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Sample records for bacterial antigen detection

  1. Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions.

    PubMed Central

    Doskeland, S O; Berdal, B P

    1980-01-01

    We sought procedures which would allow a rapid concentration in high yield of bacterial antigens from tissue fluids of patients and which could be applied also to protein-rich fluids like serum. Ethanol precipitation at a subzero temperature with albumin added as an antigen coprecipitant made it possible to achieve a more than 20-fold concentration of antigen in 15 min and a 200-fold concentration in 45 min. Heat-stable antigens could be concentrated from protein-rich fluids (like serum) after the sample had been deproteinized by boiling. Such heating (100 degrees C, 3 min) also liberated bacterial polysaccharides from antibody complexes and elminated the nonspecific interference of serum in enzyme-linked immunosorbent assay. PMID:7372801

  2. Comparison of staphylococcal coagglutination, latex agglutination, and counterimmunoelectrophoresis for bacterial antigen detection.

    PubMed Central

    Thirumoorthi, M C; Dajani, A S

    1979-01-01

    Soluble antigens of Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and group B streptococcus were looked for in cerebrospinal fluid, serum, and urine by using the staphylococcal coagglutination test, latