approach we undertook can be adopted not only for axenic bacterial populations but also for mixed communities as well. Furthermore, we were able to achieve the rapid detection of multiple bacteria a single in sample. In addition, UPPCR-DGGE was shown to be better than previously reported UPPCR-single-stranded conformation polymorphism (SSCP)-based methods for the rapid detection of bacterial pathogens. PMID:15134888

  1. 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.

  2. 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.

  3. Multiplexed immunoassay for the rapid detection of anti-tumor-associated antigens antibodies.

    Science.gov (United States)

    Desmet, C; Le Goff, G C; Brès, J-C; Rigal, D; Blum, L J; Marquette, C A

    2011-07-21

    TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors). PMID:21666912

  4. Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

    Directory of Open Access Journals (Sweden)

    Elenice Stroparo

    2010-12-01

    Full Text Available Adenovirus (AdV respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF, and a specific nested polymerase chain reaction (PCR, to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%. In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.

  5. Detection of a human intracisternal A-type retroviral particle antigenically related to HIV

    Science.gov (United States)

    Garry, R. F.; Fermin, C. D.; Hart, D. J.; Alexander, S. S.; Donehower, L. A.; Luo-Zhang, H.

    1990-01-01

    Sjogren's syndrome is an autoimmune disease that is characterized by dryness of the mouth and eyes. The loss of salivary and lacrimal gland function is accompanied by lymphocytic infiltration. Because similar symptoms and glandular pathology are observed in certain persons infected with human immunodeficiency virus (HIV), a search was initiated for a possible retroviral etiology in this syndrome. A human intracisternal A-type retroviral particle that is antigenically related to HIV was detected in lymphoblastoid cells exposed to homogenates of salivary tissue from patients with Sjogren's syndrome. Comparison of this retroviral particle to HIV indicates that they are distinguishable by several ultrastructural, physical, and enzymatic criteria.

  6. Evaluation of an Enzyme Immunoassay for Detection of Dengue Virus NS1 Antigen in Human Serum▿

    OpenAIRE

    Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R. T.; Rodrigues, Sueli G.; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

    2006-01-01

    We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the ...

  7. Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

    OpenAIRE

    Kryger, P; Mathiesen, L R; Møller, A M; Aldershvile, J; Hansson, B G; Nielsen, J O

    1981-01-01

    An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with l...

  8. Immunohistologic detection of antigen related to primate type C retrovirus p30 in normal human placentas.

    OpenAIRE

    Maeda, S.; Mellors, R C; Mellors, J. W.; Jerabek, L. B.; Zervoudakis, I. A.

    1983-01-01

    This study reports the immunohistologic detection of SSAV/GaLV type C retrovirus p30-related antigen in unfixed cryostat sections of normal human term placentas by the indirect immunofluorescence method. Goat anti-SSAV p28 serum reacted specifically with 10 of 10 anatomic specimens of human placenta. Goat anti-GaLV p29 serum reacted similarly with 8 of 10 specimens. Goat anti-BaEV p28, anti-RD-114 p28, anti-FeLV p27, anti-R-MuLV p30, and anti-MPMV p27 gave no specific reaction with placenta. ...

  9. Detection and identification of bacterial DNA in serum from patients with acute pancreatitis

    OpenAIRE

    E. de Madaria; Martínez, J.; Lozano, B; L. Sempere; S. Benlloch; J. Such; Uceda, F; Francés, R; Pérez-Mateo, M

    2005-01-01

    Background and aims: Bacterial infections are common complications in patients with acute pancreatitis, and translocation of bacteria from the intestinal lumen is probably the first step in the pathogenesis of these infections. As blood cultures in afebrile patients are usually negative, more sensitive methods to investigate this hypothesis in patients are needed. Our group has recently developed a method to detect the presence of bacterial DNA in biological fluids, and we aimed to detect bac...

  10. Bioinformatic detection of horizontally transferred DNA in bacterial genomes

    OpenAIRE

    Langille, Morgan GI; Brinkman, Fiona SL

    2009-01-01

    We highlight a selection of recent research on computational methods and associated challenges surrounding the prediction of bacterial horizontal gene transfer. This research area continues to face controversy, but is becoming more critical as the importance of horizontal gene transfer in medically and ecologically important prokaryotic evolution is further appreciated.

  11. 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.

  12. Salmonella typhimurium infection in calves: specific immune reactivity against O-antigenic polysaccharide detectable in in vitro assays.

    OpenAIRE

    Robertsson, J A; Fossum, C; Svenson, S B; Lindberg, A A

    1982-01-01

    Peripheral blood lymphocytes collected from calves infected experimentally with Salmonella typhimurium (O antigens 4,5,12) or Salmonella sp. serotype dublin (O 9,12) were stimulated with various bacterial cell envelope components, and their [3H]thymidine incorporation was measured. It was found that peripheral blood lymphocytes from infected calves incorporated significantly more [3H]thymidine than peripheral blood lymphocytes from uninfected controls (P values ranged from less than 0.05 to l...

  13. A 125I-protein A-binding assay detecting antibodies to cell surface antigens

    International Nuclear Information System (INIS)

    A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were than investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells. (orig.)

  14. Enzyme-Linked Immunosorbent Assay Employing a Recombinant Antigen for Detection of Protective Antibody against Swine Erysipelas

    OpenAIRE

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-01-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher y...

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

    OpenAIRE

    Sofía Duque-Beltrán; Rubén Santiago Nicholls-Orejuela; Adriana Arévalo-Jamaica; Rafael Guerrero-Lozano; Sonia Montenegro; James, Mark A

    2002-01-01

    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 pu...

  16. 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. PMID:26596552

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

    International Nuclear Information System (INIS)

    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)

  18. Detection of dengue NS1 antigen using long-range surface plasmon waveguides.

    Science.gov (United States)

    Wong, Wei Ru; Sekaran, Shamala Devi; Adikan, Faisal Rafiq Mahamd; Berini, Pierre

    2016-04-15

    The non-structural 1 (NS1) protein of the dengue virus circulates in infected patients' blood samples and can be used for early diagnosis of dengue infection. In this paper, we present the detection of naturally-occurring dengue NS1 antigen in infected patient blood plasma using straight long-range surface plasmon waveguides. Three commercially-available anti-NS1 monoclonal antibodies were used for recognition and their performance was compared and discussed. A similar figure of merit to the one used in conventional dengue NS1 capture using an enzyme-linked immunosorbent assay (ELISA) was applied to our results. In general, the positive patient samples can be clearly differentiated from the negative ones and the results agree with those obtained using ELISA. The largest signal-to-noise ratio observed during the experiments was 356 and the best detection limit observed is estimated as 5.73 pg/mm(2). PMID:26599483

  19. Iridium Oxide Film-Enhanced Impedance Immunosensor for Rapid Detection of Carcinoembyronic Antigen

    Institute of Scientific and Technical Information of China (English)

    DING,Yan-Jun; WANG,Hua; JIANG,Jian-Hui; SHEN,Guo-Li; YU,Ru-Qin

    2007-01-01

    A simple, rapid and sensitive impedance immunosensor based on iridium oxide (IrOx) thin film for the detection of carcinoembyronic antigen (CEA) in human sera has been proposed. Gold electrode was electrochemically modified with IrOx thin film and simultaneously functionalized with protein A (PA) to bind anti-CEA antibodies in an orientated way. It has been found that the antibody loading amount was dependent on the PA concentration and the deposition time of IrOx matrix. Under the optimized experimental conditions, the electron transfer resistances obtained were linearly related to the CEA concentration ranging from 36.2 to 460.0 ng/mL, with a detection limit of 28.0 ng/mL. Analytical results of clinical samples from cancer patients show that the proposed immunoassay is reasonably comparable with the chemiluminescence immunoassay (CLIA), indicating the feasibility of using the proposed method for CEA immunoassay in clinical laboratory.

  20. Carcinoma-associated antigens

    International Nuclear Information System (INIS)

    This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125I-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)

  1. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    Directory of Open Access Journals (Sweden)

    Jake E Lowry

    Full Text Available Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA. All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence

  2. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    Science.gov (United States)

    Lowry, Jake E; Isaak, Dale D; Leonhardt, Jack A; Vernati, Giulia; Pate, Jessie C; Andrews, Gerard P

    2011-01-01

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of

  3. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    Science.gov (United States)

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples. PMID:26693887

  4. Detection of specific IgE antibodies to major and minor antigenic determinants in sera of penicillin allergic patients

    Institute of Scientific and Technical Information of China (English)

    赵永星; 乔海灵

    2003-01-01

    Objective To investigate the mechanism (s) of penicillins allergic reaction.Methods The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test.Results Nineteen of 32 patients (59.4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34.38%), followed by BPO-IgE (31.25%). The positive rate of FLUO-IgE was the lowest (15.63%). Of the total patient group, 53.13% were positive to one or more minor antigenic determinants, while 37.5% (12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63.16%) was significantly higher than observed in the anaphylactic shock group (38.5%, P<0.05).Conclusions The minor antigenic determinant was important in allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule.

  5. Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

    Science.gov (United States)

    A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

  6. 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.

  7. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    Science.gov (United States)

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. PMID:26899824

  8. 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.

  9. Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection

    Directory of Open Access Journals (Sweden)

    Datta S

    2010-01-01

    Full Text Available Purpose: This study was undertaken to evaluate the efficacy of NS1 antigen (Ag assay as an early marker for dengue virus (DV infection. Materials and Methods: Group I evaluated the performance of NS1 antigen (Ag assay in comparison to MAC-ELISA and their detection rate when performed together in a single sample. Six hundred acute/early convalescent sera were screened by both the assays. Group II evaluated the efficacy of a single assay in 30 acute phase sera of paediatric OPD patients screened only by NS1 Ag assay. Group III evaluated the specificity of NS1 assay in comparison to MAC-ELISA on 40 samples included as controls. Results: In Group I, 140 (23.3% and 235 (39.1% samples were positive by NS1 assay and MAC-ELISA respectively. The detection rate increased to 320 (53.3% when both the assays were used together on a single sample. NS1 Ag positivity varied from 71.42% to 28.4% in acute and early convalescent sera, conversely IgM detection rate was 93.61% and 6.38% in early convalescent and acute phase sera respectively (P < 0.0001. In Group II, 66.66% (20 samples were positive by NS1 assay. All the samples in Group III were negative showing 100% specificity of both the assays. Conclusion: NS1 Ag assay holds promise in early diagnosis of dengue infection. When used in combination with MAC-ELISA on a single sample it significantly improves the diagnostic algorithm without the requirement of paired sera.

  10. 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. PMID:26229437

  11. Detection of antibody-antigen reaction by silicon nitride slot-ring biosensors using protein G

    Science.gov (United States)

    Taniguchi, Tomoya; Hirowatari, Anna; Ikeda, Takeshi; Fukuyama, Masataka; Amemiya, Yoshiteru; Kuroda, Akio; Yokoyama, Shin

    2016-04-01

    Biosensors using ring resonators with silicon nitride (SiN) slot waveguides have been fabricated. The temperature coefficient of the resonance wavelength of the SiN resonator is 0.006 nm/°C, which is one order of magnitude smaller than that of Si. The sensitivity of the biosensor has been improved by using slot waveguide together with Si-binding protein (designated as Si-tag), which bonds to SiN or SiO2 surface, as an anchoring molecule to immobilize bioreceptors on the SiN rings in an oriented manner. Furthermore, the protein G, which strongly bonds to many kinds of mammalian antibodies only by mixing the antibody solution, is used to efficiently immobilize the antigen on the sensor surface. By means of these devises the sensitivity of the biosensor has been improved by factor of 10-100 compared with that of normal Si ring resonator sensors without slot. Then the detection of prostate specific antigen (PSA) with the sensitivity of ~1×10-8 g/ml, which is the concentration of strongly suspicious for the prostate cancer, has been achieved.

  12. Detection of heat stable mycobacterial antigen in cerebrospinal fluid by Dot-Immunobinding assay

    Directory of Open Access Journals (Sweden)

    Mathai A

    2003-01-01

    Full Text Available Background: Isolation of Mycobacterium tuberculosis in cerebrospinal fluid (CSF specimen in patients with tuberculous meningitis (TBM is infrequent and carries low sensitivity. Thus development of an alternative laboratory diagnostic test is essential for the early diagnosis and treatment of TBM. Objective: A simple, rapid Dot immunobinding assay (Dot-Iba, for the laboratory diagnosis of TBM is devised. This method minimizes the risk of handling infectious material in the laboratory. Method: The Dot-Iba was standardized with heat-inactivated M tuberculosis antigen (PPD. The heat-inactivated CSF from TBM and non-TBM patients was similarly assayed and it can detect antigen upto 1ng/ml in CSF. Result: A positive result was obtained in all the five culture positive patients with TBM and in 20/25 probable TBM. A negative result was obtained in 38/40 CSF from disease control group. The overall sensitivity and specificity of Dot-Iba was 83.3% and 95% respectively. Conclusion: Dot-Iba can be used as an adjunct for the laboratory diagnosis of TBM, particularly in culture negative TBM patients and also in those clinical situations where no laboratory tests are available to distinguish between TBM and partially treated pyogenic meningitis.

  13. Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens†

    OpenAIRE

    Chen, Yi-Wen; Wang, Hong; Hupert, Mateusz; Witek, Makgorzata; Dharmasiri, Udara; Pingle, Maneesh R.; BARANY, FRANCIS; Soper, Steven A.

    2012-01-01

    The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular proc...

  14. Detection of bacterial DNA in painful degenerated spinal discs in patients without signs of clinical infection

    OpenAIRE

    Fritzell, Peter; Bergström, Tomas; Welinder-Olsson, Christina

    2004-01-01

    A local inflammatory and potentially painful response, of which the ultimate cause is unknown, has been described in nervous tissues in contact with degenerated disc material in patients with low back and leg pain. With the rationale that a possible cause of such inflammation could be bacterial infection, we utilized PCR (polymerase chain reaction) amplification of the 16S rRNA (ribosomal RNA) gene followed by gene sequencing, to investigate whether bacterial DNA might be detected in the dege...

  15. Use of radiolabeled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning

    International Nuclear Information System (INIS)

    To determine whether tumors containing carcinoembryonic antigen could be detected by administration of a radiolabeled, affinity-purified, goat IgG having 70% immunoreactivity against carcinoembryonic antigen, 18 patients with a history of cancer of diverse histopathology received an average total dose of 1.0 mCi of 131I-labeled IgG. Total-body photoscans were performed with a gamma scintillation camera at various intervals after administration of the radioactive antibody. Ordinary photoscans proved difficult to interpret because of blood-pool background radioactivity, thus necessitating the computer subtraction of radioactive blood-pool agents from the antibody's 131I activity. Tumor location could be demonstrated at 48 hours after injection in almost all cases studied. The scans were negative in patients without demonstrable tumors or with tumors apparently devoid of carcinoembryonic antigen. Circulating antigen levels of up to 350 ng per milliliter did not prevent successful tumor imaging after injection of the radioantibody

  16. Detection of intracellular bacterial communities in human urinary tract infection.

    Directory of Open Access Journals (Sweden)

    David A Rosen

    2007-12-01

    Full Text Available BACKGROUND: Urinary tract infections (UTIs are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC. While UTIs are typically considered extracellular infections, it has been recently demonstrated that UPEC bind to, invade, and replicate within the murine bladder urothelium to form intracellular bacterial communities (IBCs. These IBCs dissociate and bacteria flux out of bladder facet cells, some with filamentous morphology, and ultimately establish quiescent intracellular reservoirs that can seed recurrent infection. This IBC pathogenic cycle has not yet been investigated in humans. In this study we sought to determine whether evidence of an IBC pathway could be found in urine specimens from women with acute UTI. METHODS AND FINDINGS: We collected midstream, clean-catch urine specimens from 80 young healthy women with acute uncomplicated cystitis and 20 asymptomatic women with a history of UTI. Investigators were blinded to culture results and clinical history. Samples were analyzed by light microscopy, immunofluorescence, and electron microscopy for evidence of exfoliated IBCs and filamentous bacteria. Evidence of IBCs was found in 14 of 80 (18% urines from women with UTI. Filamentous bacteria were found in 33 of 80 (41% urines from women with UTI. None of the 20 urines from the asymptomatic comparative group showed evidence of IBCs or filaments. Filamentous bacteria were present in all 14 of the urines with IBCs compared to 19 (29% of 66 samples with no evidence of IBCs (p < 0.001. Of 65 urines from patients with E. coli infections, 14 (22% had evidence of IBCs and 29 (45% had filamentous bacteria, while none of the gram-positive infections had IBCs or filamentous bacteria. CONCLUSIONS: The presence of exfoliated IBCs and filamentous bacteria in the urines of women with acute cystitis suggests that the IBC pathogenic pathway characterized in the murine model may occur in humans. The

  17. Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens

    Directory of Open Access Journals (Sweden)

    María Susana Imaz

    2008-06-01

    Full Text Available The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B, singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50, smear-negative pulmonary TB (pTBsm- (60 and extrapulmonary TB (51 and 214 control patients (mycobacteriosis (14, mycoses(14, leprosy(4, other underlying diseases (82 and healthy people (100]. The individual antigens ranged from 25% to 42% in sensitivity and from 93% to 96% in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55%. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76% sensitivity and 83% specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63% but with a good specificity (95%, even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70% of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.

  18. Detection of virus and cellular-determined antigens in situ using [125I]protein A and autoradiography

    International Nuclear Information System (INIS)

    The present paper describes the use of [125I]Protein A, isolated from Staphylococcus aureus, in detecting antigen-antibody complexes by autoradiography on single cells. The method is relatively quick, reproducibile, potentially more sensitive than immunofluorescence, and should be useful in combination with conventional radioimmuno-assays. The authors have used it to detect the cellular expression of IgM, kappa, lambda, and β2-micro globulin, as well as the expression of Epstein-Barr Virus (EBV) associated antigens expressed in human lymphoblastoid cell lines. (Auth.)

  19. Field evaluation of latex agglutination test for detecting urinary antigens in visceral leishmaniasis in Sudan.

    Science.gov (United States)

    El-Safi, S H; Abdel-Haleem, A; Hammad, A; El-Basha, I; Omer, A; Kareem, H G; Boelaert, M; Chance, M; Hommel, M

    2003-07-01

    A latex agglutination test to detect urinary antigens for visceral leishmaniasis (VL) was studied. In 204 patients with suspected VL, KAtex had a sensitivity of 95.2% with good agreement with microscopy smears but poor agreement with 4 different serology tests. It was also positive in 2 confirmed VL cases co-infected with HIV. In all K4tex-positive confirmed cases actively followed up after treatment, the test became negative 1 month after completion of treatment. While IC4tex had a specificity of 100% in healthy endemic and non-endemic controls, the direct agglutination test (DAT) was positive in 14% of the KAtex-negative healthy endemic controls. KAtex is a simple addition to the diagnostics of VL particularly at field level and as a complementary test for the diagnosis of VL in smear-negative cases with positive DAT results. PMID:15748081

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

    International Nuclear Information System (INIS)

    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 (r2 = 0.1) between OCA and CEA levels in these patients, as determined by radioimmunoassay

  1. 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.

  2. Solid phase radioimmunoassay for detection of malaria antigen. Comparison of monoclonal and polyclonal antibodies

    International Nuclear Information System (INIS)

    A solid phase competitive binding radioimmunoassay (RIA) was developed for the detection of Plasmodium falciparum in infected blood. A suspension of NP40 treated red blood cells was mixed with labelled antimalarial IgG, incubated and then added to malarial antigen coated microtitre plate. Antimalarial IgGs were purified either from high titre sera from individuals living in a malaria endemic area in Thailand or from a locally produced monoclonal antibody (MAB) which showed a bright generalized immunofluorescent staining pattern against all blood stages of P. falciparum, including gametocytes. This MAB reacted with 27 of 31 P. falciparum isolates from Thailand. Using dilution of red blood cells from in vitro cultures of P. falciparum, the test was found to detect parasites at levels equivalent to 13 and 2.2 parasites/106 red blood cells with labelled polyclonal IgG (PIgG) and labelled monoclonal IgG (MIgG), respectively. No false positive results were obtained among samples from non-malarial subjects. Of the samples that gave negative results upon microscopic examination, 50 and 35% were still positive with RIA using MIgG and PIgG, respectively. There was a correlation between RIA and the number of parasites, especially when MIgG was used. The results indicate that the IgG fraction of sera from individuals with natural acquired immunity to malaria showed a lower degree of sensitivity in parasite detection than the IgG from monoclonal antibody. (author)

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

    Directory of Open Access Journals (Sweden)

    Sarwitri Endah Estuningsih

    2006-10-01

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

  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

    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...

  5. Detection of malarial parasite by blood smear examination and antigen detection: A comparative study

    Directory of Open Access Journals (Sweden)

    Erumalla Naveen, Dimple Arora, Vinod Agarwal, Neelam sharma, Anuradha B, Vijay Durga S

    2013-01-01

    Full Text Available At present about 100 countries in the world are considered malarious, is thought to kill between 1.1 and 2.7 million people worldwide each year, of which about 1 million are children under the age of 5 years in these areas. Under ideal circumstances, the clinical suspicion of malaria would be confirmed by a laboratory test that is simple to perform, rapid, sensitive, specific and expensive. At the present time, no such test exists. The most common test for malaria diagnosis remains the microscopic examination of giemsa or the fields –stained blood smears. The test is based on the detection of Plasmodium falciparum specific histidine rich protein ii (hrp and a pan malarial species specific enzyme aldolose, produced by the respective parasites and released into the blood and the test is based on immune chromatography, the test is highly sensitive. Method: In this study included 100 patients, 60% of patients had history suggestive of malaria, another 40% gave the history of irregular fever; For each patient peripheral blood sample was collected, thin and thick smear blood films were made immediately after blood collection, stained with Leishman stain and examined for malaria parasite by light microscopy. Results: The blood films results indicated that 40 (20% patients were infected with malaria and the rest 171 (85.5% were malaria negative. Among positive patients Plasmodium vivax was detected in 24 cases (60% and Plasmodium falciparum in 10 cases (31% and 6 cases mixed infection (PV + PF (15% correspondingly, the Para HIT Test results indicated that 29 (14.5% of the patient sample were positive for malaria parasites and 171 (85.5 were malaria negative out 29 patients cases. Infection with Plasmodium vivax accounted for 17 (58.6% while infection with Plasmodium falciparum accounted for 9 (25% and 3 (1.3% with mixed infection of Plasmodium vivax and Plasmodiumfalciparum

  6. 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

  7. [Elaboration of new adjuvant lipid-saponin complex and its use at experimental immunization by bacterial antigen].

    Science.gov (United States)

    Tsybul'skiĭ, A V; Sanina, N M; Li, I A; Popov, A M; Kostetskiĭ, E Ia; Portniagina, O Iu; Shnyrov, V L

    2007-01-01

    Results of experiments on modification of immunostimulating complexes (ISCOM's) matrix by the replacement of the phospholipid for the glycolipid (monogalactosyldiacylglycerol) from sea macrophytes, and saponin QuillA to triterpene glycoside of cucumarioside A2-2 from Cucumaria japonica are shown. The resultant complexes include the morphological structures of two types: ISCOM-like structures with the characteristic morphology and sizes and also the tubular structures with diameter of approximately 40 nm and length of 150-400 nm. We have named these structures as TI-complexes. These TI-complexes exhibit considerably lower toxicity than ISCOM. They may include an amphiphilic protein antigen and provide immunoadjuvant effect during experimental vaccination. Under conditions of experimental immunization of mice by a weak immunogen--(subunit membrane pore protein from Y. pseudotuberculosis), TI-complexes with antigen provided stronger humoral immune response to antigen than the complexes of porin with classical ISCOM, liposomes and Freund's adjuvant. Thus, it's shown the prospect of the use of TI-complexes as a new type of adjuvant carriers for antigens. PMID:17722580

  8. 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.

  9. Elevated IgG levels against specific bacterial antigens in obese patients with diabetes and in mice with diet-induced obesity and glucose intolerance.

    Science.gov (United States)

    Mohammed, Nadeem; Tang, Lihua; Jahangiri, Anisa; de Villiers, Willem; Eckhardt, Erik

    2012-09-01

    High fat diets increase the risk for insulin resistance by promoting inflammation. The cause of inflammation is unclear, but germfree mouse studies have implicated commensal gut bacteria. We tested whether diet-induced obesity, diabetes, and inflammation are associated with anti-bacterial IgG. Blood from lean and obese healthy volunteers or obese patients with diabetes were analyzed by ELISA for IgG against extracts of potentially pathogenic and pro-biotic strains of Escherichia coli (LF-82 and Nissle), Bacteroides thetaiotaomicron, and Lactobacillus acidophilus, and for circulating tumor necrosis factor α (TNFα). C57Bl/6 mice were fed low- or high-fat diets (10% or 60% kcal from fat) for 10 weeks and tested for anti-bacterial IgG, bodyweight, fasting glucose, and inflammation. Obese diabetic patients had significantly more IgG against extracts of E. coli LF-82 compared with lean controls, whereas IgG against extracts of the other bacteria was unchanged. Circulating TNFα was elevated and correlated with IgG against the LF-82 extract. Mice fed high-fat diets had increased fasting glucose levels, elevated TNFα and neutrophils, and significantly more IgG against the LF-82 extracts. Diabetes in obesity is characterized by increased IgG against specific bacterial antigens. Specific commensal bacteria may mediate inflammatory effects of high-fat diets. PMID:22424821

  10. 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 ...

  11. Prostate Cancer Detection and Dutasteride : Utility and Limitations of Prostate-Specific Antigen in Men with Previous Negative Biopsies

    NARCIS (Netherlands)

    van Leeuwen, Pim J.; Koelble, Konrad; Huland, Hartwig; Hambrock, Thomas; Barentsz, Jelle; Schroder, Fritz H.

    2011-01-01

    Context: We addressed the question whether the change of serum prostate-specific antigen (PSA) in men who use 5 alpha-reductase inhibitor (5-ARI) dutasteride is sensitive for the detection of aggressive prostate cancer (PCa). Objective: The case of a man using dutasteride diagnosed with Gleason 7 tr

  12. Evaluation of three commercial latex agglutination kits and a commercial enzyme immunoassay for the detection of cryptococcal antigen.

    Science.gov (United States)

    Babady, Ngolela Esther; Bestrom, Jean E; Jespersen, Deborah J; Jones, Mary F; Beito, Elaine M; Binnicker, Matthew J; Wengenack, Nancy L

    2009-05-01

    We compared the performance of the Meridian CALAS, Wampole Crypto-LA, Murex Cryptococcus latex agglutination assay, and the Meridian Premier EIA for the detection of cryptococcal antigen in serum and CSF. The assays demonstrated similar performance characteristics based on concordance values > or = 93% but important differences were noted in endpoint titers. PMID:19194818

  13. A ‘Magnetic’ Gram Stain for Bacterial Detection

    OpenAIRE

    Budin, Ghyslain; Chung, Hyun Jung; Lee, Hakho; Weissleder, Ralph

    2012-01-01

    Magnetic stain. Bacteria are often classified into Gram-positive and Gram-negative strains by their visual staining properties using crystal violet (CV), a triarylmethane dye. Here we show, that bioorthogonal modification of crystal violet with transcyclooctene (TCO) can be used to render Gram-positive bacteria magnetic with magneto-nanoparticles-Tetrazine (MNP-Tz). This allows for class specific automated magnetic detection, magnetic separation or other magnetic manipulations.

  14. 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.

  15. Aptasensor based on tripetalous cadmium sulfide-graphene electrochemiluminescence for the detection of carcinoembryonic antigen.

    Science.gov (United States)

    Shi, Gui-Fang; Cao, Jun-Tao; Zhang, Jing-Jing; Huang, Ke-Jing; Liu, Yan-Ming; Chen, Yong-Hong; Ren, Shu-Wei

    2014-11-21

    A facile label-free electrochemiluminescence (ECL) aptasensor, based on the ECL of cadmium sulfide-graphene (CdS-GR) nanocomposites with peroxydisulfate as the coreactant, was designed for the detection of carcinoembryonic antigen (CEA). Tripetalous CdS-GR nanocomposites were synthesized through a simple onepot solvothermal method and immobilized on the glassy carbon electrode surface. L-Cystine (L-cys) could largely promote the electron transfer and enhance the ECL intensity. Gold nanoparticles (AuNPs) were assembled onto the L-cys film modified electrode for aptamer immobilization and ECL signal amplification. The aptamer modified with thiol was adsorbed onto the surface of the AuNPs through a Au-S bond. Upon hybridization of the aptamer with the target protein, the sequence could conjugate CEA to form a Y architecture. With CEA as a model analyte, the decreased ECL intensity is proportional to the CEA concentration in the range of 0.01-10.0 ng mL(-1) with a detection limit of 3.8 pg mL(-1) (S/N = 3). The prepared aptasensor was applied to the determination of CEA in human serum samples. The recoveries of CEA in the human serum samples were between 85.0% and 109.5%, and the RSD values were no more than 3.4%. PMID:25209409

  16. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  17. Evaluation of Leishmania (Leishmania) infantum excreted-secreted antigens for detection of canine leishmaniasis.

    Science.gov (United States)

    Pinedo-Cancino, Viviana; Laurenti, Márcia Dalastra; Kesper, Norival; Umezawa, Eufrosina Setsu

    2016-09-01

    The efficacy of tests with L. (L.) infantum excreted-secreted antigens (ESA) to detect canine leishmaniasis (CanL) was evaluated using immunoblotting (ESA-blot), ELISA (ESA-ELISA) and ELISA with alkaline extract from promastigotes (PAE). Of one hundred fifty-five domestic dogs tested, 100 were suspected of CanL, 23 had other diseases and 32 were healthy. Sera from the dogs suspected of CanL were tested by immunohistochemistry (IHC), and 54% were confirmed to be infected by L. (L.) infantum (38 symptomatic and 16 asymptomatic). Of these, 100% were positive by ESA-blot, ESA-ELISA and PAE-ELISA. In the ESA-blot their sera recognized polypeptides in the 26.5-31.5kDa region. Of the 46% of dogs with negative IHC, 44-53% tested positive in all three tests irrespective of clinical status. The twenty-three dogs with other diseases were negative by ESA-blot, but sera from 9% and 26% of them reacted with ESA-ELISA and PAE-ELISA, respectively. The 32 healthy dogs were negative in all the tests. ESA-blot showed good correlation with IHC in the detection of CanL and a high specificity index. PMID:27212707

  18. Application of functional microsphere in human hepatitis B virus surface antigen detection.

    Science.gov (United States)

    Ma, Ningning; Ma, Chao; Wang, Nianyue; Li, Chuanyan; Elingarami, Sauli; Mou, Xianbo; Tang, Yongjun; Zheng, Shuang; He, Nongyue

    2014-05-01

    A novel and simple emulsifier-free emulsion polymerization technique was developed for preparation of mono-dispersed amino functionalized polymer microspheres with well defined diameters (about 400 nm). Various characterization methods demonstrated that the obtained amino microspheres had a uniform size and good dispersity which were confirmed by scanning electron microscope (SEM). Zeta potential and Fourier transform infrared spectrometer (FT-IR) demonstrated that amino groups have been successfully introduced to the microsphere surface. These functionalized microspheres have been shown to be efficient and controllable carriers capable of immobilizing and enriching monoclonal antibodies. Moreover, a newest chemiluminescent enzyme-linked immunoassay (ELISA) approach has been developed for human Hepatitis B virus surface antigen (HBsAg) detection. HBsAg was sandwiched between goat anti-HBsAg polyclonal antibody and mouse anti-HBsAg antibody. Alkaline phosphatase (ALP) conjugated horse anti-mouse immunnogloblin was used to bond with monoclonal antibody. Finally, chemiluminesent (CL) signals were recorded after adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) which was used as a chemiluminescent substrate reagent of ALP. This novel chemiluminescent ELISA assay was proved to be of excellent specificity and high sensitivity when using ALP and AMPPD luminescence systems for specific HBsAg detection. PMID:24734551

  19. A handheld real time thermal cycler for bacterial pathogen detection.

    Science.gov (United States)

    Higgins, James A; Nasarabadi, Shanavaz; Karns, Jeffrey S; Shelton, Daniel R; Cooper, Mary; Gbakima, Aiah; Koopman, Ronald P

    2003-08-15

    The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002. PMID:12788554

  20. Serological detection of H-Y antigen in humans with a cellular radioimmunobinding assay and monoclonal antibody

    International Nuclear Information System (INIS)

    A sensitive and reliable serological assay for the detection of H-Y antigen is described which uses monoclonal H-Y antibody and a cellular radioimmunobinding assay on cultured human fibroblasts. Cell lines from 2 normal human females, 2 normal human males and one XX human male were tested in 3 separate assays. Cells from XY and XX males were found to contain H-Y antigen; however, the reaction against the XX male cells was found to be intermediate between the XY male and normal XX female cells. (Auth.)

  1. Detection of IgG antibody against Crimean-Congo haemorrhagic fever virus using ELISA with recombinant nucleoprotein antigens from genetically diverse strains.

    Science.gov (United States)

    Rangunwala, A; Samudzi, R R; Burt, F J

    2014-10-01

    Crimean-Congo haemorrhagic fever virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Handling the virus requires biosafety level-4 facilities, limiting accessibility for many laboratories. Advances in molecular techniques have allowed preparation of safe recombinant antigens that have application in diagnosis and serosurveillance of CCHFV. The aim of this study was to determine genetic diversity in CCHFV based on all available complete sequence data for the S gene encoding CCHFV nucleoprotein (NP) and antibody cross-reactivity between the NP of a South African isolate and the NP of a Greek isolate (AP92), the most genetically diverse CCHFV strain. The nucleotide sequence diversity and amino-acid diversity between genotypes, within genotypes and the pairwise distances were calculated for a dataset of 45 CCHFV isolates retrieved from GenBank. The most diverse virus, AP92, isolated from a tick in Greece, displayed the highest amino-acid difference (8·7%) with SPU415/85, isolated from a human infection in South Africa. Recombinant NP encoded for by codon-optimized S genes of SPU415/85 and AP92 were expressed in a bacterial host system and used to develop an in-house ELISA to detect IgG antibody against CCHFV in South African patients who survived infection. A total of 14/14 sera reacted with the South African recombinant NP and 13/14 reacted with the Greek recombinant NP. The serological cross-reactivity of the two NP antigens suggests that recombinant antigens prepared from geographically distinct CCHFV will have diagnostic and epidemiological applications worldwide. PMID:24330947

  2. Bacterial Antigen Expression Is an Important Component in Inducing an Immune Response to Orally Administered Salmonella-Delivered DNA Vaccines

    OpenAIRE

    Gahan, Michelle E.; Webster, Diane E.; Wesselingh, Steven L.; Richard A. Strugnell; Yang, Ji

    2009-01-01

    Background The use of Salmonella to deliver heterologous antigens from DNA vaccines is a well-accepted extension of the success of oral Salmonella vaccines in animal models. Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology. An important aspect of the basic biology of the Salmonella/DNA vaccine platform is the relative contribu...

  3. 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. PMID:27094421

  4. Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

    Directory of Open Access Journals (Sweden)

    Carl A. Batt

    2009-05-01

    Full Text Available The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform.

  5. Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

    Science.gov (United States)

    Fukuma, Aiko; Fukushi, Shuetsu; Yoshikawa, Tomoki; Tani, Hideki; Taniguchi, Satoshi; Kurosu, Takeshi; Egawa, Kazutaka; Suda, Yuto; Singh, Harpal; Nomachi, Taro; Gokuden, Mutsuyo; Ando, Katsuyuki; Kida, Kouji; Kan, Miki; Kato, Nobuyuki; Yoshikawa, Akira; Kitamoto, Hiroaki; Sato, Yuko; Suzuki, Tadaki; Hasegawa, Hideki; Morikawa, Shigeru; Shimojima, Masayuki; Saijo, Masayuki

    2016-01-01

    Background Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. Methodology/Principal Findings We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. Conclusions The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. PMID:27045364

  6. Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on “Membrane-engineered” Fibroblast Cells with Virus-Specific Antibodies and Antigens

    OpenAIRE

    Antonios Perdikaris; Nikos Alexandropoulos; Spiridon Kintzios

    2009-01-01

    A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV)-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe) or antigens (HBsAg) in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, resp...

  7. Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

    Science.gov (United States)

    Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

    2016-07-01

    Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. PMID:27018063

  8. Development of a sandwich Dot-ELISA for detecting bovine viral diarrhea virus antigen with E2 recombinant protein

    Institute of Scientific and Technical Information of China (English)

    Yuelan ZHAO; Yuzhu ZUO; Lei ZHANG; Jinghui FAN; Hanchun YANG; Jianhua QIN

    2009-01-01

    The IgG antibodies of rabbit anti-E2 protein of the bovine viral diarrhea virus were prepared by a general method from high efficiency serum immunized by E2 recombinant protein antigen expressed in E. coli prokaryotic expression system and were labeled to make enzymelabeled antibody with the method of NaIO4. A sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the detection of BVDV was developed. The optimal reaction conditions of Dot-ELISAwere determined. The results show that optimal coating antibody was 300 μg·mL-1, the working concentration of HRP-labeled antibody was 1:50. The optimal blocking reagent and time were 5% bovine serum and 45 rain. The minimum detection of the content of antigen reached 1.35μg·mL-1. Compared with the routine IDEXX ELISA test kit with the whole virus, its specificity, sensitivity and coincidence rate were 90.48%, 96.55% and 95.24%, respectively. Compared with the sandwich Dot-ELISA with the negative staining electron microscope and RT-PCR, the coincidence rates were 90.9% and 93.1%, respectively. In addition, Bovine viral diarrhea virus (BVDV) antigen of 178 samples collected from cow farms in the Hebei Province, China, were detected by the developed Dot-ELISA and the IDEXX BVDV antigen Test Kit simultaneously, BVDV antigen positive rate was 39.89%-41.01%. The result of detecting clinical samples demonstrated that the established method showed its specificity, sensitivity and repeatability, whereas the results were easily interpreted without an ELISA reader.

  9. 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; Petersen, E L; Aagaard, M; Hansen, Dorte; Christensen, 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...

  10. 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. PMID:27132007

  11. Serological detection of circulating Angiostrongylus vasorum antigen and specific antibodies in dogs from central and northern Italy.

    Science.gov (United States)

    Guardone, L; Schnyder, M; Macchioni, F; Deplazes, P; Magi, M

    2013-02-18

    The most frequently employed method for the diagnosis of Angiostrongylus vasorum in dogs is the detection of first stage larvae (L1) in faeces. The sensitivity of coproscopy, however, is limited in case of low parasite load, intermittent larval excretion, and during pre-patency. An epidemiological survey on dogs was conducted applying serological methods in two Italian regions where angiostrongylosis is endemic in foxes. 265 dog serum samples from Tuscany (central Italy - site A) and 447 from Liguria (north-western Italy - site B) were tested with a sandwich-ELISA for detection of circulating antigen, and with an ELISA using A. vasorum adult somatic antigen purified by monoclonal antibodies for specific antibody detection. During previous examinations dogs naturally infected with Leishmania infantum (n=149), Dirofilaria immitis (n=40), Dirofilaria repens (n=30), Acanthocheilonema reconditum (n=27), Crenosoma vulpis (n=1), A. vasorum (n=2), Capillaria aerophila (n=35), Capillaria boehmi (n=3), Toxocara canis (n=68), Toxascaris leonina (n=5), hookworms (n=37) and Trichuris vulpis (n=39) were detected. Sera of these dogs were used to evaluate cross reactions. In site A, 2 dogs (0.8%) were seropositive for antibody and antigen detection and 4 (1.5%) for antibody detection only. From site B, 4 dogs (0.9%) were seropositive for both tests, while other 4 dogs (0.9%) for antigen detection only and 9 dogs (2%) for antibody detection only. Considering a subgroup of 347 dogs from site B which had also been tested with the Baermann technique, 2 (0.6%) were positive for both tests, 4 (1.2%) for antigen detection only and 9 (2.6%) for antibody detection only. The two dogs which were positive for both serological tests were also positive for A. vasorum L1 in the faeces. No significant difference in seropositivities was observed in the group of dogs with other proven parasitic infections. A. vasorum serology presents significant advantages (diagnosis before patency, single serum

  12. TO EVALUATE THE ROLE OF NS1 ANTIGEN FOR EARLY DETECTION OF DENGUE FEVER

    Directory of Open Access Journals (Sweden)

    Chithambaram

    2014-12-01

    Full Text Available In India, Dengue epidemics are becoming more frequent and are straining the limited resources of the public health system. Diagnosing dengue early is challenging because the initial symptoms of dengue infection are often non-specific. It is for this reason diagnosing dengue infection early becomes important. AIM: To evaluate the role of NS1 antigen for early detection of dengue fever. SETTING AND DESIGN: Prospective Study done in a tertiary care hospital between January 2013 to September 2014. MATERIAL AND METHODS: Patients in the age group between 6 months to 14 yrs with onset of fever up to 7 days and patients fulfilling the WHO clinical criteria of diagnosis dengue fever. STATISTICAL ANALYSIS: sensitivity, specificity, PPV and NPV. RESULTS: Of the 143 patients enrolled, 100 were serologically proved to have dengue illness and the rest 43 were non dengue patients. Of the 100 dengue patients NS1, IgM and IgG was positive in 62, 53 and 15 patients respectively. The sensitivity, specificity, PPV and NPV of NS1 Ag were found to be 62%, 100%, 100% and 53% respectively. CONCLUSION: The present study showed that dengue serological tests have a significant role in the early diagnosis of dengue fever. Hence it is recommended to do the serological tests (NS1 and IgM early in all suspected dengue cases so that we can diagnose early and initiate necessary treatment.

  13. Detecting antibody-antigen reaction using nano ripple gold LSPR based biosensor

    Science.gov (United States)

    Saleem, Iram; Wijesundera, Dharshana; Tilakaratne, Buddhi; Widger, William; Chu, Wei-Kan

    We introduce a simple and cost-effective scheme for bio-sensing using nano-ripple structures. One-dimension metallic nano-ripple structures formed by gas cluster ion beam irradiation have shown polarization of light as well as the localized surface plasmon resonance. These localized surface plasmon resonance (LSPR) based bio sensors not only are capable of label free real time analytical detection but also show high sensitivity. The nano surface morphology determines the changes in the plasmonic properties of nanostructures hence the plasmonic response is tunable. By immobilizing a stable and sterically accessible monolayer of antibody on the surface of these substrates and loading different concentrations of the specific antigen we identified the shift in the LSPR peaks triggered by the change of dielectric function in the neighborhood of the structures. These plasmonic nano-metallic structures can be utilized to observe the shift in the LSPR resonance frequency due to the cycle of adsorption, re-adsorption and reactions taking place on the surface that can potentially be mapped in to reaction mechanics. The bio-sensor has monolayer molecule-coating sensitivity and specific selectivity.

  14. Characterization of a Leishmania tropica antigen that detects immune responses in Desert Storm viscerotropic leishmaniasis patients.

    Science.gov (United States)

    Dillon, D C; Day, C H; Whittle, J A; Magill, A J; Reed, S G

    1995-08-15

    A chronic debilitating parasitic infection, viscerotropic leishmaniasis (VTL), has been described in Operation Desert Storm veterans. Diagnosis of this disease, caused by Leishmania tropica, has been difficult due to low or absent specific immune responses in traditional assays. We report the cloning and characterization of two genomic fragments encoding portions of a single 210-kDa L. tropica protein useful for the diagnosis of VTL in U.S. military personnel. The recombinant proteins encoded by these fragments, recombinant (r) Lt-1 and rLt-2, contain a 33-amino acid repeat that reacts with sera from Desert Storm VTL patients and with sera from L. tropica-infected patients with cutaneous leishmaniasis. Antibody reactivities to rLt-1 indicated a bias toward IgG2 in VTL patient sera. Peripheral blood mononuclear cells from VTL patients produced interferon gamma, but not interleukin 4 or 10, in response to rLt-1. No cytokine production was observed in response to parasite lysate. The results indicate that specific leishmanial antigens may be used to detect immune responses in VTL patients with chronic infections. PMID:7644524

  15. Bacterial Toxin Fusion Proteins Elicit Mucosal Immunity against a Foot-and-Mouth Disease Virus Antigen When Administered Intranasally to Guinea Pigs

    Directory of Open Access Journals (Sweden)

    Sreerupa Challa

    2011-01-01

    Full Text Available Peptides corresponding to the foot-and-mouth disease virus VP1 G-H loop are capable of inducing neutralizing antibodies in some species but are considered relatively poor immunogens, especially at mucosal surfaces. However, intranasal administration of antigens along with the appropriate delivery vehicle/adjuvant has been shown to induce mucosal immune responses, and bacterial enterotoxins have long been known to be effective in this regard. In the current study, two different carrier/adjuvant approaches were used to augment mucosal immunity to the FMDV O1 BFS G-H loop epitope, in which the G-H loop was genetically coupled to the E. coli LT-B subunit and coexpressed with the LTA2 fragment (LTA2B-GH, or the nontoxic pseudomonas exotoxin A (ntPE was fused to LTA2B-GH at LT-A2 to enhance receptor targeting. Only guinea pigs that were inoculated intranasally with ntPE-LTA2B-GH and LTA2B-GH induced significant anti-G-H loop IgA antibodies in nasal washes at weeks 4 and 6 when compared to ovalbumin or G-H loop immunized animals. These were also the only groups that exhibited G-H loop-specific antigen-secreting cells in the nasal mucosa. These data demonstrate that fusion of nonreplicating antigens to LTA2B and ntPE-LTA2B has the potential to be used as carriers/adjuvants to induce mucosal immune responses against infectious diseases.

  16. Immunomagnetic concentration of antigens and detection based on a scanning force microscopic immunoassay.

    Science.gov (United States)

    Perrin, A; Theretz, A; Lanet, V; Vialle, S; Mandrand, B

    1999-04-22

    Scanning Force Microscopy has already been shown to be a convenient and rapid method for sensitive antigen detection and quantification. Here, we describe different improvement steps brought to a TSH Scanning Force Microscopic ImmunoAssay (SFMIA), each of them aiming to solve a previous limitation of the solid phase test format and leading to a significant sensitivity enhancement. First, superparamagnetic nanoparticles conjugated to monoclonal anti alphaTSH antibodies were used for the specific TSH capture step. Their magnetic properties allowed easy separation of the complexes obtained from relatively large reaction volumes by application of a High Gradient Magnetic Field System. As a consequence, complex formation could proceed in a stirred solution, which greatly enhances binding rates compared to previous 'static' conditions of solid-phase reactions. It was established that, despite their small size, magnetic complexes could be moved over short distances by a NdFeB magnet magnetic field. This property was exploited to overcome diffusion barrier and boundary layer constraints and to drive magnetic complexes through the liquid, towards anti-betaTSH antibodies immobilized on silica wafers. Finally, we significantly increased the complex number/surface area by a stepwise reduction of the biospecific solid phase area. The proposed steps permitted a 3-fold improvement in the TSH SFMIA dynamic range. Moreover, as little as 0.02 pg/ml (0.1 nIU/ml or 0.8 amol/ml) of TSH could be detected using 1 ml sample volumes. This is over 100 times more sensitive than the current performance of commercialized automated systems. PMID:10357209

  17. Antigen detection and apoptosis in Mongolian gerbil's kidney experimentally intraperitoneally infected by swine hepatitis E virus.

    Science.gov (United States)

    Soomro, Majid Hussain; Shi, Ruihan; She, Ruiping; Yang, Yifei; Hu, Fengjiao; Li, Heng

    2016-02-01

    We examined the effect of hepatitis E virus (HEV) on the renal tissue pathogenesis, morphological damages and related molecular mechanisms following swine HEV suspension intraperitoneally inoculation in Mongolian gerbils. The microscopic and ultramicroscopic analyses of kidney tissue structure were carried out at different points after inoculation of HEV. The immunohistochemistry, real-time PCR and Western blot were performed to explore the molecular mechanisms associated with HEV presence in the renal tissues. Real-time PCR revealed that the copies of HEV RNA in the kidney were detected at 7 dpi, and peaked at 14 dpi at a concentration was 7.18 logs g(-1), with detection of HEV ORF2 antigen by immunohistochemistry. Hematoxylin and eosin (HE) staining showed pathological lesions including glomerular atrophy, degeneration, edema and necrosis of renal tubular epithelial cells and Mallory and Sirius red staining indicated the presence of collagen fibers and fibrosis in kidney tissues of inoculated gerbils. Ultrastructural studies of basal membrane of renal tubules demonstrated the rough and uneven with mitochondria swelling and vacuolation in the tissues of HEV inoculated animals. Similarly, significantly higher number of (TUNEL)-positive cells were seen in renal tubule tissues compared to control group. Moreover, immuno histochemical results indicated that significant increase expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), FAS and Caspase-3 in HEV inoculated Mongolian gerbils at each time points. Relative mRNA expression by real-time PCR revealed a significantly higher (P<0.05) mRNA level of BAX, Bcl-2 and caspase-3 transcription in HEV inoculated Mongolian gerbils. Our results demonstrates that activation of mitochondria and Caspase-3 protease might be induced the apoptosis which subsequently cause the necrosis and cell death of renal epithelial cells during acute phase of HEV infection in HEV inoculated Mongolian gerbils. PMID

  18. A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay to detect antibodies that react with antigens derived from human melanoma cells is described. A soluble preparation derived from Nonidet P-40 lysates of tissue-cultured melanoma cells was dried on the surfaces of wells of polyvinyl chloride microtiter plates and fixed with 0.02% glutaraldehyde. Antibody preparations were added and incubated for 18 h at 40C. The wells were washed and bound antibodies were detected using radioactive Staphyloccoccal protein A (125I-SpA). Optimal conditions are described for all the steps employed. Concentrations of antigen selected, the amount of 125I-SpA employed and the duration of incubation of antibodies with antigen were found to be critical. The assay was sensitive and reproducible, and lent itself to the simultaneous evaluation of many individual antibody samples in a short period of time. The assay was particularly valuable for rapid screening of hybridoma supernatants for antibodies to antigens derived from melanoma cells and from a panel of other tumor and normal cells. (orig.)

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Dong Huahuang

    2012-08-01

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

  1. 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 BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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

  2. Mass-sensing BioCD Protein Array towards Clinical Application: Prostate Specific Antigen Detection in Patient Sera

    CERN Document Server

    Wang, Xuefeng; Nolte, David D; Ratliff, Timothy L

    2009-01-01

    Mass-sensing biosensor arrays for protein detection require no fluorophores or enzyme labels. However, few mass biosensor protein arrays have demonstrated successful application in high background samples, such as serum. In this paper, we test the BioCD as a mass biosensor based on optical interferometry of antibodies covalently attached through Schiff-base reduction. We use the BioCD to detect prostate specific antigen (PSA, a biomarker of prostate cancer) in patient sera in a 96-well anti-PSA microarray. We have attained a 4 ng/ml detection limit in full serum and have measured PSA concentrations in three patient sera.

  3. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR expression by flow cytometry

    Directory of Open Access Journals (Sweden)

    Zheng Zhili

    2012-02-01

    Full Text Available Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig-binding protein that binds to the variable light chains (kappa chain of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2, two murine antibody derived CARs (anti-CSPG4, and anti

  4. Biotin-avidin amplified magnetic immunoassay for hepatitis B surface antigen detection using GoldMag nanoparticles

    International Nuclear Information System (INIS)

    Using GoldMag (Fe3O4/Au) nanoparticles as a carrier, a biotin-avidin amplified ELISA was developed to detect hepatitis B surface antigen (HBsAg). A specific antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag nanoparticles by a sandwich ELISA assay. The results showed that 5mol of biotin were surface bound per mole of antibody. The biotin-avidin amplified ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at dilution of 1:4000 is 5.98% (n=11)

  5. Aspergillus fumigatus Antigen Detection in Sera from Patients at Risk for Invasive Aspergillosis

    OpenAIRE

    Chumpitazi, Bernabé F. F.; Pinel, Claudine; Lebeau, Bernadette; Ambroise-Thomas, Pierre; Grillot, Renee

    2000-01-01

    We have developed an inhibition enzyme immunoassay (inhibition-EIA) to monitor for the occurrence of invasive aspergillosis (IA) in sera from 45 immunocompromised (IC) patients. The test uses rabbit polyclonal antibodies and a mixture of components from Aspergillus fumigatus, containing three predominant antigens with molecular weights of 18,000, 33,000, and 56,000. Circulating antigens were found in five of seven proven cases of IA due to A. fumigatus. In two of the five positive cases, anti...

  6. Rift Valley fever in Central Africa: Serological evidence and virus antigen detection in cattle in the Democratic Republic of Congo

    International Nuclear Information System (INIS)

    In order to assess the disease status within the cattle population in the Democratic Republic of Congo, a survey was carried out using two investigation strategies, i.e. 1) anti - RVF virus (RVFV) Ig G antibodies (Abs) detection, and 2) virus detection. Five provinces were selected due to their high bovine exploitation activity. 962 sera samples randomly collected in 26 locations from the 5 mentioned provinces were tested exploiting the recombinant N protein indirect ELISA (I - ELISA) for antibodies detection and prevalence estimate and for virus detection, two diagnostic methods were exploited: a) - virus antigen (Ag) detection in tissues using the immunoperoxidase (IMP) staining with Avidin Biotin Complex (ABC) technique and, b) -virus cDNA detection using RT- PCR. Only some tissues from syndromically suspected cases from one location with the highest prevalence (20%) were analysed for virus detection. The structural, nucleo . capsid (N) protein that the small segment (S) of the RVFV genome encodes with forms the key antigen targeted in all the 3 diagnostic methods. As a matter of fact, N protein is the most immunocompetent and most expressed protein of RVFV; it is also the highest conserved protein amongst members of the Bunyaviridae family

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

    OpenAIRE

    Kellermann, Gottfried H.; Lehmann, Paul V; Diana R. Roen; Chenggang Jin

    2013-01-01

    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....

  8. Detection of Ebola Viral Antigen by Enzyme-Linked Immunosorbent Assay Using a Novel Monoclonal Antibody to Nucleoprotein

    OpenAIRE

    Niikura, Masahiro; Ikegami, Tetsuro; Saijo, Masayuki; Kurane, Ichiro; Miranda, Mary E.; Morikawa, Shigeru

    2001-01-01

    With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope ...

  9. 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

  10. PCR detection of bacterial genes provides evidence of death by drowning.

    Science.gov (United States)

    Suto, Miwako; Kato, Naho; Abe, Sumiko; Nakamura, Masahide; Tsuchiya, Reo; Hiraiwa, Kouichi

    2009-04-01

    We have developed a sensitive and specific PCR method for detecting plankton DNA in cases of death by drowning. However, this PCR method could not be used for cases of drowning in water containing no plankton. Bacteria species are normally localized in the throat and trachea and they may invade into blood through the respiratory tract in people who have drowned as well as species localized in water. The aim of this study was to establish a novel and expedient PCR method for detecting bacterial genes in samples from drowning cases. We designed primer pairs for Streptococcus salivarius (SL1) and Streptococcus sanguinis (SN1), which are common species in the throat, and for Aeromonas hydrophila (AH1), which has been found in various water samples. With SL1, SN1, and AH1, we detected 10, 0.1, and 1 pg of target DNA, respectively. Among 19 drowned cases within 3 days postmortem, SL-DNA was detected in all of the blood samples from hearts with SL1 and AH-DNA was detected in several samples with AH1. In a case of drowning in a bathtub, use of the conventional acid digestion method for diatom analyses and the PCR method for identifying plankton DNA revealed no plankton, but our PCR method for detecting bacterial DNA showed a positive result for SL-DNA in a blood sample from the heart. In conclusion, our novel PCR method is highly specific and sensitive for detecting bacterial DNA and is useful for cases of death by drowning in water containing no plankton. PMID:19264526

  11. Antigen detection of rabies virus in brain smear using direct Rapid Immunohistochemistry Test

    Directory of Open Access Journals (Sweden)

    Damayanti R

    2014-03-01

    Full Text Available Rabies is zoonotic disease caused by a fatal, neurotropic virus. Rabies virus is classified into the Genus of Lyssavirus under the yang family of Rhabdoviridae. Rabies affecting hot- blooded animals, as well as human. Dogs, cats, monkeys are the vectors or reservoirs for rabies and the virus was transmitted through the saliva after infected animal’s bites. The aim of this study was to conduct rapid diagnosis to detect rabies viral antigen in brain smear using immunohistochemical (IHC method namely direct Rapid Immunohistochemical Test (dRIT. A total number of 119 brain samples were achieved from Bukittinggi Veterinary Laboratory, West Sumatra. Standardisation and validation of the method were compared to Fluorescent Antibody Test (FAT as a golden standard for rabies diagnosis. Results show that dRIT was a very good method, it can be performed within two hours without the need of fluorescent microscope. The samples were tested using FAT and from 119 samples tested, 80 (67.23% samples were positive for rabies and 39 (32.77% samples were negative for rabies whereas using dRIT showed that 78 (65.54% samples were positive for rabies and 41 (34.45% samples were negative for rabies. The dRIT results were validated by comparing them with FAT results as a golden standard for rabies. The relative sensitivity of dRIT to FAT was 97.5% and the relative specificity to FAT was 100% (with Kappa value of 0.976, stated as excellent. The achievement showed that dRIT is very potential diagnostic tool and is highly recommended to be used widely as a rapid diagnosis tool for rabies.

  12. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    Science.gov (United States)

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  13. Use of 4-aminophenol and p-phenylenediamine derivatives for the detection of bacterial hydrolyases

    OpenAIRE

    Ford, Michael

    2004-01-01

    A range of novel substrates for the detection of bacterial hydrolyases have been examined in both liquid and solid media. The potential inhibitory effect of these substrates on both Gram-positive and Gram-negative organisms has been evaluated. In addition a range of alternative substrates possessing an appropriate chemical configuration have also been evaluated as substrates in this thesis. It has been demonstrated that substrates based on 4-aminophenol, particularly the dichloro derivative p...

  14. Rapid detection of malto-oligosaccharide-forming bacterial amylases by high performance anion-exchange chromatography

    DEFF Research Database (Denmark)

    Duedahl-Olesen, Lene; Larsen, K. L.; Zimmermann, W.

    2000-01-01

    High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto-oligosaccharide-formi......-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7.0 and 10....

  15. Fluorescence in situ hybridization for the tissue detection of bacterial pathogens associated with porcine infections

    DEFF Research Database (Denmark)

    Jensen, Henrik Elvang; Jensen, Louise Kruse; Barington, Kristiane;

    2015-01-01

    sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue......Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target...

  16. Fluorescence In Situ Hybridization for the Tissue Detection of Bacterial Pathogens Associated with Porcine Infections

    DEFF Research Database (Denmark)

    Elvang Jensen, Henrik; Jensen, Louise Kruse; Barington, Kristiane;

    2015-01-01

    sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue......Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target...

  17. Detection of Renibacterium salmoninarum, the Causative Agent of Bacterial Kidney Disease in Salmonid Fish, from Pen-Cultured Coho Salmon.

    Science.gov (United States)

    Sakai, M; Kobayashi, M

    1992-03-01

    The detection of Renibacterium salmoninarum antigen from pen-cultured coho salmon was attempted. Flounder (Limanda herzensteini) (n = 24), greenling (Hexagrammos otakii) (n = 5), Japanese sculpin (Cottus japonicus) (n = 1), and flathead (Platycephalus indicus) (n = 22) captured by fishing around coho salmon net pens were examined for the presence of R. salmoninarum antigen by an indirect dot blot assay and by an indirect fluorescent-antibody technique using polyclonal and monoclonal antibodies. R. salmoninarum antigen was detected from kidney samples of one greenling and six flathead. Moreover, 86 scallops (Patinopecten yessoensis) were hung from the edge of the net pen for 50 days, and R. salmoninarum antigen was demonstrated in 31 samples by the indirect dot blot assay and the indirect fluorescent-antibody technique. PMID:16348666

  18. Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on “Membrane-engineered” Fibroblast Cells with Virus-Specific Antibodies and Antigens

    Directory of Open Access Journals (Sweden)

    Antonios Perdikaris

    2009-03-01

    Full Text Available A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe or antigens (HBsAg in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA. The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg. The observed response was rapid (45 sec and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca2+]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Decho, Alan W; Beckman, Erin M; Chandler, G Thomas; Kawaguchi, Tomohiro [Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208 (United States)

    2008-06-11

    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.

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

    Science.gov (United States)

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

    2008-06-01

    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.

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

    International Nuclear Information System (INIS)

    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

  2. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sendra, H [Laboratorio de Laser. Facultad de Ingenieria. Universidad Nacional de Mar del Plata, Juan B. Justo 4302. (7600) Mar del Plata (Argentina); Murialdo, S [Grupo de Ingenieria BioquImica. Departamento de Quimica. Facultad de Ingenieria. Universidad Nacional de Mar del Plata, Juan B. Justo 4302. (7600) Mar del Plata (Argentina); Passoni, L [Laboratorio de BioingenierIa. Facultad de Ingenieria. Universidad Nacional de Mar del Plata, Juan B. Justo 4302. (7600) Mar del Plata (Argentina)

    2007-11-15

    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.

  4. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife.

    Directory of Open Access Journals (Sweden)

    Maria Razzauti

    Full Text Available Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations.We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq. In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454. In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles.We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of

  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. Clinical value of Helicobacter pylori stool antigen test, ImmunoCard STAT HpSA, for detecting H pylori infection

    Institute of Scientific and Technical Information of China (English)

    Yi-Hui Li; Hong Guo; Peng-Bin Zhang; Xiao-Yan Zhao; Si-Ping Da

    2004-01-01

    AIM: To evaluate the reliability of the Helicobacter pylori stool antigen test, ImmunoCard STAT HpSA, for detecting H pylori infection.METHODS: Stool specimens were collected from 53 patients who received upper endoscopy examination due to gastrointestinal symptoms. ImmunoCard STAT HpSA wasused to detect H pylori stool antigens. H pyloriinfection wasdetected based on three different tests: the urease test, Warthin-Starry staining and culture. H pylori status wasdefined as positive when both the urease test and histology or culture alone was positive.RESULTS: Sensitivity, specificity, positive predictive and negative predictive values and the total accuracy of ImmunoCard STAT HpSA for the diagnosis of H pylorinfection were 92.6% (25/27), 88.5% (23/26), 89.3% (25/28), 92%(23/25) and 90.6% (48/53), respectively.CONCLUSION: The stool antigen test, ImmunoCard STAT HpSA, is a simple noninvasive and accurate test for the diagnosis of H pyloriinfection.

  7. Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

    Directory of Open Access Journals (Sweden)

    Alfonso Rosalba

    2002-01-01

    Full Text Available In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR amplification fragments for the precise tuberculosis (TB diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients. Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

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

    International Nuclear Information System (INIS)

    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

  9. Development of a mixed antigen agar gel enzyme assay (AGEA) for the detection of antibodies to poxvirus in chicken and turkey sera.

    Science.gov (United States)

    Tadese, Theodros; Potter, E A; Reed, W M

    2003-02-01

    A mixed-antigen agar gel enzyme assay (AGEA) was developed to detect antibodies to poxviruses in chicken and turkey sera. The assay combines the principles of immunodiffusion and enzyme assay. For the detection of antibodies to fowl poxvirus (FP), pigeon poxvirus (PP) and turkey poxvirus (TP) in turkey serum samples, the three antigens were combined to form a mixed-antigen assay. To screen for antibodies to FP and PP in chicken serum samples, the two antigens were combined. When FP and PP viruses were combined as antigens, the sensitivity for chicken sera was 64% but the sensitivity of the agar gel precipitation test (AGPT) was 34% (PAGEA had a sensitivity of 66.4% while that of AGPT was 25% (P<0.001). PMID:12655123

  10. ELISA for the detection of toxic antigens in experimental and clinical envenoming by Tityus serrulatus scorpion venom.

    Science.gov (United States)

    Chávez-Olórtegui, C; Fonseca, S C; Campolina, D; Amaral, C F; Diniz, C R

    1994-12-01

    An ELISA was developed for identification of circulating toxic antigens from Tityus serrulatus scorpion venom. The toxic fraction from the scorpion venom was purified by Sephadex G-50 chromatography and immunoaffinity techniques were used for identifying antibodies that reacted with this fraction. These antibodies were used to develop a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity for identifying mice that were experimentally inoculated with T. serrulatus venom from those inoculated with Phoneutria nigriventer spider venom, Apis mellifera bee venom and Bothrops atrox, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.1 ng of venom per assay. The ELISA also detected antigens in the sera of patients systemically envenomed by T. serrulatus. Therefore, this ELISA could be a valuable tool for clinicians and epidemiologists, owing to its sensitivity and specificity. PMID:7725332

  11. 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 sampling methods to permit detection of low-level bacterial infections of rainbow trout.

  12. Standardization of natural mycolic acid antigen composition and production for use in biomarker antibody detection to diagnose active tuberculosis.

    Science.gov (United States)

    Ndlandla, F L; Ejoh, V; Stoltz, A C; Naicker, B; Cromarty, A D; van Wyngaardt, S; Khati, M; Rotherham, L S; Lemmer, Y; Niebuhr, J; Baumeister, C R; Al Dulayymi, J R; Swai, H; Baird, M S; Verschoor, J A

    2016-08-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches

  13. Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody.

    OpenAIRE

    Gallarda, J L; Henrard, D R; Liu, D.; Harrington, S.; Stramer, S L; Valinsky, J E; Wu, P

    1992-01-01

    Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14...

  14. Elaboration of optical immunosensors based on the surface plasmon resonance for detecting specific antibodies and antigens of Epstein-Barr virus and human adenovirus.

    Science.gov (United States)

    Nesterova, N V; Nosach, L M; Zagorodnya, S D; Povnitsa, O Y; Boltovets, P M; Baranova, G V; Golovan, A V

    2008-01-01

    The study of antigen-antibody interaction on the model of Epstein-Barr virus (EBV) and second type adenovirus (Ad2) based on the surface plasmon resonance (SPR) was carried out. Kinetic and concentration dependences between virus antigens and specific antisera to them at different pH were determined. Experimental samples of biosensors for the detection by SPR method of virus (EBV and Ad2) antigens using monospecific antibodies, immobilized on the surface of gold, and also for detection of specific antibodies in the blood sera of patients with EBV or adenovirus infection were elaborated PMID:19351051

  15. Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

    Science.gov (United States)

    Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y.

    2016-03-01

    We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

  16. 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...

  17. Near-infrared spectroscopy for the detection and quantification of bacterial contaminations in pharmaceutical products.

    Science.gov (United States)

    Quintelas, Cristina; Mesquita, Daniela P; Lopes, João A; Ferreira, Eugénio C; Sousa, Clara

    2015-08-15

    Accurate detection and quantification of microbiological contaminations remains an issue mainly due the lack of rapid and precise analytical techniques. Standard methods are expensive and time-consuming being associated to high economic losses and public health threats. In the context of pharmaceutical industry, the development of fast analytical techniques able to overcome these limitations is crucial and spectroscopic techniques might constitute a reliable alternative. In this work we proved the ability of Fourier transform near infrared spectroscopy (FT-NIRS) to detect and quantify bacteria (Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Salmonella enterica, Staphylococcus epidermidis) from 10 to 10(8) CFUs/mL in sterile saline solutions (NaCl 0.9%). Partial least squares discriminant analysis (PLSDA) models showed that FT-NIRS was able to discriminate between sterile and contaminated solutions for all bacteria as well as to identify the contaminant bacteria. Partial least squares (PLS) models allowed bacterial quantification with limits of detection ranging from 5.1 to 9 CFU/mL for E. coli and B. subtilis, respectively. This methodology was successfully validated in three pharmaceutical preparations (contact lens solution, cough syrup and topic anti-inflammatory solution) proving that this technique possess a high potential to be routinely used for the detection and quantification of bacterial contaminations. PMID:26151105

  18. Peroxide test strips detect added hydrogen peroxide in raw milk at levels affecting bacterial load.

    Science.gov (United States)

    Martin, Nicole H; Friedlander, Adam; Mok, Allen; Kent, David; Wiedmann, Martin; Boor, Kathryn J

    2014-10-01

    Hydrogen peroxide (H2O2) has a long-established history of use as a preservative in milk worldwide. The use of H2O2 to activate the inherent lactoperoxidase enzyme system has dramatically improved the quality of raw dairy products in areas in which cooling is not widely available. In the United States, however, where refrigeration is widely available, the addition of H2O2 to milk is not permitted, with the exception of certain applications prior to cheesemaking and during the preparation of modified whey. Due to the relatively quick deterioration of H2O2 in fluid milk, the detection of raw milk adulterated with the compound can be challenging. In this study we evaluated (i) total aerobic bacterial counts and (ii) ability of peroxide test strips to detect H2O2 in raw milk with various concentrations (0, 100, 300, 500, 700, and 900 ppm) of added H2O2, incubated at both 6 and 21°C for 0, 24, and 48 h. Results showed that at both 6 and 21°C the H2O2 concentration and time had a significant effect on bacterial loads in raw milk. Additionally, commercially available test strips were able to detect H2O2 in raw milk, with predicted probability of >90%, immediately after addition and after 24 and 48 h for the higher concentrations used, offering a viable method for detecting raw milk adulteration with H2O2. PMID:25285503

  19. 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 func

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

    NARCIS (Netherlands)

    P. Koraka (Penelopie); 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

  1. Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

    Science.gov (United States)

    Othman, Nurulhasanah; Mohamed, Zeehaida; Yahya, Maya Mazuwin; Leow, Voon Meng; Lim, Boon Huat; Noordin, Rahmah

    2013-08-01

    Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band. PMID:23680184

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

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker; Schumacher, Nana Maria Pii

    2010-01-01

    The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different...

  3. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

    Science.gov (United States)

    Baumann, D; Gottstein, B

    1987-06-01

    A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system. PMID:2888183

  4. Diagnostic performances of antigen detection compared to conventional and nucleic acid detection of Entamoeba histolytica in a non-endemic setting.

    Science.gov (United States)

    Calderaro, Adriana; Piergianni, Maddalena; Piccolo, Giovanna; Rossi, Sabina; Montecchini, Sara; Buttrini, Mirko; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora

    2016-04-01

    This study evaluated the immunochromatographic (IC) assay "TECHLAB(®) E. HISTOLYTICA QUIK CHEK™" analysing 36 faecal samples and 7 cultured strains. This assay was compared to the methods performed in our laboratory for the diagnosis of amoebiasis. The IC assay revealed a detection limit of 103 trophozoites/g faeces and no cross-reactivity with other parasites and failed to detect E. histolytica antigen in frozen faeces. In our laboratory located in a non-endemic setting this assay could not replace the methods currently used for the diagnosis of amoebiasis. PMID:27196557

  5. 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)

    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)

  6. Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing

    OpenAIRE

    Siala, Mariam; Jaulhac, Benoit; Gdoura, Radhouane; Sibilia, Jean; Fourati, Hela; Younes, Mohamed; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Znazen, Abir; Barthel, Cathy; Collin, Elody; Hammami, Adnane; Sghir, Abdelghani

    2008-01-01

    Introduction Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reacti...

  7. Pretreatment of serum samples to reduce interference of colostrum-derived specific antibodies with detection of Bovine viral diarrhea virus antigen by ELISA in young calves.

    Science.gov (United States)

    Lanyon, Sasha R; Reichel, Michael P

    2016-05-01

    Antigen enzyme-linked immunosorbent assay (ELISA) is used for the detection of Bovine viral diarrhea virus persistently infected (BVDV PI) cattle; however, colostrum-derived antibodies may interfere with antigen detection in serum from young PI calves. Our study aimed to assess serum pretreatment methods for reducing such interference. Dilution of PI serum with serum containing specific antibody showed that antibody levels equivalent to those observed in colostrum-fed calves were able to eliminate all antigen signals in a serum sample. Serum was treated with ethylenediamine tetra-acetic acid at pH 4.5, 5.5, 6.5, and 7.5, then boiled, centrifuged, and the supernatant-recovered. BVDV antibody was undetectable by ELISA in supernatants from treated samples, and the antigen ELISA signal was improved. Maximum antigen signal recovery of >90% was achieved at pH 5 ± 0.5. When this optimal treatment method was applied to field samples from 3 PI calves (which were negative in the antigen-capture ELISA without treatment), the antigen signal improved and gave a positive result in each case. Pretreatment may provide an improvement in the detection of young PI calves. PMID:27016723

  8. Value of Prostate-Specific Antigen and Prostate-Specific Antigen Density in Detection of Prostate Cancer in an Iranian Population of Men

    Directory of Open Access Journals (Sweden)

    Mahyar Ghafoori

    2009-08-01

    Full Text Available

    Introduction: The objective of this study was to evaluate the value of serum prostate-specific antigen (PSA and prostate-specific antigen density (PSAD in the diagnosis of prostate cancer.

    Materials and Methods: A total of 330 consecutive patients suspected of having prostate cancer due to either abnormal digital rectal examination or elevated serum PSA levels underwent transrectal ultrasonography-guided sextant biopsy of the prostate. The PSA and PSAD values were assessed based on the biopsy results.

    Results: One hundred and twenty-one patients (36.7% had prostate cancer. In this group, the mean PSA was 31.60 ± 30.85 ng/mL (range, 1.9 ng/mL to 166.0 ng/mL and the mean PSAD was 0.83 ± 1.01 (range, 0.04 ng/mL/cm3 to 6.38 ng/mL/cm3. In those without prostate cancer the mean PSA and PSAD levels were 13.80 ± 18.72 ng/mL (range, 0.4 ng/mL to 130.0 ng/mL; P < .001 and 0.24 ± 0.32 (range of 0.01 ng/mL/cm3 to 2.29 ng/mL/cm3; P < .001. The receiver operating characteristic curve analysis revealed that the discriminating power of serum PSA for detecting prostate cancer, as estimated by the area under the curve, was 0.74 while that for PSAD was 0.81 (P < .001. For the PSA range of 3.5 ng/mL to 41 ng/mL (gray zone the areas under the curve was 0.68 for PSA, while it was 0.78 for PSAD (P < .001.

    Conclusion: The use of PSAD instead of PSA in the diagnosis of prostatic cancer improves the diagnostic accuracy.

  9. Combined Serological Detection of Circulating Angiostrongylus vasorum Antigen and Parasite-specific Antibodies in Dogs from Hungary.

    Science.gov (United States)

    Schnyder, Manuela; Schaper, Roland; Lukács, Zoltán; Hornok, Sándor; Farkas, Róbert

    2015-08-01

    The occurrence of the nematode Angiostrongylus vasorum, also known as the French heartworm, is increasingly being reported from various European countries. The adults of this parasite species live in the pulmonary arteries and right cardiac ventricle of wild canids and domestic dogs. Larval stages and eggs in the lungs induce inflammatory verminous pneumonia, causing severe respiratory disease in dogs. Furthermore, haematological and neurological signs and even death may occur. In Hungary, A. vasorum has been identified in red foxes, golden jackals and in two dogs and some slugs. In this first large-scale survey, 1247 sera from pet dogs were collected and tested by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA to detect specific antibodies against the parasite. A total of 1.36% (n = 17, 95 % confidence intervals, CI: 0.80 - 2.17 %) of the animals were positive in both ELISAs, while 1.76 % (n = 22, CI: 1.11 - 2.66 %) of the tested dogs were antigen-positive only and 2.73 % (n = 34, CI: 1.90 - 3.79 %) were positive for specific antibodies only. Regions with antigen- and antibody-positive animals overlapped and were distributed over nearly the whole sampled areas of the country. A considerable number of cases was observed in Budapest and also in the southern part of the country bordering Croatia, while in the most eastern part bordering Ukraine no positive samples were detected. These results confirm the endemic occurrence of A. vasorum in dogs originating from different parts of Hungary and the significant advantages of A. vasorum serology in epidemiological studies. PMID:26152415

  10. A simple bacterial turbidimetric method for detection of some radurized foods

    International Nuclear Information System (INIS)

    A simple and quick method for detection of irradiated food is proposed. The method is based on the principle of microbial contribution to the development of turbidity in a clear medium. It employs measurement of absorbance at 600 nm of the medium after the test commodity has been suspended and shaken in it for a fixed interval. The differences in the bacterial turbidity from irradiated and nonirradiated samples are quite marked so as to allow identification of the irradiated foods like fish, lamb meat, chicken and mushroom. (author)

  11. A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera.

    Science.gov (United States)

    Osawa, T; Wastling, J; Maley, S; Buxton, D; Innes, E A

    1998-09-01

    Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity. PMID:9777723

  12. 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.

  13. DETECTION OF CYTOMEGALOVIRUS(CMV) IMMEDIATE EARLY ANTIGEN IN KIDNEY BIOPSIES AND TRANSPLANT NEPHRECTOMIES

    Institute of Scientific and Technical Information of China (English)

    燕航; 薛武军; 田普训; 郭奇; 何晓丽

    2004-01-01

    Objective To investigate the relationship between CMV infection and renal allograft rejection. Methods 39 kidney biopsies and transplant nephrectomies were collected and investigated for CMV immediate early antigen by immunohistochemistry. Results In 14 out of 39 tissue specimens CMV immediate early antigen were found. 8 biopsies from normal donor kidneys were negative; only 1 (10%) in 10 tissue specimens with early stage acute rejection was positive; 5(55.6%) in 9 biopsies with late stage acute rejection and 8 (66.7%) in 12 tissue blocks with chronic rejection were positive. Compared with normal kidney tissues, the infections in tissues with early stage acute rejection didn't increase obviously, but increased obviously in kidney tissue specimens with late stage rejection and with chronic rejection (P<0.05). Conclusion CMV infection appears to contribute to late stage acute rejection and chronic rejection after renal transplantation.

  14. 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.

  15. A sandwich-type immunosensor using Pd–Pt nanocrystals as labels for sensitive detection of human tissue polypeptide antigen

    International Nuclear Information System (INIS)

    A sandwich-type immunosensor was developed for the detection of human tissue polypeptide antigen (hTPA). In this work, a graphene sheet (GS) was synthesized to modify the surface of a glassy carbon electrode (GCE), and Pd–Pt bimetallic nanocrystals were used as secondary-antibody (Ab2) labels for the fabrication of the immunosensor. The amperometric response of the immunosensor for catalyzing hydrogen peroxide (H2O2) was recorded. And electrochemical impedance spectroscopy was used to characterize the fabrication process of the immunosensor. The anti-human tissue polypeptide antigen primary antibody (Ab1) was immobilized onto the GS modified GCE via cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). With Ab1 immobilized onto the GS modified GCE and Ab2 linked on Pd–Pt bimetallic nanocrystals, the immunosensor demonstrated a wide linear range (0.0050–15 ng ml−1), a low detection limit (1.2 pg ml−1), good reproducibility, good selectivity and acceptable stability. This design strategy may provide many potential applications in the detection of other cancer biomarkers. (paper)

  16. Development and evaluation of an immunochromatographic strip for rapid detection of capsid protein antigen p27 of avian leukosis virus.

    Science.gov (United States)

    Qian, Kun; Liang, You-zhi; Yin, Li-ping; Shao, Hong-xia; Ye, Jian-qiang; Qin, Ai-jian

    2015-09-01

    A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease. PMID:25977186

  17. Detection of hidden nephritogenic antigen determinants in human renal and nonrenal basement membranes.

    OpenAIRE

    Yoshioka, K; Michael, A F; Velosa, J; Fish, A. J.

    1985-01-01

    The reactivity of 10 human anti-glomerular basement membrane (GBM) autoantibodies with basement membrane antigens of human adult and infant kidney, lung, placenta, and skin was examined by ELISA and immunofluorescence microscopy. All autoantibodies were previously shown to react with adult kidney by indirect immunofluorescence and with collagenase-digested adult GBM by ELISA. Four antibodies (group A) were positive on infant and fetal kidney sections by immunofluorescence, and six antibodies ...

  18. Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis

    OpenAIRE

    Cao, Liang; Chen, Da-Liang; Lee, Cindy; Chan, Che-Man; Chan, King-Man; Vanittanakom, Nongnuch; Tsang, Dominic N.C.; Yuen, Kwok-Yung

    1998-01-01

    The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marne...

  19. Multiple antigen peptide dendrimer elicits antibodies for detecting rat and mouse growth hormone binding proteins

    OpenAIRE

    Aguilar, Roberto M.; Talamantes, Frank J.; Bustamante, Juan J.; Muñoz, Jesus; Treviño, Lisa R.; Martinez, Andrew O.; Haro, Luis S.

    2009-01-01

    The membrane-bound rat growth hormone receptor (GH-R) and an alternatively spliced isoform, the soluble rat GH binding protein (GH-BP), are comprised of identical N-terminal GH binding domains, however, their C-terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent multiple antigen peptide (MAP) dendrimer with four identical branches of a C-ter...

  20. Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

    OpenAIRE

    Martins, Thomas B; Burlingame, Rufus; von Mühlen, Carlos A.; Jaskowski, Troy D; Litwin, Christine M.; Hill, Harry R.

    2004-01-01

    Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus ...

  1. Direct fluorescent-antibody confirmation of chlamydial antigen below the detection threshold of the chlamydiazyme enzyme-linked immunosorbent assay.

    OpenAIRE

    Kellogg, J A; Seiple, J W; Stroll, E S

    1993-01-01

    Of 4,000 endocervical specimens tested with the Chlamydiazyme enzyme-linked immunosorbent assay (Abbott Laboratories), 233 (5.8%) gave positive results (A492 above the cutoff), which were confirmed with a blocking reagent (Abbott). An additional 34 specimens (14.6%) with chlamydial antigen were detected and confirmed with the direct fluorescent-antibody test (Syva) from among those 66 Chlamydiazyme-negative specimens which had A492s that ranged from 0.030 to the cutoff and that could be block...

  2. 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.

  3. 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.

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

    International Nuclear Information System (INIS)

    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)

  5. 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)

    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)

  6. 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.

  7. A broadband capacitive sensing method for label-free bacterial LPS detection.

    Science.gov (United States)

    Rydosz, Artur; Brzozowska, Ewa; Górska, Sabina; Wincza, Krzysztof; Gamian, Andrzej; Gruszczynski, Slawomir

    2016-01-15

    In this paper, the authors present a new type of highly sensitive label-free microwave sensor in a form of interdigital capacitor coated with T4 bacteriophage gp37 adhesin. The adhesin binds Escherichia coli B (E. coli B) by precise recognizing its bacterial host lipopolysaccharide (LPS). The C-terminal part of the adhesin consists of the receptor-binding amino acid residues which are involved in a specific interaction with two terminal glucose residues of the bacterial LPS. The change of the sensors' capacitance and conductance as a subject to LPS presence is an indicator of the detection. The measurements in the frequency range of 0-3GHz utilizing vector network analyzer have been carried out at different concentrations to verify experimentally the proposed method. The measured capacitance change between the reference and the biofunctionalized sensor equals 15% in the entire frequency range and the measured conductance change exceeds 19%. The changes of both parameters can be used as good indicators of the LPS detection. The selectivity has been confirmed by the ELISA experiments and tested by sensor measurements with lipopolysaccharide (LPS) from E. coli B, E. coli 056, E. coli 0111, Pseudomonas aeruginosa NBRC 13743 and Hafnia alvei 1185. PMID:26339930

  8. gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Heydorn, Arne; Hentzer, Morten; Eberl, Leo; Geisenberger, Otto; Christensen, Bjarke Bak; Molin, Søren; Givskov, Michael Christian

    2001-01-01

    In order to perform single-cell analysis and online studies of N-acyl homoserine lactone (AHL)-mediated communication among bacteria, components of the Vibrio fischeri quorum sensor encoded by luxR-P-luxI have been fused to modified versions of gfpmut3* genes encoding unstable green fluorescent p...... detection at the single-cell level and allowed for real-time measurements of fluctuations in AHL concentrations. This green fluorescent AHL sensor provides a state-of-the art tool for studies of communication between the individuals present in mixed bacterial communities.......In order to perform single-cell analysis and online studies of N-acyl homoserine lactone (AHL)-mediated communication among bacteria, components of the Vibrio fischeri quorum sensor encoded by luxR-P-luxI have been fused to modified versions of gfpmut3* genes encoding unstable green fluorescent...... proteins. Bacterial strains harboring this green fluorescent sensor detected a broad spectrum of AHL molecules and were capable of sensing the presence of 5 nM N-3-oxohexanoyl-L-homoserine lactone in the surroundings. In combination with epifluorescent microscopy, the sensitivity of the sensor enabled AHL...

  9. 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...... different bacteriological media. While 65% reacted positive; in the PCR only 23% were positive by culture, thereby suggesting a superior sensitivity of the PCR test to that of culture. The use of selective media, large inoculum and incubation for 48 h gave the highest number of positive PCR reactions from......A PCR for the detection of Actinobacillus pleuropneumoniae was evaluated. All of 102 field isolates of A. pleuropneumoniae reacted in the PCR by amplification of a 985 bp product. No PCR amplification product was observed when examining strains of A. ureae, A. capsulatus, A. hominis, A. equuli, A...

  10. 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)

    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 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-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

  11. 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.

  12. 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.

  13. An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

    OpenAIRE

    Nicholson, W L; Comer, J A; Sumner, J W; Gingrich-Baker, C; Coughlin, R T; Magnarelli, L A; Olson, J G; Childs, J. E.

    1997-01-01

    An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with ac...

  14. The Diagnostic and Prognostic Value of Dengue Non-Structural 1 Antigen Detection in a Hyper-Endemic Region in Indonesia

    OpenAIRE

    Herman Kosasih; Bachti Alisjahbana; Susana Widjaja; Nurhayati,; Quirijn de Mast; Ida Parwati; Blair, Patrick J; Burgess, Timothy H.; Andre van der Ven; Maya Williams

    2013-01-01

    As dengue fever is undifferentiated from other febrile illnesses in the tropics and the clinical course is unpredictable, early diagnosis is important. Several commercial assays to detect dengue NS1 antigen have been developed; however, their performances vary and data is lacking from hyper-endemic areas where all four serotypes of dengue are equally represented. To assess the sensitivity of the Bio-Rad platelia Dengue NS1 antigen assay according to virus serotype, immune status, gender, and ...

  15. Synthesis of a peptide-universal nucleotide antigen: towards next-generation antibodies to detect topoisomerase I-DNA covalent complexes.

    Science.gov (United States)

    Perkins, Angela L; Peterson, Kevin L; Beito, Thomas G; Flatten, Karen S; Kaufmann, Scott H; Harki, Daniel A

    2016-04-26

    The topoisomerase (topo) I-DNA covalent complex represents an attractive target for developing diagnostic antibodies to measure responsiveness to drugs. We report a new antigen, peptide , and four murine monoclonal antibodies raised against that exhibit excellent specificity for recognition of in comparison to structurally similar peptides by enzyme-linked immunosorbent assays. Although topo I-DNA complex detection was not achieved in cellular samples by these new antibodies, a new strategy for antigen design is reported. PMID:27113574

  16. 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...... been shown to recognise Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This protein is inserted by the parasite into the host cell membrane and mediates the adhesion to the venular endothelium of the host organism in vivo. METHODS: Erythrocytes infected at high parasitaemias with...... subsequently detected by two-colour flow cytometry. RESULTS: Binding of human antibodies to the surface of erythrocytes infected with adhesive strains of Plasmodium falciparum can be measured by the two-colour flow cytometry (FCM) assay described. In addition, we demonstrate that the adhesive capacity of a...

  17. An Improved K-means Clustering Based Approach to Detect a DNA Structure in H&E Image of Mouse Tissue Reacted with CD4-Green Antigen

    OpenAIRE

    Prashanth, B U V; Sastry, P Narahari; V. Rajesh

    2013-01-01

    In this manuscript we present the technique to detect and analyze the DNA rich structure in Haemotoxylin & Eosin (H&E) image of a tissue treated with anti CD4 green antigen. The detection of DNA rich structure can be considered as a detection of blue nuclei present through the biomedical signal/image processing technique performed on the image of the tissue obtained by the Scanning Electron Microscope(SEM). Earlier the tissue treated with the anti CD4 green antigen, is stained with the H&E st...

  18. 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.

  19. Identification and analysis of the antigens detected by two commercial Bacillus cereus diarrheal enterotoxin immunoassay kits.

    OpenAIRE

    Beecher, D J; Wong, A C

    1994-01-01

    The usefulness of two commercial immunoassays for the detection of diarrheal enterotoxin of Bacillus cereus is unclear because the identity of the enterotoxin(s) has not been proven and the kits detect different proteins. We found that the Bacillus cereus Enterotoxin-Reversed Passive Latex Agglutination kit (Oxoid) detects the L2 component from hemolysin BL, and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra) detects two apparently nontoxic proteins.

  20. 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.

  1. Single-cell-based sensors and synchrotron FTIR spectroscopy: a hybrid system towards bacterial detection.

    Science.gov (United States)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C; Bertozzi, Carolyn; Zhang, Miqin

    2007-09-30

    Microarrays of single macrophage cell-based sensors were developed and demonstrated for potential real-time bacterium detection by synchrotron FTIR microscopy. The cells were patterned on gold electrodes of silicon oxide substrates by a surface engineering technique, in which the gold electrodes were immobilized with fibronectin to mediate cell adhesion and the silicon oxide background was passivated with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. Cell morphology and IR spectra of single, double, and triple cells on gold electrodes exposed to lipopolysaccharide (LPS) of different concentrations were compared to reveal the detection capability of this cell-based sensing platform. The single-cell-based system was found to generate the most significant and consistent IR spectrum shifts upon exposure to LPS, thus providing the highest detection sensitivity. Changes in cell morphology and IR shifts upon cell exposure to LPS were found to be dependent on the LPS concentration and exposure time, which established a method for the identification of LPS concentration and infected cell population. Possibility of using this single-cell system with conventional IR spectroscopy as well as its limitation was investigated by comparing IR spectra of single-cell arrays with gold electrode surface areas of 25, 100, and 400 microm2 using both synchrotron and conventional FTIR spectromicroscopes. This cell-based platform may potentially provide real-time, label-free, and rapid bacterial detection, and allow for high-throughput statistical analyses, and portability. PMID:17560777

  2. Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

    Science.gov (United States)

    Henihan, Grace; Schulze, Holger; Corrigan, Damion K; Giraud, Gerard; Terry, Jonathan G; Hardie, Alison; Campbell, Colin J; Walton, Anthony J; Crain, Jason; Pethig, Ronald; Templeton, Kate E; Mount, Andrew R; Bachmann, Till T

    2016-07-15

    Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps. PMID:27016627

  3. Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR.

    Science.gov (United States)

    Jansson, E; Lindberg, L; Säker, E; Aspán, A

    2008-10-01

    Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated. PMID:18681904

  4. Detection of carcinoembryonic antigen mRNA in peritoneal washes from gastric cancer patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    Yan-Song Zhang; Jun Xu; Guang-Hua Luo; Rong-Chao Wang; Jiang Zhu; Xiao-Ying Zhang; Peter Nilsson-Ehle; Ning Xu

    2006-01-01

    AIM: To establish a more sensitive method for detection of free cancer cells in peritoneal washes from gastric cancer patients during surgery and to evaluate its clinical significance.METHODS: The carcinoembryonic antigen (CEA) mRNA levels in peritoneal washes from 65 cases of gastric cancer were detected by real-time RT-PCR. Peritoneal lavage cytology (PLC) was applied simultaneously to detection of free cancer cells. Negative controls included peritoneal washes from 5 cases of benign gastric disease and blood samples from 5 adult healthy volunteers.RESULTS: There was no CEA mRNA in peritoneal washes from benign gastric disease patients and in blood of adult healthy volunteers. The positive percentage of free cancer cells detected by real-time RT-PCR was 47.7% and only 12.3% by PLC. The positive rate of CEA mRNA was significantly related with serosa invasion between peritoneal metastasis and stage of gastric cancer.CONCLUSION: Real-time RT-PCR is a sensitive and rapid method for the detection of free cancer cells in peritoneal washes. The presence of free cancer cells in peritoneal washes is related to the pathologic stage of gastric cancer.

  5. Eosinophils and filarial parasites. Factors affecting eosinophils in animal models and the detection of filarial antigens by radioimmunoassay

    International Nuclear Information System (INIS)

    The effects of drugs, such as diethylcarbamazine (DEC), hydrocortisone and an immunologically stimulated state of the host on the sites of localization and behaviour of 113Insup(m)-eosinophils, were studied in guinea pigs. The sites of localization of 113Insup(m)-eosinophils were in the lungs, liver, spleen, bone and muscle. Administration of hydrocortisone did not change the normal distribution. After administration of DEC higher levels of 113Insup(m)-eosinophils were seen in the circulation, bone and muscle. When animals were actively immunized there was an avid concentration of cells in the liver and spleen. Preliminary experiments were undertaken to demonstrate the existence of an eosinophilopoetic factor in tropical eosinophilia (TE), using a rat model. Human gammaglobulin (HGG)-coated latex particles when injected intravenously induced a rise of eosinophils in the bone marrow on the fourth post-injection day, followed by a rise in peripheral eosinophils on the sixth day. When sera of stimulated rats were re-injected into normal rats there was again an eosinophilic response, suggesting the presence of a humoral transferable factor. Sera of two patients of TE also induced a rise of eosinophils in the bone marrow of rats injected with the sera, suggesting the presence of some circulating factor in TE which can stimulate eosinophilopoesis in rats. Serological diagnosis of filariasis by testing for antibodies does not necessarily detect active infection. However, determination of circulating antigens may prove to be more useful. Owing to the difficulty of obtaining W. bancrofti antigens, heteroantigens of Settaria cervii, which is cross-active with W. bancrofti, are used in the development of a radioimmunoassay for filarial antigens. (author)

  6. Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens†

    Science.gov (United States)

    Chen, Yi-Wen; Wang, Hong; Hupert, Mateusz; Witek, Makgorzata; Dharmasiri, Udara; Pingle, Maneesh R.; Barany, Francis

    2015-01-01

    The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft3). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic system

  7. Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens.

    Science.gov (United States)

    Chen, Yi-Wen; Wang, Hong; Hupert, Mateusz; Witek, Makgorzata; Dharmasiri, Udara; Pingle, Maneesh R; Barany, Francis; Soper, Steven A

    2012-09-21

    The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft(3)). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic

  8. 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

  9. Beetroot-pigment-derived colorimetric sensor for detection of calcium dipicolinate in bacterial spores.

    Directory of Open Access Journals (Sweden)

    Letícia Christina Pires Gonçalves

    Full Text Available 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 × 10(5 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× 10(6 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.

  10. 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 × 10(5) 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)× 10(6) 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

  11. Detection of prostate-specific antigen with biomolecule-gated AlGaN/GaN high electron mobility transistors

    International Nuclear Information System (INIS)

    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)

  12. Detection of IgG Anti-Leishmania Antigen by Flow Cytometry as a Diagnostic Test for Cutaneous Leishmaniasis.

    Science.gov (United States)

    Pedral-Sampaio, Geraldo; Alves, Jessé S; Schriefer, Albert; Magalhães, Andréa; Meyer, Roberto; Glesby, Marshall J; Carvalho, Edgar M; Carvalho, Lucas P

    2016-01-01

    Diagnosis of cutaneous leishmaniasis (CL) relies on clinical presentation, parasite isolation, histopathologic evaluation and positive Montenegro skin test. However, the low amounts of parasites in the lesion of these individuals make parasite isolation and histopatologic diagnosis unreliable, often leading to false-negative results. Also, 15% of people living in endemic areas have sub-clinical infection characterized by positive Montenegro skin test, which may contribute to misdiagnosis. Although the main Leishmania killing mechanism is through cell-mediated immune response, antibodies against Leishmania antigens are found in infected individuals. Here our goal was to develop a new serological technique using polystyrene microspheres sensitized with soluble Leishmania antigens as a tool for the detection of IgG in serum from CL patients by flow cytometry. To validate the assay we carried out a comparative test (ELISA) commonly used as a diagnostic test for parasitic diseases. To determine cross-reactivity we used serum from patients with Chagas disease, caused by a trypanosome that has several proteins with high homology to those of the Leishmania genus. We observed that the flow cytometry technique was more sensitive than the ELISA, but, less specific. Our results show that the flow cytometry serologic test can be used to confirm CL cases in L. braziliensis transmission areas, however, presence of Chagas disease has to be ruled out in these individuals. PMID:27622535

  13. Bacterial ghosts provided with antigens

    NARCIS (Netherlands)

    Leenhouts Cornelis, Johannes; Ramasamy, Ranjan; Steen, Anton; Kok, Jan; Buist, Girbe; Kuipers, Oscar

    2003-01-01

    Methods for improving binding of a proteinaceous substance to cell-wall material of a Gram-positive bacterium are disclosed. The proteinaceous substance includes an AcmA cell-wall binding domain, homolog or functional derivative thereof. The method includes treating the cell-wall material with a sol

  14. Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

    Science.gov (United States)

    Wang, Z-Y; Cai, J-P; Qiu, L-W; Hao, W; Pan, Y-X; Tung, E T K; Lau, C C Y; Woo, P C Y; Lau, S K P; Yuen, K-Y; Che, X-Y

    2012-11-01

    Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis. PMID:22669560

  15. Detection of bacterial endospores by means of ultrafast coherent Raman spectroscopy

    Science.gov (United States)

    Pestov, Dmitry Sergeyevich

    This work is devoted to formulation and development of a laser spectroscopic technique for rapid detection of biohazards, such as Bacillus anthracis spores. Coherent anti-Stokes Raman scattering (CARS) is used as an underlying process for active retrieval of species-specific characteristics of an analyte. Vibrational modes of constituent molecules are Raman-excited by a pair of ultrashort, femtosecond laser pulses, and then probed through inelastic scattering of a third, time-delayed laser field. We first employ the already known time-resolved CARS technique. We apply it to the spectroscopy of easy-to-handle methanol-water mixtures, and then continue building our expertise on solutions of dipicolinic acid (DPA) and its salts, which happen to be marker molecules for bacterial spores. Various acquisition schemes are evaluated, and the preference is given to multi-channel frequency-resolved detection, when the whole CARS spectrum is recorded as a function of the probe pulse delay. We demonstrate a simple detection algorithm that manages to differentiate DPA solution from common interferents. We investigate experimentally the advantages and disadvantages of near-resonant probing of the excited molecular coherence, and finally observe the indicative backscattered CARS signal from DPA and NaDPA powders. The possibility of selective Raman excitation via pulse shaping of the preparation pulses is also demonstrated. The analysis of time-resolved CARS experiments on powders and B. subtilis spores, a harmless surrogate for B. anthracis, facilitates the formulation of a new approach, where we take full advantage of the multi-channel frequency-resolved acquisition and spectrally discriminate the Raman-resonant CARS signal from the background due to other instantaneous four-wave mixing (FWM) processes. Using narrowband probing, we decrease the magnitude of the nonresonant FWM, which is further suppressed by the timing of the laser pulses. The devised technique, referred to as

  16. The diagnosis of malaria infection using a solid-phase radioimmunoassay for the detection of malaria antigens

    International Nuclear Information System (INIS)

    A method was devised to show that malaria parasites can be detected serologically in infected blood with a high degree of sensitivity. Using a murine malaria model, parasites were demonstrated in a solid-phase radio-immunoassay which measured antibody-binding inhibition. Lysed red blood cells (r.b.c.) were incubated with labelled specific antibody and were then reacted in antigen-coated tubes. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test blood. Using homologous antisera the test detected infection at a level of 1 parasite/million r.b.c.. The specificity of the method was shown by comparison of antibody-binding inhibition in normal and infected r.b.c. and in r.b.c. from non-infected mice with induced reticulocytosis. The sensitivity was shown in vitro in tests of serially diluted blood of high parasitaemia and in vivo for the detection of early infection. The presence of antibody in the test blood did not significantly affect the sensitivity of parasite detection. (author)

  17. A 16 × 16 CMOS Capacitive Biosensor Array Towards Detection of Single Bacterial Cell.

    Science.gov (United States)

    Couniot, Numa; Francis, Laurent A; Flandre, Denis

    2016-04-01

    We present a 16 × 16 CMOS biosensor array aiming at impedance detection of whole-cell bacteria. Each 14 μm × 16 μm pixel comprises high-sensitive passivated microelectrodes connected to an innovative readout interface based on charge sharing principle for capacitance-to-voltage conversion and subthreshold gain stage to boost the sensitivity. Fabricated in a 0.25 μm CMOS process, the capacitive array was experimentally shown to perform accurate dielectric measurements of the electrolyte up to electrical conductivities of 0.05 S/m, with maximal sensitivity of 55 mV/fF and signal-to-noise ratio (SNR) of 37 dB. As biosensing proof of concept, real-time detection of Staphylococcus epidermidis binding events was experimentally demonstrated and provides detection limit of ca. 7 bacteria per pixel and sensitivity of 2.18 mV per bacterial cell. Models and simulations show good matching with experimental results and provide a comprehensive analysis of the sensor and circuit system. Advantages, challenges and limits of the proposed capacitive biosensor array are finally described with regards to literature. With its small area and low power consumption, the present capacitive array is particularly suitable for portable point-of-care (PoC) diagnosis tools and lab-on-chip (LoC) systems. PMID:25974947

  18. Preparation of Pd/Bacterial Cellulose Hybrid Nanofibers for Dopamine Detection.

    Science.gov (United States)

    Li, Dawei; Ao, Kelong; Wang, Qingqing; Lv, Pengfei; Wei, Qufu

    2016-01-01

    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. PMID:27187327

  19. 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.

  20. 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

    A murine anti-human monoclonal antibody (19-DEJ-1) has been produced that binds to basement membranes (BMs) of the dermoepidermal junction and arrector pili muscles but not to either dermal glandular or vascular BMs. 19-DEJ-1 also recognizes BMs underneath epithelia of buccal mucosa, tongue......, esophagus, cervix, and cornea, and BMs surrounding smooth muscle in medium-sized vessels, placenta, uterus, and esophagus. When 16 human fetal skins (aged 54-142 gestational days) were examined, the antigen was first detected at 81 days. Using immunoperoxidase and immunogold staining techniques, indirect...... cells, 19-DEJ-1 monoclonal antibody specifically precipitated 2.75% of the total radiolabeled proteoglycans produced in culture supernatant and isolated by anion exchange chromatography. On the basis of our present findings, we conclude that 19-DEJ-1 monoclonal antibody defines a unique primate...

  1. Evaluation of dengue NS1 antigen detection for diagnosis in public health laboratories, São Paulo State, 2009

    Directory of Open Access Journals (Sweden)

    Ivani Bisordi

    2011-12-01

    Full Text Available The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%, and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.

  2. Detection of proteins antigenically related to Bothrops asper myotoxin in crotaline snake venoms.

    Science.gov (United States)

    Lomonte, B; Moreno, E; Gutiérrez, J M

    1987-01-01

    The presence of components antigenically related to Bothrops asper myotoxin was investigated by Western blotting and immunoelectrophoretic techniques. B. asper myotoxin is a non-glycosylated monomeric phospholipase A with a molecular weight by SDS-PAGE of 16,000 and isoelectric point of pH 9.8-10.0. Results showed that proteins in the venoms of B. nummifer, B. godmani, B. schlegelii, B. picadoi, and Agkistrodon bilineatus were recognized by monospecific antibodies to B. asper myotoxin raised in rabbit and sheep. Western blotting indicated that cross-reacting proteins have a molecular weight of 16,000, with the exception of that of B. picadoi, which is of 24,000 mol. wt. However, immunoelectrophoresis indicated that these components are highly heterogeneous in charge, ranging from basic to acidic proteins. The cross-reacting component(s) present in newborn B. asper venom has a different charge from that of the 'adult-type'. Venoms from newborn specimens showed an additional cross-reacting band of 18,000 mol. wt. Myotoxin is an abundant component in adult B. asper venom. Myotoxin-antimyotoxin complexes had different electrophoretic mobilities in rocket immunoelectrophoresis depending upon the species in which monospecific immune sera were produced. PMID:2448918

  3. Detection of human leukocyte antigen compatibility and antibodies in liver transplantation in China

    Institute of Scientific and Technical Information of China (English)

    Xue-Qin Meng; Xuan Zhang; Jun Fan; Lin Zhou; Bing Hao; Xiao-Ming Chen; Wei-Hang Ma; Shu-Sen Zheng

    2009-01-01

    BACKGROUND: The exact roles of human leukocyte antigen (HLA) compatibility, HLA antibodies and underlying diseases in acute rejection of liver transplants are not clear. Moreover, cytomegalovirus (CMV) infection, one of the most common infections after transplantation, is related to HLA genotype and the incidence of acute rejection. METHODS: Since there are controversial reports, we analyzed the impact of HLA matching, HLA antibodies and underlying diseases in 38 liver transplant recipients in China, and assessed the association of CMV infection and HLA compatibility. RESULTS: The frequency of no HLA compatibility was high in patients without antigenemia (P=0.019). All 17 patients with HLA-A matching developed antigenemia (P0.05). In patients with acute rejection, no differences were found in the incidence of acute rejection in transplants for hepatitis B, tumors, or combined hepatitis B and tumors (P>0.05).CONCLUSIONS: There are fewer acute rejections in transplants with more HLA compatibilities. Speciifc investigations of underlying diseases and HLA typing may be necessary in liver transplantation. The mechanisms of CMV infection and HLA matching should be further studied. HLA before transplantation should be examined for the prevention of acute rejection and CMV infection.

  4. 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.

  5. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

    Science.gov (United States)

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R

    2014-06-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  6. A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.

    Directory of Open Access Journals (Sweden)

    Kailash P Patra

    2014-06-01

    Full Text Available 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.

  7. 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.

  8. Diagnosis of Paracoccidioidomycosis by Detection of Antigen and Antibody in Bronchoalveolar Lavage Fluids▿

    OpenAIRE

    Marques-da-Silva, Silvia Helena; Colombo, Arnaldo Lopes; Blotta, Maria Heloisa Souza Lima; Queiroz-Telles, Flávio; Balthazar, Alípio Barbosa; Lopes, José Daniel; Camargo, Zoilo Pires

    2006-01-01

    Paracoccidioidomycosis (PCM) is a systemic infection caused by the fungus Paracoccidioides brasiliensis and is believed to be the leading cause of fungal pulmonary infection. In this study, we used an inhibition enzyme-linked immunosorbent assay to diagnose pulmonary PCM based on the detection of 43-kDa and 70-kDa molecules in bronchoalveolar lavage fluids. The results were compared with results obtained by classical methods for antibody detection.

  9. Polyhydroquinone-graphene composite as new redox species for sensitive electrochemical detection of cytokeratins antigen 21-1.

    Science.gov (United States)

    Wang, Huiqiang; Rong, Qinfeng; Ma, Zhanfang

    2016-01-01

    Polyhydroquinone-graphene composite as a new redox species was synthesized simply by a microwave-assisted one-pot method through oxidative polymerization of hydroquinone by graphene oxide, which exhibited excellent electrochemical redox activity at 0.124 V and can remarkably promote electron transfer. The as-prepared composite was used as immunosensing substrate in a label-free electrochemical immunosensor for the detection of cytokeratins antigen 21-1, a kind of biomarker of lung cancer. The proposed immunosensor showed wide liner range from 10 pg mL(-1) to 200 ng mL(-1) with a detection limit 2.3 pg mL(-1), and displayed a good stability and selectivity. In addition, this method has been used for the analysis of human serum sample, and the detection results showed good consistence with those of ELISA. The present substrate can be easily extended to other polymer-based nanocomposites. PMID:27464571

  10. 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.

  11. 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.

  12. How free of germs is germ-free? Detection of bacterial contamination in a germ free mouse unit.

    Science.gov (United States)

    Fontaine, Clinton A; Skorupski, Anna M; Vowles, Chriss J; Anderson, Natalie E; Poe, Sara A; Eaton, Kathryn A

    2015-07-01

    Management of germ free animals has changed little since the beginning of the 20th century. The current upswing in their use, however, has led to interest in improved methods of screening and housing. Traditionally, germ free colonies are screened for bacterial colonization by culture and examination of Gram stained fecal samples, but some investigators have reported using PCR-based methods of microbial detection, presumably because of perceived increased sensitivity. The accuracy and detection limit for traditional compared to PCR-based screening assays are not known. The purpose of this study was to determine the limit of detection of bacterial contamination of mouse feces by aerobic and anaerobic culture, Gram stain, and qPCR, and to compare the accuracy of these tests in the context of a working germ free mouse colony. We found that the limit of detection for qPCR (approximately 10(5) cfu/g of feces) was lower than for Gram stain (approximately 10(9) cfu/g), but that all 3 assays were of similar accuracy. Bacterial culture was the most sensitive, but the least specific, and qPCR was the least sensitive and most specific. Gram stain but not qPCR detected heat-killed bacteria, indicating that bacteria in autoclaved diet are unlikely to represent a potential confounding factor for PCR screening. We conclude that as a practical matter, bacterial culture and Gram stain are adequate for screening germ free mouse colonies for bacterial contaminants, but that should low numbers of unculturable bacteria be present, they would not be detected with any of the currently available means. PMID:26018301

  13. 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.

  14. Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

    Science.gov (United States)

    Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

    2013-08-01

    Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals. PMID:23761660

  15. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    International Nuclear Information System (INIS)

    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

  16. Simultaneous Detection of Bacterial Fish Pathogens, Edwardsiella ictaluri, Flavobacterium columnnare and Aeromonas Hydrophila by Multiplex-PCR

    Science.gov (United States)

    Edwardsiella ictaluri, Flavobacterium columnare and Aeromonas hydrophila are three major bacterial pathogens of fish that cause diseases with significant economic impact on the aquaculture industry world-wide. Rapid detection of multiple infections with these bacteria in the same host is important f...

  17. Immune responses to the enduring hypoxic response antigen Rv0188 are preferentially detected in Mycobacterium bovis infected cattle with low pathology.

    Directory of Open Access Journals (Sweden)

    Gareth J Jones

    Full Text Available The DosR regulon and the Enduring Hypoxic Response (EHR define a group of M. tuberculosis genes that are specifically induced in bacilli exposed in vitro to conditions thought to mimic the environment encountered by Mycobacteria during latent infection. Although well described in humans, latent mycobacterial infection in cattle remains poorly understood. Thus, the aim of this study was to identify antigens that may potentially disclose cattle with latent M. bovis infection. To this end, we initially screened 57 pools of overlapping peptides representing 4 DosR regulon and 29 EHR antigens for their ability to stimulate an immune response in whole blood from TB-reactor cattle using IFN-γ and IL-2 as readouts. All 4 DosR regulon proteins were poorly recognized (maximum responder frequency of 10%. For the EHR antigens, both IFN-γ and IL-2 revealed similar response hierarchies, with responder frequencies ranging from 54% down to 3% depending on the given EHR antigen. Furthermore, these results demonstrated that responses in the infected cattle were largely IFN-γ biased. To support the concept for their role in latency, we evaluated if EHR antigen responses were associated with lower pathology. The EHR antigen Rv0188 was recognised predominantly in animals presenting with low pathology scores, whereas responses to ESAT-6/CFP-10 or the other EHR antigens tested were prevalent across the pathology spectrum. However, when we determined the production of additional cytokines induced by the M. bovis antigens PPD-B or ESAT-6/CFP-10, we detected significantly greater PPD-B-induced production of the pro-inflammatory cytokine IL-1β in animals recognizing Rv0188 (i.e. those with limited or no pathology. Thus, these results are consistent with the idea that responses to Rv0188 may identify a subset of animals at early stages of infection or in which disease progression may be limited.

  18. Construction and comparison of fluorescence and bioluminescence bacterial biosensors for the detection of bioavailable toluene and related compounds

    International Nuclear Information System (INIS)

    Environmental pollution with petroleum products such as benzene, toluene, ethylbenzene, and xylenes (BTEX) has garnered increasing awareness because of its serious consequences for human health and the environment. We have constructed toluene bacterial biosensors comprised of two reporter genes, gfp and luxCDABE, characterized by green fluorescence and luminescence, respectively, and compared their abilities to detect bioavailable toluene and related compounds. The bacterial luminescence biosensor allowed faster and more-sensitive detection of toluene; the fluorescence biosensor strain was much more stable and thus more applicable for long-term exposure. Both luminescence and fluorescence biosensors were field-tested to measure the relative bioavailability of BTEX in contaminated groundwater and soil samples. The estimated BTEX concentrations determined by the luminescence and fluorescence bacterial biosensors were closely comparable to each other. Our results demonstrate that both bacterial luminescence and fluorescence biosensors are useful in determining the presence and the bioavailable fractions of BTEX in the environment. - The choice of reporter genes for toluene bacterial biosensors to determine BTEX bioavailability is case-specific

  19. Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus.

    OpenAIRE

    K. Keegan; Collett, M S

    1986-01-01

    Four distinct antigenic determinants along the G2 glycoprotein encoded by the M segment RNA of the Phlebovirus Rift Valley fever virus were localized. These epitopes were defined by four monoclonal antibodies, three of which were capable of neutralizing virus infectivity; one was nonneutralizing. Immunoprecipitation by these monoclonal antibodies of either denatured or native antigen characterized the epitopes as having linear or higher order structure. Molecular cloning of G2 glycoprotein-co...

  20. Clinical score and rapid antigen detection test to guide antibiotic use for sore throats: randomised controlled trial of PRISM (primary care streptococcal management)

    OpenAIRE

    P. Little; Hobbs, F D R; Moore, M.; Mant, D; Williamson, I.; McNulty, C; Cheng, Y.E.; Leydon, G; McManus, R; Kelly, J; Barnett, J; Glasziou, P.; Mullee, M

    2013-01-01

    OBJECTIVE: To determine the effect of clinical scores that predict streptococcal infection or rapid streptococcal antigen detection tests compared with delayed antibiotic prescribing. DESIGN: Open adaptive pragmatic parallel group randomised controlled trial. SETTING: Primary care in United Kingdom. PATIENTS: Patients aged ?3 with acute sore throat. INTERVENTION: An internet programme randomised patients to targeted antibiotic use according to: delayed antibiotics (the compara...

  1. The diagnostic and prognostic value of dengue non-structural 1 antigen detection in a hyper-endemic region in indonesia

    NARCIS (Netherlands)

    Kosasih, H.; Alisjahbana, B.; Widjaja, S.; Nurhayati, .; Mast, Q. de; Parwati, I.; Blair, P.J.; Burgess, T.H.; Ven, A. van der; Williams, M.

    2013-01-01

    As dengue fever is undifferentiated from other febrile illnesses in the tropics and the clinical course is unpredictable, early diagnosis is important. Several commercial assays to detect dengue NS1 antigen have been developed; however, their performances vary and data is lacking from hyper-endemic

  2. Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

    Directory of Open Access Journals (Sweden)

    Sydow Farid FO von

    2000-01-01

    Full Text Available Fluorescent activated cell sorter (FACS analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus and Vero cells (green monkey kidney. Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML. FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+ are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

  3. A portable chemiluminescence imaging immunoassay for simultaneous detection of different isoforms of prostate specific antigen in serum.

    Science.gov (United States)

    Liu, Anran; Zhao, Fang; Zhao, Yuewu; Shangguan, Li; Liu, Songqin

    2016-07-15

    A multianalyte chemiluminescence (CL) imaging immunoassay strategy for sensitive detection of different isoforms of prostate specific antigen (PSA) was developed. The microtiter plates were fabricated by simultaneously immobilizing of free-PSA (f-PSA) and total-PSA (t-PSA) capture antibody on nitrocellulose (NC) membrane. Each of the array were spotted in replicates of six spots within a spacing of 2mm. 16 or 48 detection wells were integrated on a single NC membrane and each well could be used as a microreactor and microanalysis chamber. Under a sandwiched immunoassay, the CL signals on each sensing site were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging. Soybean peroxidase (SBP) was used to label f-PSA or t-PSA monoclonal antibody. With the amplification effects of two enhancers, 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP), the CL intensity could significantly enhanced, which improved the sensing sensitivity and detection limit. Under the optimal conditions, the linear response to the analyte concentration ranged from 0.01-36.7ng/mL and 0.02-125ng/mL for f-PSA and t-PSA, respectively. The results for the detection of forty serum samples from prostate cancer patients and cancer-free patients showed good agreement with the clinical data, suggesting that the proposed assay had acceptable accuracy. The proposed CL imaging immunoassay possess high throughput and acceptable reproducibility, stability and accuracy, which made it great potential to available to distinguish different isoforms of PSA in serum samples. PMID:26922048

  4. A Homogenous Fluorescence Quenching Based Assay for Specific and Sensitive Detection of Influenza Virus A Hemagglutinin Antigen

    Directory of Open Access Journals (Sweden)

    Longyan Chen

    2015-04-01

    Full Text Available Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs. The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs, and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs. When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human and H5 (avian. The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures.

  5. [Detection of antigen-antibody interaction of human adenovirus by the method of surface plasmon resonance].

    Science.gov (United States)

    Nosach, L M; Boltovets', P M; Povnytsia, O Iu; Zhovnovata, V L; Zakharenko, O M; Snopok, B A; Shyrshov, Iu M; Diachenko, N S

    2005-01-01

    A possibility to detect adenoviral protein--hexon, using specific antibodies by surface plasmon resonance (SPR) was demonstrated. The hexon of the human adenovirus 2 (Ad2) binds to antibodies immobilized on the sensor surface treated by KNCS and protein A Staphylococcus aureus. The specificity of antihexon antibodies was demonstrated by indirect method of fluorescent antibodies (MFA) and cellular variant of the immunoassay (cELISA). PMID:16250237

  6. The validity of rapid test to detect prostate-specific antigen (PSA in seminal fluid

    Directory of Open Access Journals (Sweden)

    Henky Henky

    2011-11-01

    Full Text Available Background: This study was conducted to determine whether the rapid test device can be used to detect PSA in seminal fluid specifically, for solving sexual assault cases.Methods: A cross sectional study has been conducted. A total of 45 samples were taken consecutively. Semen was diluted in serially up to 1:5.000.000 and male urin up to 1:200 using distilled water, whereas female urin was not diluted. Samples were analyzed using rapid test PSA.Results: The proportion of positive results of PSA in seminal fluid, male urin and female urin respectively was 100%, 6.67%, and 0%. Statistically, these differences are highly significant. The analysis revealed that the PSA rapid test device was 100% sensitive and 96.67% specific to detect seminal fluid. The test also have PPV 93.75%, NPV 100%, LR(+ 33.33, LR(- 0, and AUC 0.983.Conclusion: PSA Rapid Test is very specific and sensitive to detect PSA in seminal fluid. Therefore, this device is suggested for forensic use in sexual assault cases. (Med J Indones 2011; 20:278-82Keywords: PSA, rapid test, sexual assault

  7. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

    OpenAIRE

    Zheng Zhili; Chinnasamy Nachimuthu; Morgan Richard A

    2012-01-01

    Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymp...

  8. 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.

  9. 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.

  10. 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. PMID:27102680

  11. Validation of Serological Antibody Profiles Against Human Papillomavirus Type 16 Antigens as Markers for Early Detection of Cervical Cancer.

    Science.gov (United States)

    Salazar-Piña, Dolores Azucena; Pedroza-Saavedra, Adolfo; Cruz-Valdez, Aurelio; Ortiz-Panozo, Eduardo; Maldonado-Gama, Minerva; Chihu-Amparan, Lilia; Rodriguez-Ocampo, Angelica Nallelhy; Orozco-Fararoni, Emilia; Esquivel-Guadarrama, Fernando; Gutierrez-Xicotencatl, Lourdes

    2016-02-01

    Cervical cancer (CC) is the second most frequent neoplasia among women worldwide. Cancer prevention programs around the world have used the Papanicolaou (Pap) smear as the primary diagnostic test to reduce the burden of CC. Nevertheless, such programs have not been effective in developing countries, thus leading to research on alternative tests for CC screening. During the virus life cycle and in the process toward malignancy, different human papillomavirus (HPV) proteins are expressed, and they induce a host humoral immune response that can be used as a potential marker for different stages of the disease. We present a new Slot blot assay to detect serum antibodies against HPV16 E4, E7, and VLPs-L1 antigens. The system was validated with sera from a female population (n = 485) aged 18 to 64 years referred to the dysplasia clinic at the General Hospital in Cuautla, Morelos, Mexico. To evaluate the clinical performance of the serological markers, the sensitivity, specificity, positive, and negative predictive values and receiver-operating characteristic curves (for antibodies alone or in combination) were calculated in groups of lesions of increasing severity. The results showed high prevalence of anti-E4 (73%) and anti-E7 (80%) antibodies in the CC group. Seropositivity to 1, 2, or 3 antigens showed associations of increasing magnitude with CC (odds ratio [OR] = 12.6, 19.9, and 58.5, respectively). The highest association with CC was observed when the analysis was restricted to only anti-E4+E7 antibodies (OR = 187.7). The best clinical performance to discriminate CC from cervical intraepithelial neoplasia 2 to 3 was the one for the combination of anti-E4 and/or anti-E7 antibodies, which displayed high sensitivity (93.3%) and moderate specificity (64.1%), followed by anti-E4 and anti-E7 antibodies (73.3% and 80%; 89.6% and 66%, respectively). In addition, the sensitivity of anti-E4 and/or anti-E7 antibodies is high at any time of sexual activity (TSA

  12. Case-control comparison of bacterial and protozoan microorganisms associated with gastroenteritis: application of molecular detection.

    Science.gov (United States)

    Bruijnesteijn van Coppenraet, L E S; Dullaert-de Boer, M; Ruijs, G J H M; van der Reijden, W A; van der Zanden, A G M; Weel, J F L; Schuurs, T A

    2015-06-01

    The introduction of molecular detection of infectious organisms has led to increased numbers of positive findings, as observed for pathogens causing gastroenteritis (GE). However, because little is known about the prevalence of these pathogens in the healthy asymptomatic population, the clinical value of these additional findings is unclear. A case-control study was carried out in a population of patients served by general practitioners in the Netherlands. A total of 2710 fecal samples from case and matched control subjects were subjected to multiplex real-time PCR for the 11 most common bacterial and four protozoal causes of GE. Of 1515 case samples, 818 (54%) were positive for one or more target organisms. A total of 49% of the controls were positive. Higher positivity rates in cases compared to controls were observed for Campylobacter spp., Salmonella spp., Clostridium difficile, enteroinvasive Escherichia coli/Shigella spp., enterotoxigenic E. coli, enteroaggregative E. coli, atypical enteropathogenic E. coli (EPEC), Cryptosporidium parvum/hominis, and Giardia lamblia. However, Dientamoeba fragilis and Shiga-like toxigenic E. coli were detected significantly less frequent in cases than in controls, while no difference in prevalence was found for typical EPEC and enterohemorrhagic E. coli. The association between the presence of microorganisms and GE was the weakest in children aged 0 to 5 years. Higher relative loads in cases further support causality. This was seen for Campylobacter spp., Salmonella spp., enterotoxigenic E. coli, and C. parvum/hominis, and for certain age categories of those infected with C. difficile, enteroaggregative E. coli, and atypical EPEC. For D. fragilis and Shiga-like toxigenic E. coli/enterohemorrhagic E. coli, pathogen loads were lower in cases. Application of molecular diagnostics in GE is rapid, sensitive and specific, but results should be interpreted with care, using clinical and additional background information. PMID:25700890

  13. Direct detection of fatty acid ethyl esters using low temperature plasma (LTP) ambient ionization mass spectrometry for rapid bacterial differentiation.

    Science.gov (United States)

    Zhang, J Isabella; Costa, Anthony B; Tao, W Andy; Cooks, R Graham

    2011-08-01

    Low temperature plasma mass spectrometry (LTP-MS) was employed to detect fatty acid ethyl esters (FAEE) from bacterial samples directly. Positive ion mode FAEE mass spectrometric profiles of sixteen different bacterial samples were obtained without extraction or other sample preparation. In the range m/z 200-300, LTP mass spectra show highly reproducible and characteristic patterns. To identify the FAEE's associated with the characteristic peaks, accurate masses were recorded in the full scan mode using an LTQ/Orbitrap instrument, and tandem mass spectrometry was performed. Data were examined by principal component analysis (PCA) to determine the degree of differentiation possible amongst different bacterial species. Gram-positive and gram-negative bacteria are readily distinguished, and 11 out of 13 Salmonella strains show distinctive patterns. Growth media effects are observed but do not interfere with species recognition based on the PCA results. PMID:21706093

  14. 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

  15. COMPARISION OF DIAGNOSTIC EFFICACY OF NS1 ANTIGEN B ASED IMMUNOCHROMATOGRAPHIC TEST WITH IMMUNO-SORBENT ASSA Y AND ITS ROLE IN DETECTION OF EARLY DENGUE INFECTION

    Directory of Open Access Journals (Sweden)

    Trinain Kumar

    2012-12-01

    Full Text Available ABSTRACT:INTRODUCTION: Dengue is an acute viral infection with potential f atal complications. Rapid and easy diagnosis of dengue can help in patient triage and care- management. The detection of dengue virus NS1 antig en by rapid lateral flow tests offers a faster method to a presumptive diagnosis in the periphe ral centers of developing countries like India where the laboratory has no great technologic al backup. MATERIALS AND METHODS: A total of 351 sera from patients suspected of having dengue virus infection were tested for dengue NS1 antigen and dengue IgM and IgG antibodie s by both ELISA and immunochromatographic test (ICT. RESULTS: From the 351 samples tested, 249 (70.94% of the sera were found to be positive for DENV infectio n based on the IgM antibody or IgG antibody or NS1 antigen. Of the 249 samples 105 (42.16% were positive for only IgM or only IgG or both antibodies by ELISA and 101 (40.56% by ICT. Based on dengue NS1 antigen along with IgM and / or IgG tests, 144 (57.8% were positive for dengue infections by ELISA and 139 (55.8% by ICT. Among these 144 samples, only dengue NS1 antige n was detected in 67 (26.90% by ELISA and 65 (26.10% by ICT. CONCLUSIONS: Inclusion of NS1 in the diagnosis of dengue increa ses the detection rate significantly. The sensitivity o f ICT for both antigen and antibody detection are almost equal to ELISA. Thus, the potential use o f the NS1 antigen along with antibody tests in an ICT could increase the diagnostic efficiency for early diagnosis of dengue infection.

  16. A robust electrochemiluminescence immunoassay for carcinoembryonic antigen detection based on a microtiter plate as a bridge and Au@Pd nanorods as a peroxidase mimic.

    Science.gov (United States)

    Zhang, Yong; Pang, Xuehui; Wu, Dan; Ma, Hongmin; Yan, Zhaoqing; Zhang, Jiatao; Du, Bin; Wei, Qin

    2016-01-01

    The common drawbacks of most traditional electrochemiluminescence (ECL) immunoassays are the strict storage conditions for the ECL electrode and the steric hindrance caused by bovine serum albumin and antigen. The strict storage conditions require that the modified electrode must be stored at 4 °C before measurement, which may cause the degradation of protein molecules and low reproducibility as the time goes by. The steric hindrance can hinder electron transfer between the electrode and the electrochemical active substance unable to transmit proteins on the electrode surface. The current study takes a 96-well microtiter plate (MTP) as a bridge for analyte pre-treatment and Au@Pd nanorods as a peroxidase mimic to assemble a simple and robust ECL immunoassay. Advantages of such assay include not only high sensitivity but also robust detection circumstance. We demonstrated the method by detecting carcinoembryonic antigen from human serum and obtained a good detection limit of 3 fg mL(-1). PMID:26609799

  17. Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads.

    Science.gov (United States)

    Shen, Jun; Zhou, Yaofeng; Fu, Fen; Xu, Hengyi; Lv, Jiaofeng; Xiong, Yonghua; Wang, Andrew

    2015-09-01

    Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X-0.0059 (R(2)=0.9983) for low HBsAg concentrations between 75 pg mL(-1) and 4.8 ng mL(-1), and Y=0.8404 X-2.9364 (R(2)=0.9939) for high HBsAg concentrations in the range from 4.8 ng mL(-1) to 75 ng mL(-1). The detection limit of the proposed ICA sensor achieved was 75 pg mL(-1), which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75 pg mL(-1), 3.75 ng mL(-1) and 18.75 ng mL(-1) ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R(2)=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers. PMID:26003704

  18. Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.

    Science.gov (United States)

    Eng, Hui-Yan; Wang, Cheng-I; Xue, Yuezhen; Lee, Chia-Yin; Zulkifli, Sarah Binte; Chiam, Poh-Cheang; Ghadessy, Farid J; Lane, David P

    2016-01-01

    The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality. PMID:26508747

  19. Developments of sensitive immunoassays for detection of antibodies against hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Three micro solid phase immunoassays (a micro-SPRIA and two ELISA techniques) were developed and tested for the detection of anti-HBs antibodies. Two different crosslinkers (glutaraldehyde and N-succinimidyl 3-(2-pyridyldithio) propionate) were used to couple a goat anti-mouse IgG reagent to alkaline phosphatase for use as enzyme-labeled probes in the two ELISA tests. With the latter cross-linker, a defined conjugate with a 1 : 1 antibody-enzyme molar ratio was obtained. The sensitivities of micro-SPRIA and the two types of ELISA were compared to that of the commercial solid phase radioimmunoassay AUSAB test. All three microtests were significantly more sensitive than the AUSAB test. The ELISA using the glutaraldehyde cross-linked conjugate was 3-5 times less sensitive than micro-SPRIA, while the ELISA using the disulfide-linked conjugate was 2.6-4.0 times more sensitive than micro-SPRIA. (Auth.)

  20. Evaluation of bacterial contamination rate of the anterior chamber during phacoemulsification surgery using an automated microbial detection system

    Institute of Scientific and Technical Information of China (English)

    Ibrahim; Kocak; Funda; Kocak; Bahri; Teker; Ali; Aydin; Faruk; Kaya; Hakan; Baybora

    2014-01-01

    ·AIM: To assess the incidence of anterior chamber bacterial contamination during phacoemulsification surgery using an automated microbial detection system(BacT/Alert).·METHODS: Sixty-nine eyes of 60 patients who had uneventful phacoemulsification surgery, enrolled in this prospective study. No prophylactic topical or systemic antibiotics were used before surgery. After antisepsis with povidone-iodine, two intraoperative anterior chamber aqueous samples were obtained, the first whilst entering anterior chamber, and the second at the end of surgery. BacT/Alert culture system was used to detect bacterial contamination in the aqueous samples.·RESULTS: Neither aqueous samples obtained at the beginning nor conclusion of the surgery was positive for microorganisms on BacT/Alert culture system. The rate of bacterial contamination during surgery was 0%. None of the eyes developed acute-onset endophthalmitis after surgery.· CONCLUSION: In this study, no bacterial contamination of anterior chamber was observed during cataract surgery. This result shows that meticulous surgical preparation and technique can prevent anterior chamber contamination during phacoemulsification cataract surgery.

  1. Detection of leishmanial antigen in the urine of patients with visceral leishmaniasis by a latex agglutination test.

    Science.gov (United States)

    Sundar, Shyam; Agrawal, Shrinkhla; Pai, Kalpana; Chance, Michael; Hommel, Marcel

    2005-08-01

    Diagnosis of visceral leishmaniasis (VL) is usually done by demonstration of parasites in tissue smears. However, obtaining these smears may be risky, painful, and difficult. Antibody-based diagnostics are limited by their inability to predict active disease. In this study, a new latex agglutination test (KAtex), which detects parasite antigen in freshly voided and boiled urine, was evaluated in patients with VL before the start (n = 382) and at the end of treatment (n = 273); 185 healthy controls from leishmaniasis-endemic region were also studied. The KAtex result was positive in 87% (95% confidence interval [CI] = 83.3-90.3). However, at the end of treatment only 3% (95% CI = 1.6-6.2) patients were positive. The specificity of the test was 99% and 2 of 185 healthy controls tested positive. Positive and negative predictive values were 0.994 and 0.788, respectively. KAtex is a promising test, and in a simplified and improved format it could be applied meaningfully in the diagnosis of VL. PMID:16103587

  2. Comparison of Real Time Polymerase Chain Reaction with Microscopy and Antigen Detection Assay for the Diagnosis of Malaria

    International Nuclear Information System (INIS)

    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)

  3. Efficacy of Dot-ELISA using different antigens in detecting anti-schistosome antibodies among bovines in field conditions.

    Science.gov (United States)

    Lakshmanan, Bindu; Devada, K; Joseph, Siju; Binu, M B; Kuttan, Karthik

    2016-03-01

    Schistosomosis has been recognised as one of the major parasitic diseases of livestock and human beings. Schistosoma spindale is the major cause of visceral schistosomosis among bovines of Kerala State. Besides pathology in animals, it has been long known that cercariae of S. spindale are a common cause of dermatitis in human beings in Asia. However, detection of this disease based on coprology has underestimated the prevalence of this economically important disease among cattle of the State. An efficient diagnostic tool providing unequivocal evidence of infection in living animals is perhaps, the key to formulate and deliver control measures to the target population. It is also crucial for an enhanced understanding of parasite epidemiology. The utility of excretory-secretory proteins as diagnostic and vaccine candidates for schistosomosis has been a focus of medical research since long. There exists a paucity of information with regard to analysis of ES proteins of S. spindale and their incorporation to develop sensitive and specific serodiagnostic tool. Hence a study was designed to evaluate the efficacy of Dot-ELISA incorporating different antigens of S. spindale and to validate the test under field conditions. PMID:27065623

  4. Electrochemical immunosensor for the prostate specific antigen detection based on carbon nanotube and gold nanoparticle amplification strategy

    International Nuclear Information System (INIS)

    We describe a highly sensitive electrochemical sandwich-type immunosensor for the prostate specific antigen (PSA), a biomarker related to prostate cancer. A glassy carbon electrode was covalently modified with multi-walled carbon nanotubes to which the anti-PSA was immobilized. Following addition of a sample containing PSA, a secondary antibody is added that consists of gold nanoparticles modified with secondary antibody and 6-ferrocenyl hexanethiol acting as the signal molecule. This approach represents a multiple signal amplification strategy in that the functionalized carbon nanotubes improve the electron transfer on the surface of the electrode, while the gold nanoparticles act as carriers for capturing large quantities of Ab2 and ferrocene. The modifying processes were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimal conditions, the differential pulse voltammetric signals (acquired at a typical potential of 0.31 V vs. SCE) is linearly related to the concentration of PSA in the 10 pg.mL−1 to 100 ng.mL−1 range. The detection limit is 5.4 pg.mL−1. When applied to the determination of PSA in spiked human serum, the recoveries were between 92.4 and 120.0 %. We perceive that this immunosensor holds great promise in the field of clinical screening for cancer biomarkers. (author)

  5. Grafting of a peptide probe for Prostate-Specific Antigen detection using diazonium electroreduction and click chemistry.

    Science.gov (United States)

    Strzemińska, I; Sainte Rose Fanchine, S; Anquetin, G; Reisberg, S; Noël, V; Pham, M C; Piro, B

    2016-07-15

    The main objective of this work was to validate a label-free electrochemical method of protein detection using peptides as capture probes. As a proof-of-concept, we used a 7 amino acids sequence (HSSKLQL) specific for Prostate Specific Antigen. We investigated various electrografting conditions of two anilines (2-[(4-aminophenyl)sulfanyl]-8-hydroxy-1,4-naphthoquinone and 4-azidoaniline) further converted in situ into their corresponding diazonium salts on glassy carbon electrodes. It was demonstrated that the best method to obtain a mixed layer is the simultaneous electroreduction of the two diazonium salts. 4-azidoaniline was used to covalently immobilize the ethynyl-functionalized peptide probe by click coupling, and the hydroxynaphthoquinone derivative plays the role of electrochemical transducer of the peptide-protein recognition. The proteolytic activity of PSA towards a small peptide substrate carrying streptavidin at its distal end was also investigated to design an original sensing architecture leading to a reagentless, label free, and "signal-on" PSA sensor. Without optimization, the limit of quantification can be estimated in the nM to pM range. PMID:26938492

  6. Reassessment of the Role of Rapid Antigen Detection Tests in Diagnosis of Invasive Group A Streptococcal Infections.

    Science.gov (United States)

    Gazzano, Vincent; Berger, Anne; Benito, Yvonne; Freydiere, Anne-Marie; Tristan, Anne; Boisset, Sandrine; Carricajo, Anne; Poyart, Claire; Vandenesch, François; Descours, Ghislaine

    2016-04-01

    Rapid antigen detection tests (RADTs) for group A streptococci (GAS) are widely used for diagnosing acute pharyngitis, which has led to a considerable reduction in antibiotic prescriptions over the past decade. Beyond this intended use, their reassessment on invasive samples may be relevant in the management of life-threatening GAS infections. To this end, we evaluated the performances of three RADTs, culture, GAS PCR, and 16S rRNA gene PCR assays, and compared them with a composite gold standard (GAS-PCR assay and/or culture) for the diagnosis of severe GAS infection. A total of 192 specimens from deep-tissue (mostly normally sterile) sites enriched for 75 GAS-positive samples were enrolled in the study. The three evaluated RADTs showed sensitivities ranging from 88.0% to 94.7% versus 98.7% for GAS PCR, 84% for 16S rRNA gene PCR, and 77.3% for culture. The sensitivities of the ImmunoCardSTAT! Strep A test (Meridian Bioscience) and the NADAL Strep A strip (Nal Von Minden) were similar to that of GAS PCR (P= 0.25 and 0.03, respectively) and higher than that of culture (P= 0.001 and 0.006, respectively), whereas the SD Bioline Strep A test strip (Standard Diagnostics) showed a performance similar to that of culture (P= 0.02). The three RADTs detected 10 distinctemmtypes, including a predominance ofemm1 (33.3%),emm89 (10.6%), andemm12 (7.6%). No false-positive results were observed, leading to a specificity of 100% for all the evaluated RADTs. The GAS RADTs turned out to be sensitive, specific, and easy-to-use tools that may aid in the management of invasive GAS infections in 24/7 point-of-care laboratories by enabling early diagnosis and focused therapy. PMID:26818671

  7. Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

    Science.gov (United States)

    Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

    2002-01-01

    Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

  8. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens.

    Science.gov (United States)

    Meermeier, Erin W; Laugel, Bruno F; Sewell, Andrew K; Corbett, Alexandra J; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J; Franks, Tamera; Gold, Marielle C; Lewinsohn, David M

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  9. P48 Major Surface Antigen of Mycoplasma agalactiae Is Homologous to a malp Product of Mycoplasma fermentans and Belongs to a Selected Family of Bacterial Lipoproteins

    OpenAIRE

    Rosati, Sergio; Pozzi, Sarah; Robino, Patrizia; Montinaro, Barbara; Conti, Amedeo; Fadda, Manlio; Pittau, Marco

    1999-01-01

    A major surface antigenic lipoprotein of Mycoplasma agalactiae, promptly recognized by the host's immune system, was characterized. The mature product, P48, showed significant similarity and shared conserved amino acid motifs with lipoproteins or predicted lipoproteins from Mycoplasma fermentans, Mycoplasma hyorhinis, relapsing fever Borrelia spp., Bacillus subtilis, and Treponema pallidum.

  10. Detection of ST Segment Elevation Myocardial Infarction (STEMI Using Bacterial Foraging Optimization Technique

    Directory of Open Access Journals (Sweden)

    Bensujin

    2014-05-01

    Full Text Available The rife of heart disease (HD is a comprehensive phenomenon, and the scale of the cardiovascular disease (CVD increases in prevalence in the developed world. Cardio vascular disease (CVD is the foremost cause of death worldwide; the World Health Organization (WHO estimates that globally 17.3 million people died from Heart Disease in 2008, representing 30% of global deaths. The forecast of heart disease is a multi-layered problem, which is not free from false assumptions. The eminence of the clinical decisions and the effect of the stratagems should optimize the patient’s outcomes and to lessen the risk of disease factors, if the methods are applied effectively and properly grounded on the expert analysis on the presented data. The major clinical information related to heart disease can be obtained by the analysis for electrocardiograph (ECG signal. The ST segment Myocardial Infarction (STEMI is the severe type and the elevated ST segment on the ECG data represents that large amount of heart muscle mutilation is stirring. In this paper we recommend a constructive approach to identify the STEMI in the ECG signal of a person. The sample ECG data’s are acquired from the MIT-BIH databases. Those data’s are subsequently pre-processed; the ST segment is extracted and then measured to identify the availability of the disease. During the ST segment analysis stage the beats generated by the ventricular in origin or ventricular paced are resolute. The fine-tuned data set is converted into a formatted data set and conceded to the Bacterial Foraging Optimization Algorithm (BFOA to detect the approximate solution. The proposed system overcomes the superseded algorithms by a focussed update in the methodology with reliable algorithms and techniques.

  11. 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.

  12. Lack of detectable DNA uptake by bacterial gut isolates grown in vitro and by Acinetobacter baylyi colonizing rodents in vivo.

    Science.gov (United States)

    Nordgård, Lise; Nguyen, Thuy; Midtvedt, Tore; Benno, Yoshimi; Traavik, Terje; Nielsen, Kaare M

    2007-01-01

    Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA containing a kanamycin resistance gene (nptII). Strain BD413 was found in various gut locations in germ-free mice at 10(3)-10(5) CFU per gram GIT content 24-48 h after administration. However, subsequent DNA exposure of the colonized mice did not result in detectable bacterial transformants, with a detection limit of 1 transformant per 10(3)-10(5) bacteria. Further attempts to increase the likelihood of detection by introducing weak positive selection with kanamycin of putative transformants arising in vivo during a 4-week-long feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) fed either purified DNA (50 microg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of germfree mice was also highly inhibitory to transformation of A. baylyi, indicating that microbially-produced nucleases are not responsible for the sharp 500- to 1,000,000-fold reduction of transformation frequencies seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was examined for competence expression in vitro, without yielding any transformants. In conclusion, model choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can be measured experimentally in the GIT. Our observations suggest the contents of the GIT shield or

  13. Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems.

    OpenAIRE

    Faude, U C; Höfle, M.G.

    1997-01-01

    Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plusssee) in the spring of 1990. The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718. These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plusssee by the direct analysi...

  14. 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 ...

  15. Detection of irradiated chicken and fish meats by the determination of Gram negative bacterial count and bacterial endotoxins

    International Nuclear Information System (INIS)

    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

  16. Paraneoplastic antigen Ma2 autoantibodies as specific blood biomarkers for detection of early recurrence of small intestine neuroendocrine tumors.

    Directory of Open Access Journals (Sweden)

    Tao Cui

    Full Text Available BACKGROUND: Small intestine neuroendocrine tumors (SI-NETs belong to a rare group of cancers. Most patients have developed metastatic disease at the time of diagnosis, for which there is currently no cure. The delay in diagnosis is a major issue in the clinical management of the patients and new markers are urgently needed. We have previously identified paraneoplastic antigen Ma2 (PNMA2 as a novel SI-NET tissue biomarker. Therefore, we evaluated whether Ma2 autoantibodies detection in the blood stream is useful for the clinical diagnosis and recurrence of SI-NETs. METHODOLOGY/PRINCIPAL FINDINGS: A novel indirect ELISA was set up to detect Ma2 autoantibodies in blood samples of patients with SI-NET at different stages of disease. The analysis was extended to include typical and atypical lung carcinoids (TLC and ALC, to evaluate whether Ma2 autoantibodies in the blood stream become a general biomarker for NETs. In total, 124 blood samples of SI-NET patients at different stages of disease were included in the study. The novel Ma2 autoantibody ELISA showed high sensitivity, specificity and accuracy with ROC curve analysis underlying an area between 0.734 and 0.816. Ma2 autoantibodies in the blood from SI-NET patients were verified by western blot and sequential immunoprecipitation. Serum antibodies of patients stain Ma2 in the tumor tissue and neurons. We observed that SI-NET patients expressing Ma2 autoantibody levels below the cutoff had a longer progression and recurrence-free survival compared to those with higher titer. We also detected higher levels of Ma2 autoantibodies in blood samples from TLC and ALC patients than from healthy controls, as previously shown in small cell lung carcinoma samples. CONCLUSION: Here we show that high Ma2 autoantibody titer in the blood of SI-NET patients is a sensitive and specific biomarker, superior to chromogranin A (CgA for the risk of recurrence after radical operation of these tumors.

  17. Label-free and real-time detection of antigen-antibody interactions by Oblique-incidence Reflectivity Difference (OIRD) method

    Science.gov (United States)

    He, LiPing; Sun, Yue; Dai, Jun; Wang, JingYi; Lü, HuiBin; Wang, ShuFang; Jin, KuiJuan; Zhou, YueLiang; Yang, GuoZhen

    2012-09-01

    We label-free and real-time detected three interaction processes of antigen-antibodies, Human Immunoglobulin G (IgG), Rabbit IgG, and Mouse IgG as the targets, and Goat Anti-human IgG, Goat Anti-rabbit IgG, and Goat Anti-mouse IgG as the probe, by the Oblique-incidence Reflectivity Difference (OIRD) method. The interaction dynamic curves of the OIRD signal, corresponding to the interaction processes of antigen-antibodies, are generated. The reaction times from beginning to equilibrium state are about 1800, 900, and 1200 s for Human IgG, Rabbit IgG, and Mouse IgG, respectively. The experimental results demonstrate that the OIRD method not only can distinguish biomolecular interactions, but also can be used in real-time detection of interactions and dynamic processes of biomolecules.

  18. Laboratory and Field Evaluation of a New Rapid Test for Detecting Wuchereria bancrofti Antigen in Human Blood

    OpenAIRE

    Weil, Gary J; Curtis, Kurt C.; Fakoli, Lawrence; Fischer, Kerstin; Gankpala, Lincoln; Lammie, Patrick J; Majewski, Andrew C; Pelletreau, Sonia; Kimberly Y Won; Bolay, Fatorma K.; Fischer, Peter U.

    2013-01-01

    Global Program to Eliminate Lymphatic Filariasis (GPELF) guidelines call for using filarial antigen testing to identify endemic areas that require mass drug administration (MDA) and for post-MDA surveillance. We compared a new filarial antigen test (the Alere Filariasis Test Strip) with the reference BinaxNOW Filariasis card test that has been used by the GPELF for more than 10 years. Laboratory testing of 227 archived serum or plasma samples showed that the two tests had similar high rates o...

  19. Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

    OpenAIRE

    Alfonso Rosalba; Romero Rosa Elena; Patarroyo Manuel Elkin; Murillo Luis Angel

    2002-01-01

    In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR) amplification fragments for the precise tuberculosis (TB) diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture) test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients). T...

  20. 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

    Directory of Open Access Journals (Sweden)

    Matovina Emil

    2015-01-01

    Full Text Available Introduction. 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. Material and Methods. 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. Results. 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. Conclusion. 18F- fluorodeoxyglucose positron emission tomography-computed tomography is a powerful tool that could be used in determining colorectal cancer recurrence in

  1. 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. PMID:27156997

  2. Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

    Science.gov (United States)

    Talha, Sheikh M; Saviranta, Petri; Hattara, Liisa; Vuorinen, Tytti; Hytönen, Jukka; Khanna, Navin; Pettersson, Kim

    2016-02-01

    Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections. PMID:26711310

  3. Reduced accuracy of 14C-D-xylose breath test for detecting bacterial overgrowth in gastrointestinal motility disorders

    International Nuclear Information System (INIS)

    The accuracy of the 14C-D-xylose breath test in the diagnosis of small-bowel bacterial overgrowth was prospectively evaluated in 10 patients with motility disorders: 6 myopathic, 3 neuropathic, and 1 mechanical obstruction. 6 of 10 patients had small-bowel bacterial overgrowth on culture of small-bowel aspirate. Increased breath 14CO2 levels were documented in 3 of 6 patients with positive cultures and in 2 of 4 with negative cultures. 2 patients with positive results by both methods and 1 of 2 with positive breath 14CO2 but negative cultures had previously undergone gastric surgery. 3 patients with myopathic dysmotility had positive cultures but negative breath tests. Cultures of duodenal aspirates and the D-xylose test had sensitivities of 80% and 40%, respectively, for the finding of hypoalbuminemia. Compared with cultures, the sensitivity and specificity of the breath test were 60% and 40%, respectively. Impaired delivery of 14C-D-xylose for bacterial metabolism may result from postprandial antral hypomotility or low amplitude small-bowel motility, contributing to the false-negative breath tests. Thus, cultures is the optimal method to detect small-bowel bacterial overgrowth in patients with motility disorders. 25 refs., 1 fig., 4 tabs

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

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva

    2003-07-01

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

  5. A stable live bacterial vaccine.

    Science.gov (United States)

    Kunda, Nitesh K; Wafula, Denis; Tram, Meilinn; Wu, Terry H; Muttil, Pavan

    2016-06-01

    Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most. PMID:27020530

  6. Detection of NS1 antigen, IgM antibody for the diagnosis of dengue infection in patients with acute febrile illness

    OpenAIRE

    Arvind Neralwar; Bimala Banjare; Barapatre R.

    2015-01-01

    Background: Dengue is an endemic viral disease affecting tropical and subtropical regions around the world. Infection with any 1 of 4 dengue viruses produces with spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue haemorrhagic fever (DHF), a potentially life threatening disease. The mortality and morbidity of DHF can be reduced by early diagnosis, hospitalisation and careful supportive care. Detection of non-structural antigen (NS1...

  7. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    OpenAIRE

    M Anthony Moody; Mattia Bonsignori

    2012-01-01

    The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities ha...

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

    OpenAIRE

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

    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 9...

  9. Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid.

    OpenAIRE

    Kiska, D L; Orkiszewski, D R; Howell, D; Gilligan, P H

    1994-01-01

    We evaluated the performance of CRYPTO-LEX (Trinity Laboratories, Inc., Raleigh, N. C.), a new mouse immunoglobulin M monoclonal antibody latex agglutination reagent which reacts with the capsular polysaccharide of the four serogroups of Cryptococcus neoformans. This test was compared with CALAS (Meridian Diagnostics, Cincinnati, Ohio) for the ability to detect cryptococcal antigen in serum and cerebrospinal fluid (CSF). A total of 580 clinical specimens (327 serum and 253 CSF samples), prima...

  10. Detection of GAD-Ab index in diabetic patients using 35S labeled recombinant human GAD65 antigen

    International Nuclear Information System (INIS)

    Objective: To establish a novel method for measuring glutamic acid decarboxylase autoanti-bodies(GAD-Ab). Methods: Recombinant human GAD65 was used as the antigen, in vitro transcribed and translated 35S-GAD65 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 35S-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

  11. Validation in Uganda of antibody-detection ELISA's using plates precoated with denatured Trypanosoma congolense and Trypanosoma vivax antigen

    International Nuclear Information System (INIS)

    Indirect enzyme-linked immunosorbent assays (ELISA) for detection of trypanosomal antibodies using denatured Trypanosoma congolense and Trypanosoma vivax antigens were evaluated in Uganda. A total of 400 bovine sera were analyzed, consisting of 120 parasitologically negative samples from a tsetse-free area, 120 parasitologically positive samples, 80 samples from an area of low tsetse challenge (Kiboga) and 80 samples from of an area of medium to high tsetse challenge (Arua) of unknown disease status. Using the modified ROC analysis, cut-off points of 35% and 25% positivity were determined for the T. congolense and T. vivax assay, respectively. The T. congolense assay had a diagnostic specificity of 74%, a sensitivity of 52.5% for infections due to all trypanosome species, 81% for homogenous infections and 48% for heterogenous infections. The T. vivax assay had a diagnostic specificity of 81.3%, sensitivity of 81.3% for infections due to all trypanosome species, 76.5% for homogenous infections and 81.3% for heterogenous infections. The Buffy Coat Technique (BCT) revealed trypanosomes in none of the samples from Kiboga and in 15% from Arua. In contrast, the T. congolense assay revealed a sero-prevalence of 52.5% in Kiboga and 30% in Arua while the T. vivax assay revealed 21.3% in Kiboga and 46.3% in Arua. The T. vivax assay had a higher negative predictive value (78%) than the T. congolense assay (47%) in the area of low tsetse challenge (Kiboga) and a higher positive predictive value (75%) than the T. congolense assay (66%) in the area of medium to high tsetse challenge (Arua). The T. vivax assay appears to be more efficient than the T. congolense assay and it is potentially useful in determining the distribution bovine of trypanosomosis and targeting appropriate control measures in different areas of Uganda. (author)

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

    International Nuclear Information System (INIS)

    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

  13. The use of an antigen-detection ELISA for the diagnosis of trypanosome infections in N'dama cattle in Gambia

    International Nuclear Information System (INIS)

    An antigen-detection enzyme-linked immunosorbent assay (Ag-ELISA) for the detection of circulating trypanosomal antigens was evaluated for use in the diagnosis of trypanosomiasis in N'dama cattle in Gambia. Initial evaluation was carried out in cattle kept in areas of low or high tsetse challenge. Overall, approximately 12% of samples were parasite, but 67% showed presence of circulating antigens. The overall apparent sensitivity of the Ag-ELISA was 75%. In a separate experiment, the effects of treatment of antigenaemic N'dama cattle on weight gain and packed cell volume (PCV) was determined in animals kept under low level natural tsetse challenge. No differences were found in PCVs between drug treated and untreated animals but there was a significant weight gain in the drug treated group. Comparison of the same parameters in cattle in which parasites were detected by buffy coat technique (BCT), cattle which were Ag-ELISA positive but BCT negative, and cattle which were Ag-ELISA negative and BCT negative revealed little differences in weight, but PCVs were significantly lower in animals with a patent parasitaemia. (author). 4 refs, 4 figs, 3 tabs

  14. 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

    2016-03-16

    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. PMID:26932233

  15. Enteric bacterial pathogen detection in southern sea otters (Enhydra lutris nereis) is associated with coastal urbanization and freshwater runoff.

    Science.gov (United States)

    Miller, Melissa A; Byrne, Barbara A; Jang, Spencer S; Dodd, Erin M; Dorfmeier, Elene; Harris, Michael D; Ames, Jack; Paradies, David; Worcester, Karen; Jessup, David A; Miller, Woutrina A

    2010-01-01

    Although protected for nearly a century, California's sea otters have been slow to recover, in part due to exposure to fecally-associated protozoal pathogens like Toxoplasma gondii and Sarcocystis neurona. However, potential impacts from exposure to fecal bacteria have not been systematically explored. Using selective media, we examined feces from live and dead sea otters from California for specific enteric bacterial pathogens (Campylobacter, Salmonella, Clostridium perfringens, C. difficile and Escherichia coli O157:H7), and pathogens endemic to the marine environment (Vibrio cholerae, V. parahaemolyticus and Plesiomonas shigelloides). We evaluated statistical associations between detection of these pathogens in otter feces and demographic or environmental risk factors for otter exposure, and found that dead otters were more likely to test positive for C. perfringens, Campylobacter and V. parahaemolyticus than were live otters. Otters from more urbanized coastlines and areas with high freshwater runoff (near outflows of rivers or streams) were more likely to test positive for one or more of these bacterial pathogens. Other risk factors for bacterial detection in otters included male gender and fecal samples collected during the rainy season when surface runoff is maximal. Similar risk factors were reported in prior studies of pathogen exposure for California otters and their invertebrate prey, suggesting that land-sea transfer and/or facilitation of pathogen survival in degraded coastal marine habitat may be impacting sea otter recovery. Because otters and humans share many of the same foods, our findings may also have implications for human health. PMID:19720009

  16. [Development of multiplex PCR for fast detection of Paenibacillus larvae in putrid masses and in isolated bacterial colonies].

    Science.gov (United States)

    ruseinova, N V; Parvanov, P; Stanilova, S

    2013-01-01

    The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies' DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies. PMID:23662456

  17. Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.

    Science.gov (United States)

    Ghahremani, Hossein; Farshad, Shohreh; Amini Najafabadi, Hossein; Kashanian, Susan; Momeni Moghaddam, Mohammad Amin; Moradi, Nariman; Paknejad, Maliheh

    2015-02-01

    Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen) is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs) against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated. PMID:25530147

  18. Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Hossein Ghahremani

    2015-02-01

    Full Text Available Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.

  19. Beetroot-Pigment-Derived Colorimetric Sensor for Detection of Calcium Dipicolinate in Bacterial Spores

    OpenAIRE

    Letícia Christina Pires Gonçalves; Sandra Maria Da Silva; DeRose, Paul C.; Rômulo Augusto Ando; Erick Leite Bastos

    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 × 10(5) 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 t...

  20. CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

    OpenAIRE

    Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

    2016-01-01

    Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal an...

  1. Label-Free and Real-Time Detection of Antigen-Antibody Capture Processes Using the Oblique-Incidence Reflectivity Difference Technique

    International Nuclear Information System (INIS)

    We successfully label-free and real-time detect the capture processes of human immunoglobulin G (IgG)/goat anti-human IgG and mouse IgG/goat anti-mouse IgG antigen-antibody pairs with different concentrations using the oblique-incidence reflectivity difference (OIRD) method, and obtain the interaction kinetics curves and the interaction times. The experimental results prove that the OIRD method is a promising technique for label-free and real-time detection of the biomolecular interaction processes and achieving the quantitative information of interaction kinetics. (general)

  2. Label-Free and Real-Time Detection of Antigen-Antibody Capture Processes Using the Oblique-Incidence Reflectivity Difference Technique

    Science.gov (United States)

    He, Li-Ping; Dai, Jun; Sun, Yue; Wang, Jing-Yi; Lü, Hui-Bin; Wang, Shu-Fang; Jin, Kui-Juan; Zhou, Yue-Liang; Yang, Guo-Zhen

    2012-07-01

    We successfully label-free and real-time detect the capture processes of human immunoglobulin G (IgG)/goat anti-human IgG and mouse IgG/goat anti-mouse IgG antigen-antibody pairs with different concentrations using the oblique-incidence reflectivity difference (OIRD) method, and obtain the interaction kinetics curves and the interaction times. The experimental results prove that the OIRD method is a promising technique for label-free and real-time detection of the biomolecular interaction processes and achieving the quantitative information of interaction kinetics.

  3. Highly efficient SERS-based detection of cerebrospinal fluid neopterin as a diagnostic marker of bacterial infection.

    Science.gov (United States)

    Kamińska, Agnieszka; Witkowska, Evelin; Kowalska, Aneta; Skoczyńska, Anna; Gawryszewska, Iwona; Guziewicz, Elżbieta; Snigurenko, Dymitr; Waluk, Jacek

    2016-06-01

    A highly efficient recognition unit based on surface-enhanced Raman spectroscopy (SERS) was developed as a promising, fast, and sensitive tool for detection of meningococcal meningitis, which is an extremely serious and often fatal disease of the nervous system (an inflammation of the lining around the brain and spinal cord). The results of this study confirmed that there were specific differences in SERS spectra between cerebrospinal fluid (CSF) samples infected by Neisseria meningitidis and the normal CSF, suggesting a potential role for neopterin in meningococcal meningitis detection and screening applications. To estimate the best performance of neopterin as a marker of bacterial infection, principal component analysis (PCA) was performed in a selected region (640-720 cm(-1)) where the most prominent SERS peak at 695 cm(-1) arising from neopterin was observed. The calculated specificity of 95 % and sensitivity of 98 % clearly indicate the effective diagnostic efficiency for differentiation between infected and control samples. Additionally, the limit of detection (LOD) of neopterin in CSF clinical samples was estimated. The level of neopterin was significantly higher in CSF samples infected by N. meningitidis (48 nmol/L), compared to the normal (control) group (4.3 nmol/L). Additionally, this work presents a new type of SERS-active nanostructure, based on polymer mats, that allows simultaneous filtration, immobilization, and enhancement of the Raman signal, enabling detection of spectra from single bacterial cells of N. meningitidis present in CSF samples. This provides a new possibility for fast and easy detection of bacteria in CSF and other clinical body fluids on a time scale of seconds. This method of detection produces consistent results faster and cheaper than traditional laboratory techniques, demonstrates the powerful potential of SERS for detection of disease, and shows the viability of future development in healthcare applications. PMID

  4. Vaginal Microflora Associated With Bacterial Vaginosis in Nonpregnant Women: Reliability of Sialidase Detection

    Directory of Open Access Journals (Sweden)

    Hebe Bianchini

    2001-01-01

    Full Text Available Objective: To determine the prevalence of Gardnerella vaginalis, anaerobic bacteria and Mycoplasma hominis in vaginal specimens of women with and without bacterial vaginosis (BV as well as to determine the sensitivity and specificity of the direct sialidase assay of vaginal fluid as a rapid test for diagnosing this syndrome.

  5. System automation for a bacterial colony detection and identification instrument via forward scattering

    International Nuclear Information System (INIS)

    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

  6. Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

    OpenAIRE

    Jake E Lowry; Isaak, Dale D.; Leonhardt, Jack A.; Giulia Vernati; Jessie C Pate; Andrews, Gerard P.

    2011-01-01

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for ...

  7. Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r in patients with cutaneous leishmaniasis in Brazil

    Directory of Open Access Journals (Sweden)

    Felix Javier Leon Molinet

    2013-05-01

    Full Text Available The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL. This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect(r(InBios, Seattle, WA recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer's instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.

  8. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    Directory of Open Access Journals (Sweden)

    Vojnov Adrian A

    2010-06-01

    Full Text Available Abstract Background Citrus Bacterial Canker (CBC is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP, which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA

  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

    -PAGE as high molecular weight ([greater than or equal to] 70 kDa) or low molecular weight (< 70 kDa). The number of discernable low molecular weight parasite antigens detected by different IgG subclass antibodies from each plasma sample was recorded. Using Wilcoxons rank sum test these reactivities were...... compared amongst 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 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...

  10. Induction/Engineering, Detection, Selection, and Expansion of Clinical-Grade Human Antigen-Specific CD8+ Cytotoxic T Cell Clones for Adoptive Immunotherapy

    Directory of Open Access Journals (Sweden)

    Matjaž Jeras

    2010-01-01

    Full Text Available Adoptive transfer of effector antigen-specific immune cells is becoming a promising treatment option in allogeneic transplantation, infectious diseases, cancer, and autoimmune disorders. Within this context, the important role of CD8+ cytotoxic T cells (CTLs is objective of intensive studies directed to their in vivo and ex vivo induction, detection, selection, expansion, and therapeutic effectiveness. Additional questions that are being addressed by the scientific community are related to the establishment and maintenance of their longevity and memory state as well as to defining critical conditions underlying their transitions between discrete, but functionally different subtypes. In this article we review and comment latest approaches and techniques used for preparing large amounts of antigen-specific CTLs, suitable for clinical use.

  11. Predictive value of serum prostate specific antigen in detecting bone metastasis in prostate cancer patients using bone scintigraphy

    International Nuclear Information System (INIS)

    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

  12. Tuberculin Skin Testing Compared with T-Cell Responses to Mycobacterium tuberculosis-Specific and Nonspecific Antigens for Detection of Latent Infection in Persons with Recent Tuberculosis Contact

    Science.gov (United States)

    Arend, Sandra M.; Engelhard, Anrik C. F.; Groot, Gertjan; de Boer, Kirsten; Andersen, Peter; Ottenhoff, Tom H. M.; van Dissel, Jaap T.

    2001-01-01

    The tuberculin skin test (TST) is used for the identification of latent tuberculosis (TB) infection (LTBI) but lacks specificity in Mycobacterium bovis BCG-vaccinated individuals, who constitute an increasing proportion of TB patients and their contacts from regions where TB is endemic. In previous studies, T-cell responses to ESAT-6 and CFP-10, M. tuberculosis-specific antigens that are absent from BCG, were sensitive and specific for detection of active TB. We studied 44 close contacts of a patient with smear-positive pulmonary TB and compared the standard screening procedure for LTBI by TST or chest radiographs with T-cell responses to M. tuberculosis-specific and nonspecific antigens. Peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, TB10.4 (each as recombinant antigen and as a mixture of overlapping synthetic peptides), M. tuberculosis sonicate, purified protein derivative (PPD), and short-term culture filtrate, using gamma interferon production as the response measure. LTBI screening was by TST in 36 participants and by chest radiographs in 8 persons. Nineteen contacts were categorized as TST negative, 12 were categorized as TST positive, and 5 had indeterminate TST results. Recombinant antigens and peptide mixtures gave similar results. Responses to TB10.4 were neither sensitive nor specific for LTBI. T-cell responses to ESAT-6 and CFP-10 were less sensitive for detection of LTBI than those to PPD (67 versus 100%) but considerably more specific (100 versus 72%). The specificity of the TST or in vitro responses to PPD will be even less when the proportion of BCG-vaccinated persons among TB contacts evaluated for LTBI increases. PMID:11687445

  13. 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.

  14. 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.

  15. Detection of Immune-Complex Dissociated Nonstructural-1 (NS-1) Antigen in Patients with Acute Dengue Virus Infections

    NARCIS (Netherlands)

    P. Koraka (Penelopie); 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

  16. Use of Novel Recombinant Antigens in the Interferon Gamma Assay for Detection of Mycobacterium Avium Subsp. Paratuberculosis Infection in Cattle

    DEFF Research Database (Denmark)

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

    . The study included blood samples from 26 heifers of a MAP infected herd, collected three times with 4 and 5 week interval and blood samples from 60 heifers of a MAP non-infected herd collected once. The IFN-γ responses of the non-infected heifers were used to establish cut-off values for each antigen...

  17. Novel antigens for detection of cell mediated immune responses to Mycobacterium avium subsp. paratuberculosis infection in cattle

    DEFF Research Database (Denmark)

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

    2011-01-01

    included blood samples from 26 heifers from a MAP infected herd, collected three times with four to five-week intervals, and blood samples from 60 heifers of a non-infected herd collected once. Heifers of the non-infected herd were used to establish cut-off values for each antigen. The case definition was...

  18. 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;

    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...

  19. 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 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.

  20. Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model.

    Science.gov (United States)

    Tabenski, L; Maisch, T; Santarelli, F; Hiller, K-A; Schmalz, G

    2014-11-01

    Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l(-1) sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0-53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing. PMID:25119373

  1. PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

    Science.gov (United States)

    LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

    2012-01-01

    The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

  2. Development of an edema factor-mediated cAMP-induction bioassay for detecting antibody-mediated neutralization of anthrax protective antigen.

    Science.gov (United States)

    Zmuda, Jonathan F; Zhang, Linyi; Richards, Terri; Pham, Quyen; Zukauskas, David; Pierre, Jennifer L; Laird, Michael W; Askins, Janine; Choi, Gil H

    2005-03-01

    Intoxication of mammalian cells by Bacillus anthracis requires the coordinate activity of three distinct bacterial proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Among these proteins, PA has become the major focus of work on monoclonal antibodies and vaccines designed to treat or prevent anthrax infection since neither EF nor LF is capable of inducing cellular toxicity in its absence. Here, we present the development of a sensitive, precise, and biologically relevant bioassay platform capable of quantifying antibody-mediated PA neutralization. This bioassay is based on the ability of PA to bind and shuttle EF, a bacterial adenylate cyclase, into mammalian cells leading to an increase in cAMP that can be quantified using a sensitive chemiluminescent ELISA. The results of this study indicate that the cAMP-induction assay possesses the necessary performance characteristics for use as both a potency-indicating release assay in a quality control setting and as a surrogate pharmacodynamic marker for ensuring the continued bioactivity of therapeutic antibodies against PA during clinical trials. PMID:15847796

  3. Determination of the Normal Prostate Specific Antigen (PSA) Value in Iraqi Society and its Relation to Bacterial Urinary Tract Infection (UTI)

    International Nuclear Information System (INIS)

    The study was carried out by radioimmunoassay and immuno radiographic analysis in the Iraqi Ministry of Health, within the research plan of Kurdistan institution for strategic study and scientific research. A total of 793 serum samples were collected in which 50 patient samples have biopsy with positive bacterial UTI. The other 743 samples were obtained from normal healthy volunteers all were over 45 years old. The samples were gathered randomly from three regions in Iraq namely, from north (Sulaymaniyah, Erbil and Dohuk), from the middle (Baghdad and Diyala) and from south (Basra, Missan and Najaf). The total PSA was measured and the results were subjected to statistical analysis based on statistical package social science (SPSS) method. The obtained data showed that the normal PSA values are function of the age of the donors. The results were grouped and clarified that PSA was less than 3.8 ng/ml for the age 45-55 years, while it was less than 4.8 ng/ml for the volunteers from 56-65 years old and the values lower than 5.9 ng/ml for group aged 66-75 years old. On the other hand, the obtained data illustrated that there were non-significant variations in PSA values as a function of the geographic regions. The PSA values for the 50 male positive bacterial UTI samples were within the same grouping previously stated for the normal healthy volunteers. Seven cases of the 743 samples showed abnormal high PSA values (i.e. greater than 9 ng/ml) which represent 0.93% of the healthy collected samples. It could be concluded that the PSA has non-significance relation to the bacterial UTI. In addition, the radioimmunoassay has a sensitivity of about 99.04% for the normal cases and specificity of 0.96% for prostate cancer.

  4. Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients.

    Science.gov (United States)

    Onori, Manuela; Coltella, Luana; Mancinelli, Livia; Argentieri, Marta; Menichella, Donato; Villani, Alberto; Grandin, Annalisa; Valentini, Diletta; Raponi, Massimiliano; Russo, Cristina

    2014-06-01

    We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations. PMID:24656922

  5. The use of magnetic resonance and MR angiography in the detection of cerebral infarction: A complication of pediatric bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Stošić-Opinćal Tatjana

    2005-01-01

    Full Text Available Bacground. Association of both cerebral infarction and acute bacterial meningitis is more common in younger patients than in the elderly. The rate of mortality and the frequency of sequel are very high inspite of the use of modern antibiotic therapy. In more than 30% of the cases of childhood bacterial meningitis, both arterial and venous infarctions can occur. The aim of this study was to present the role of the use of magnetic resonance (MRI, and MR angiography (MRA in the detection of bacterial meningitis in children complicated with cerebral infarctions. Method. In the Centre for MR, the Clinical Centre of Serbia, 25 patients with the diagnosis of bacterial meningitis, of which 9 children with cerebral infarction whose clinical conditon deteriorated acutely, despite the antibiotic therapy, underwent MRI and MR angiography examination on a 1T scanner. Examination included the conventional spin-echo techniques with T1-weighted saggital and coronal, and T2- weighted axial and coronal images. Coronal fluid attenuated inversion recovery (FLAIR and the postcontrast T1-weighted images in three orthogonal planes were also used. The use MR angiography was accomplished by the three-dimensional time-of-flight (3D TOF technique. Results. The findings included: multiple hemorrhagic infarction in 4 patients, multiple infarctions in 3 patients, focal infarction in 1 patient and diffuse infarction (1 patient. Common sites of involvement were: the frontal lobes, temporal lobes and basal ganglia. The majority of infarctions were bilateral. In 3 of the patients empyema was found, and in 1 patient bitemporal abscess was detected. In 8 of the patients MR angiography confirmed inflammatory vasculitis. Conclusion. Infarction is the most common sequel of severe meningitis in children. Since the complication of cerebral infarction influences the prognosis of meningitis, repetitive MRI examinations are very significant for the evaluation of the time course of

  6. LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

    Directory of Open Access Journals (Sweden)

    Priya Natarajan

    2012-05-01

    Full Text Available Abstract Background Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH, is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. Results In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers while the other a secondary endosymbiont Arsenophonus (and present in less numbers. Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. Conclusion By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction

  7. Prostate cancer detection upon transrectal ultrasound-guided biopsy in relation to digital rectal examination and prostate-specific antigen level: what to expect in the Chinese population?

    Directory of Open Access Journals (Sweden)

    Jeremy YC Teoh

    2015-01-01

    Full Text Available We investigated the prostate cancer detection rates upon transrectal ultrasound (TRUS-guided biopsy in relation to digital rectal examination (DRE and prostate-specific antigen (PSA, and risk factors of prostate cancer detection in the Chinese population. Data from all consecutive Chinese men who underwent first TRUS-guided prostate biopsy from year 2000 to 2013 was retrieved from our database. The prostate cancer detection rates with reference to DRE finding and PSA level of 50 ng ml−1 were investigated. Multivariate logistic regression analyses were performed to investigate for potential risk factors of prostate cancer detection. A total of 2606 Chinese men were included. In patients with normal DRE, the cancer detection rates were 8.6%, 13.4%, 21.8%, 41.7% and 85.2% in patients with PSA 50 ng ml−1 respectively. In patients with abnormal DRE, the cancer detection rates were 12.4%, 30.2%, 52.7%, 80.6% and 96.4% in patients with PSA 50 ng ml−1 respectively. Older age, smaller prostate volume, larger number of biopsy cores, presence of abnormal DRE finding and higher PSA level were associated with increased risk of prostate cancer detection upon multivariate logistic regression analyses (P < 0.001. Chinese men appeared to have lower prostate cancer detection rates when compared to the Western population. Taking the different risk factors into account, an individualized approach to the decision of TRUS-guided biopsy can be adopted.

  8. A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads.

    Science.gov (United States)

    Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying

    2016-08-01

    A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%. PMID:27403652

  9. Clinical experience with cytomegalovirus isolation using conventional cell cultures and early antigen detection in centrifugation-enhanced shell vial cultures.

    OpenAIRE

    Leland, D S; Hansing, R L; French, M L

    1989-01-01

    A total of 1,915 clinical samples was inoculated by low-speed centrifugation into shell vials (Bartels Immunodiagnostics, Bellvue, Wash.) containing cover slip monolayers of MRC-5 fibroblasts. At 1 and 2 days postinoculation, one cover slip was stained by an indirect immunofluorescence technique using a monoclonal antibody (Biotech Research Laboratories for Dupont, Billerica, Mass.) to cytomegalovirus (CMV) early antigen (EA). Clinical samples were also inoculated into three MRC-5 or MRHF cel...

  10. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: application to diagnosis and blood screening for posttransfusion hepatitis.

    OpenAIRE

    Miyamura, T; Saito, I; Katayama, T; Kikuchi, S; Tateda, A; Houghton, M; Choo, Q L; Kuo, G

    1990-01-01

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatitis (NANBH). We have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) p...

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

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans; Lernmark, A

    1985-01-01

    detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry...... showed specific surface immunofluorescence in 1:100-1:1000 dilutions in lymphoblastoid and blood mononucleated cells. In the latter the binding was primarily confined to monocytes and a subpopulation of lymphocytes. It is concluded that locus-specific immunological reagents to distinguish between beta...

  12. Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen▿

    OpenAIRE

    Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi

    2007-01-01

    Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody ...

  13. Urinary microRNA-based signature improves accuracy of detection of clinically relevant prostate cancer within the prostate-specific antigen grey zone

    OpenAIRE

    SALIDO-GUADARRAMA, ALBERTO IVAN; MORALES-MONTOR, JORGE GUSTAVO; Rangel-Escareño, Claudia; Langley, Elizabeth; Peralta-Zaragoza, Oscar; COLIN, JOSE LUIS CRUZ; Rodriguez-dorantes, Mauricio

    2016-01-01

    At present, prostate-specific antigen (PSA) is used as a clinical biomarker for prostate cancer (PCa) diagnosis; however, a large number of patients with benign prostate hyperplasia (BPH) with PSA levels in the ʻgray areaʼ (4–10 ng/ml) are currently subjected to unnecessary biopsy due to overdiagnosis. Certain microRNAs (miRs) have been proven to be useful biomarkers, several of which are detectable in bodily fluids. The present study identified and validated a urinary miR-based signature to ...

  14. Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue

    DEFF Research Database (Denmark)

    Tingstedt, Jens Erik; Tornehave, Ditte; Lind, Peter;

    2003-01-01

    inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded...... detection of several major cell types of the porcine immune system in fixed tissue with optimal preservation of histological details.......Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with...

  15. Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.

    OpenAIRE

    Knowles, D P; Evermann, J F; Shropshire, C; VanderSchalie, J; Bradway, D; Gezon, H M; Cheevers, W P

    1994-01-01

    The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test ...

  16. Streptomycin application has no detectable effect on bacterial community structure in apple orchard soil.

    Science.gov (United States)

    Shade, Ashley; Klimowicz, Amy K; Spear, Russell N; Linske, Matthew; Donato, Justin J; Hogan, Clifford S; McManus, Patricia S; Handelsman, Jo

    2013-11-01

    Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 μg/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielou's evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria. PMID:23974143

  17. Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections.

    Science.gov (United States)

    Wohlwend, Nadia; Tiermann, Sacha; Risch, Lorenz; Risch, Martin; Bodmer, Thomas

    2016-09-01

    A total of 1,056 native or Cary-Blair-preserved stool specimens were simultaneously tested by conventional stool culturing and by enteric bacterial panel (EBP) multiplex real-time PCR for Campylobacter jejuni, Campylobacter coli, Salmonella spp., and shigellosis disease-causing agents (Shigella spp. and enteroinvasive Escherichia coli [EIEC]). Overall, 143 (13.5%) specimens tested positive by PCR for the targets named above; 3 coinfections and 109 (10.4%) Campylobacter spp., 17 (1.6%) Salmonella spp., and 20 (1.9%) Shigella spp./EIEC infections were detected. The respective positive stool culture rates were 75 (7.1%), 14 (1.3%), and 7 (0.7%). The median threshold cycle (CT) values of culture-positive specimens were significantly lower than those of culture-negative ones (CT values, 24.3 versus 28.7; P Campylobacter infections, the respective median fecal calprotectin concentrations in PCR-negative/culture-negative (n = 40), PCR-positive/culture-negative (n = 14), and PCR-positive/culture-positive (n = 15) specimens were 134 mg/kg (interquartile range [IQR], 30 to 1,374 mg/kg), 1,913 mg/kg (IQR, 165 to 3,813 mg/kg), and 5,327 mg/kg (IQR, 1,836 to 18,213 mg/kg). Significant differences were observed among the three groups (P Campylobacter spp., Salmonella spp., and Shigella spp./EIEC using the BD Max EBP assay will result in timely diagnosis and improved sensitivity. The determination of inflammatory markers, such as calprotectin, in fecal specimens may aid in the interpretation of PCR results, particularly for enteric pathogens associated with mucosal damage and colonic inflammation. PMID:27307458

  18. Total internal reflection plasmonic scattering-based fluorescence-free nanoimmunosensor probe for ultra-sensitive detection of cancer antigen 125.

    Science.gov (United States)

    Chakkarapani, Suresh Kumar; Zhang, Peng; Ahn, Sujin; Kang, Seong Ho

    2016-07-15

    Highly sensitive detection of cancer antigen 125 (CA125) on nanoarray chips was carried out by means of total internal reflection (TIR) microscopy based on fluorescent labeling (i.e., TIR fluorescence microscopy; TIRFM) and fluorescent-free labeling (TIR scattering microscopy; TIRSM). TIR plasmonic scattering of nanoparticles (NPs) as a fluorescence-free immunosensor probe potentially superior to fluorescent probes was applied to quantify CA125 on a nanoarray chip. NP-labeled CA125 (NP-CA125) was immunoreacted on chips, and the TIR scattering illumination of NP-CA125 allowed quantitative TIRSM measurement of wavelength-dependent plasmonic scattering detection of CA125. In addition, Alexafluor 488-labeled CA125 was immunoreacted on the same chips for comparison of detection sensitivity. TIRSM showed less photobleaching and higher photostability and detection sensitivity than TIRFM, as well as a lower limit of detection (LOD), 0.0018U/mL. This LOD was ~144 times lower than that of previously reported detection methods. These results demonstrated that the wavelength-dependent TIR plasmon NPs can be used as an enhanced nanoimmunosensor probe, providing ultra-sensitive fluorescence-free biomolecule detection to enable earliest-stage disease diagnosis. PMID:26913504

  19. Identification of antigenic proteins of the nosocomial pathogen Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Sebastian Hoppe

    Full Text Available The continuous expansion of nosocomial infections around the globe has become a precarious situation. Key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. Thus, new ways to rapidly detect these infections are vital. Consequently, researchers around the globe pursue innovative approaches for point-of-care devices. In many cases the specific interaction of an antigen and a corresponding antibody is pivotal. However, the knowledge about suitable antigens is lacking. The aim of this study was to identify novel antigens as specific diagnostic markers. Additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. Hence, a cDNA-based expression library was constructed and screened via microarrays to detect novel antigens of Klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (ESBL. After screening 1536 clones, 14 previously unknown immunogenic proteins were identified. Subsequently, each protein was expressed in full-length and its immunodominant character examined by ELISA and microarray analyses. Consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. After specificity analysis, homology survey and 3d structural modelling, one epitope sequence GAVVALSTTFA of KPN_00363, an ion channel protein, was identified harboring specificity for K. pneumoniae. The remaining epitopes showed ambiguous results regarding the specificity for K. pneumoniae. The approach adopted herein has been successfully utilized to discover novel antigens of Campylobacter jejuni and Salmonella enterica antigens before. Now, we have transferred this knowledge to the key nosocomial agent, K. pneumoniae. By identifying several novel antigens

  20. Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

    DEFF Research Database (Denmark)

    Mezger, Anja; Fock, Jeppe; Antunes, Paula Soares Martins;

    2015-01-01

    and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks...

  1. 确证TRFIA检测乙肝病毒表面抗体结果的研究%Detection of Hepatitis B Surface Antigen and Antibody by TRFIA

    Institute of Scientific and Technical Information of China (English)

    吉飞跃; 钱开成

    2011-01-01

    建立简易时间分辨免疫荧光法(TRFIA)检测乙型肝炎病毒表面抗体(抗-HBs)的中和试验方法,验证TRFIA检测结果.收集乙型肝炎病毒表面抗原(HBsAg)异常值血清,确定TRFIA中和抑制试验中和比为0.25-1.26ng/mL∶1mIU/mL,对抗-HBs的定量结果进行确认.结果显示,使用TRFIA定量检测的结果与中和抑制试验的结果符合率达100%.结论:使用中和抑制试验确认TRFIA定量检测乙型肝炎病毒(HBV)表面抗体的检测结果,可以排除非特异反应导致的结果.%To establish a simple neutralization test to detect hepatitis B surface antigen and antibody (anti-HBs) by TRFIA. The serum samples with outlier serum of heptatitis B surface antigen (HBsAg) were collected, and the neutralization inhibition test was carried out to confirm the quantitative result of anti-HBs which analysis by TRFIA. The results showed that the coincidence rate of anti-HBs analysis by TRFIA quantitative test and neu- tralization inhibition test was 100%. The neutralization inhibition test could be used to confirm the detection re- suits of hepatitis B surface antigen and antibody by TRFIA and to exclude the result of non-specific reaction.

  2. Schistosoma mansoni Infections in young children: when are schistosome antigens in urine, eggs in stool and antibodies to eggs first detectable?

    Directory of Open Access Journals (Sweden)

    J Russell Stothard

    Full Text Available BACKGROUND: in uganda, control of intestinal schistosomiasis with preventive chemotherapy is typically focused towards treatment of school-aged children; the needs of younger children are presently being investigated as in lakeshore communities very young children can be infected. In the context of future epidemiological monitoring, we sought to compare the detection thresholds of available diagnostic tools for Schistosoma mansoni and estimate a likely age of first infection for these children. METHODS AND FINDINGS: a total of 242 infants and preschool children (134 boys and 108 girls, mean age 2.9 years, minimum 5 months and maximum 5 years were examined from Bugoigo, a well-known disease endemic village on Lake Albert. Schistosome antigens in urine, eggs in stool and host antibodies to eggs were inspected to reveal a general prevalence of 47.5% (CI(95 41.1-54.0%, as ascertained by a positive criterion from at least one diagnostic method. Although children as young as 6 months old could be found infected, the average age of infected children was between 3¼-3¾ years, when diagnostic techniques became broadly congruent. CONCLUSION: whilst different assays have particular (disadvantages, direct detection of eggs in stool was least sensitive having a temporal lag behind antigen and antibody methods. Setting precisely a general age of first infection is problematic but if present Ugandan policies continue, a large proportion of infected children could wait up to 3-4 years before receiving first medication. To better tailor treatment needs for this younger ageclass, we suggest that the circulating cathodic antigen urine dipstick method to be used as an epidemiological indicator.

  3. A Label-Free Microelectrode Array Based on One-Step Synthesis of Chitosan–Multi-Walled Carbon Nanotube–Thionine for Ultrasensitive Detection of Carcinoembryonic Antigen

    Directory of Open Access Journals (Sweden)

    Huiren Xu

    2016-07-01

    Full Text Available Carcinoembryonic antigen (CEA has been an extensively used tumor marker responsible for clinical early diagnosis of cervical carcinomas, and pancreatic, colorectal, gastric and lung cancer. Combined with micro-electro mechanical system (MEMS technology, it is important to develop a novel immune microelectrode array (MEA not only for rapid analysis of serum samples, but also for cell detection in vitro and in vivo. In this work, we depict a simple approach to modify chitosan–multi-walled carbon nanotubes–thionine (CS–MWCNTs–THI hybrid film through one-step electrochemical deposition and the CS-MWCNTs-THI hybrid films are successfully employed to immobilize anti-CEA for fabricating simple, label-free, and highly sensitive electro-chemical immune MEAs. The detection principle of immune MEA was based on the fact that the increasing formation of the antigen-antibody immunocomplex resulted in the decreased response currents and the relationship between the current reductions with the corresponding CEA concentrations was directly proportional. Experimental results indicated that the label-free MEA had good selectivity and the limit of detection for CEA is 0.5 pg/mL signal to noise ratio (SNR = 3. A linear calibration plot for the detection of CEA was obtained in a wide concentration range from 1 pg/mL to 100 ng/mL (r = 0.996. This novel MEA has potential applications for detecting CEA for the research on cancer cells and cancer tissue slices as well as for effective early diagnosis.

  4. Detecting bacterial magnetite in sediments: strengths and limitations of FMR spectroscopy

    Science.gov (United States)

    Winklhofer, M.

    2012-04-01

    Ferromagnetic resonance spectroscopy (FMR) is increasingly being used as a diagnostic tool for identifying bacterial magnetite in sediments [e.g., Kopp et al. 2007; Kind et al. 2011, Roberts et al. 2011 ], the reason being that magnetic bacteria have a characteristic FMR fingerprint which is not known from inorganic geological samples [Kopp & Kirschvink, 2008]. The diagnostic FMR features of single-stranded magnetite chains are a g-value 2, quite opposite to what we know from single-stranded chains. Therefore, in order to better understand possible biogenic FMR fingerprints and to refine the screen, there is a clear need to acquire FMR spectra of magnetic bacteria with different chain configurations and, in particular, of greigite producing bacteria.

  5. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10{sup 3}A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

  6. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens

    International Nuclear Information System (INIS)

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 103A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater

  7. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens.

    Science.gov (United States)

    Lee, Dae-Young; Lauder, Heather; Cruwys, Heather; Falletta, Patricia; Beaudette, Lee A

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10(3)A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater. PMID:18423816

  8. A locked nucleic acid (LNA-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    Directory of Open Access Journals (Sweden)

    Lingxiang Zhu

    Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  9. Low-fouling surface plasmon resonance biosensor for multi-step detection of foodborne bacterial pathogens in complex food samples.

    Science.gov (United States)

    Vaisocherová-Lísalová, Hana; Víšová, Ivana; Ermini, Maria Laura; Špringer, Tomáš; Song, Xue Chadtová; Mrázek, Jan; Lamačová, Josefína; Scott Lynn, N; Šedivák, Petr; Homola, Jiří

    2016-06-15

    Recent outbreaks of foodborne illnesses have shown that foodborne bacterial pathogens present a significant threat to public health, resulting in an increased need for technologies capable of fast and reliable screening of food commodities. The optimal method of pathogen detection in foods should: (i) be rapid, specific, and sensitive; (ii) require minimum sample preparation; and (iii) be robust and cost-effective, thus enabling use in the field. Here we report the use of a SPR biosensor based on ultra-low fouling and functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes for the rapid and sensitive detection of bacterial pathogens in crude food samples utilizing a three-step detection assay. We studied both the surface resistance to fouling and the functional capabilities of these brushes with respect to each step of the assay, namely: (I) incubation of the sensor with crude food samples, resulting in the capture of bacteria by antibodies immobilized to the pCBAA coating, (II) binding of secondary biotinylated antibody (Ab2) to previously captured bacteria, and (III) binding of streptavidin-coated gold nanoparticles to the biotinylated Ab2 in order to enhance the sensor response. We also investigated the effects of the brush thickness on the biorecognition capabilities of the gold-grafted functionalized pCBAA coatings. We demonstrate that pCBAA-compared to standard low-fouling OEG-based alkanethiolate self-assemabled monolayers-exhibits superior surface resistance regarding both fouling from complex food samples as well as the non-specific binding of S-AuNPs. We further demonstrate that a SPR biosensor based on a pCBAA brush with a thickness as low as 20 nm was capable of detecting E. coli O157:H7 and Salmonella sp. in complex hamburger and cucumber samples with extraordinary sensitivity and specificity. The limits of detection for the two bacteria in cucumber and hamburger extracts were determined to be 57 CFU/mL and 17 CFU/mL for E. coli and 7.4 × 10

  10. Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus.

    Science.gov (United States)

    de Rezende, N A; Dias, M B; Campolina, D; Chavéz-Olortegui, C; Amaral, C F

    1995-01-01

    The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients. PMID:7569644

  11. Seven Human Immunodeficiency Virus (HIV) Antigen-Antibody Combination Assays: Evaluation of HIV Seroconversion Sensitivity and Subtype Detection

    OpenAIRE

    Ly, Thoai Duong; Martin, Lynn; Daghfal, David; Sandridge, Arnold; West, Daniel; Bristow, Richard; Chalouas, Laurence; Qiu, Xiaoxing; Lou, Sheng C.; Hunt, Jeffrey C.; Schochetman, Gerald; Devare, Sushil G.

    2001-01-01

    In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability t...

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

    Science.gov (United States)

    Yeargan, Michelle R; Howe, Daniel K

    2011-02-28

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

  13. Evaluation of prostate cancer antigen 3 for detecting prostate cancer: a systematic review and meta-analysis

    OpenAIRE

    Cui, Yong; Cao, Wenzhou; Li, Quan; Shen, Hua; Liu, Chao; Deng, Junpeng; Xu, Jiangfeng; Shao, Qiang

    2016-01-01

    Previous studies indicate that prostate cancer antigen 3 (PCA3) is highly expressed in prostatic tumors. However, its clinical value has not been characterized. The aim of this study was to investigate the clinical value of the urine PCA3 test in the diagnosis of prostate cancer by pooling the published data. Clinical trials utilizing the urine PCA3 test for diagnosing prostate cancer were retrieved from PubMed and Embase. A total of 46 clinical trials including 12,295 subjects were included ...

  14. 快速检测流感病毒抗原方法的建立%Establishment of Rapid detection Method for influenza virus antigen

    Institute of Scientific and Technical Information of China (English)

    李月越; 陈杭薇; 王萍; 彭文鸿; 王瑞娟; 霍秀青; 胡美

    2011-01-01

    目的 建立有效、简便的胶体金免疫层析方法(GICA)快速检测流感病毒感染抗原方法.方法 分别对1145例急性呼吸道疾病住院患者应用GICA法检测患者鼻咽部分泌物的流感病毒A、B(FluA、B)抗原,并同时用IFA法作对照,对GICA法做出评价.对以上患者分别应用鼻、咽拭子法和鼻咽部细导管负压吸引法两种不同方法采集患者鼻咽部分泌物,均经GICA法快速检测分泌物标本中的Flu A、B抗原,对两种不同采集方法的结果做出评价.结果 以上两种检测方法及采集方法所得结果,均有较好的一致性,之间差异无统计学意义(P>0.05).结论 鼻、咽拭子采集法配合GICA法是简便、快捷、敏感的检测流感病毒抗原的好方法.%Objective To set up an effective and simple method, GICA, for rapid detection of influenza virus infection. Methods 1145 patients with different types of respiratory diseases were selected for check. GICA assay was used to detect influenza virus A/B (FluA/B) antigen in nasopharyngeal secretions of patients ,in the meanwhile comparing with IFA ,for evaluating the diagnostic test of GICA method. Nasopharyngeal secretions from above-mentioned patients were collected by the method of nasopharynx swab or nasopharyngeal suction catheter. Both methods were applied with GICA kit to detect rapidly FluA/B antigen in secretion specimens,then the evaluation to the results of different collecting methods was assessed. Results The above two kinds of detecting methods and the results of collecting methods have good consistency. The difference between them has no statistical significance( P >0. 05 ). Conclusions Collection by nasopharynx swab with the GICA method is simple,fast,sensitive and good way to detect influenza virus antigen.

  15. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

    Science.gov (United States)

    Altinok, Ilhan; Capkin, Erol; Kayis, Sevki

    2008-10-15

    A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method. PMID:18499358

  16. Hybrid Bacterial Foraging and Particle Swarm Optimization for detecting Bundle Branch Block

    OpenAIRE

    Kora, Padmavathi; Kalva, Sri Ramakrishna

    2015-01-01

    Abnormal cardiac beat identification is a key process in the detection of heart diseases. Our present study describes a procedure for the detection of left and right bundle branch block (LBBB and RBBB) Electrocardiogram (ECG) patterns. The electrical impulses that control the cardiac beat face difficulty in moving inside the heart. This problem is termed as bundle branch block (BBB). BBB makes it harder for the heart to pump blood effectively through the heart circulatory system. ECG feature ...

  17. Detection and confirmation of PPR virus antigen in sheep and goats by sandwich-ELISA and RT-PCR in Andhra Pradesh, India

    Directory of Open Access Journals (Sweden)

    G. Saritha

    2015-06-01

    Full Text Available Peste des petits ruminants (PPR is a highly contagious disease of domestic and wild small ruminants. Rapid and accurate laboratory assay are essential to enable the implementation of appropriate control strategies to restrict the spread of PPR. The present study was designed to detect the PPR virus (PPRV antigen (N-gene in nasal swabs and tissue samples. A total of 195 samples comprising of 138 nasal swabs from PPR suspected sheep (n=72 and goats (n=66, and 57 tissue samples comprising of lymph nodes from dead sheep (n=39 and goats (n=18 were collected from certain parts of Andhra Pradesh. The samples were subjected to sandwich-ELISA followed by RT-PCR for confirmatory diagnosis. In this study, PPRV could be detected in 27.53% (n=38/138 nasal swabs and 49.12% (n=28/57 tissue samples. Data showed that PPRV infection is widespread in the Andhra Pradesh, India.

  18. Detection of ionizing radiations with the SOS chromotest, a bacterial short-term test for genotoxic agents

    International Nuclear Information System (INIS)

    The effects if 3 type of ionizing radiation, γ-rays, neutrons and accelerated α-particles, were examined usingthe SOS Chromotest, a bacterial colorimetric assay for genotoxic agents besed on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses deing in the order of 2.5-5 Gy. We discuss the possible use of these observation to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals. (Author). 43 refs.; 1 fig.; 1 tab

  19. Development of a real-time PCR method for the detection of bacterial colonization in rat models of severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    PENG Jun-sheng; LIU Zhong-hui; LI Chu-jun; WU Xiao-bin; DIAO De-chang; DU Yan-ping; CHEN Jun-rong; LI Yun; WANG Hua-she

    2010-01-01

    Background Techniques for the fast and accurate detection of bacterial infection are critical for early diagnosis, prevention and treatment of bacterial translocation in clinical severe acute pancreatitis (SAP). In this study, the availability of a real-time PCR method in detection of bacterial colonization in SAP rat models was investigated.Methods Samples of blood, mesenteric lymph nodes (MLN), pancreas and liver from 24 specific pathogen-free rats (8 in a control group, 16 in a SAP group) were detected for bacterial infection rates both by agar plate culture and a real-time PCR method, and the results were made contrast.Results Bacterial infection rates of the blood, MLN, pancreas and liver in the SAP group and the control group by the two different methods were almost the same, which were 5/16, 12/16, 15/16, 12/16 in the SAP group compared with 0/8, 1/8, 0/8, 0/8 in the control group by agar plate culture, while 5/16, 10/16, 13/16, 12/16 and 0/8, 1/8, 0/8, 0/8 respectively by a real-time PCR method. Bacterial number was estimated by real-time PCR, which showed that in the same mass of tissues, the pancreas contained more bacteria than the other three kinds of organs in SAP rats (P <0.01), that may be due to the edema, necrosis and hemorrhage existing in the pancreas, making it easier for bacteria to invade and breed.Conclusion Fast and accurate detection of bacterial translocation in SAP rat models could be carried out by a real-time PCR procedure.

  20. Detection of antibodies to bacterial cell wall peptidoglycan in human sera. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Heymer, B.; Schleifer, K.H.; Read, S.; Zabriskie, J.B.; Krause, R.M.

    1976-07-01

    A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic feaver and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten /sup 125/I-L-Ala-..gamma..-D-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis.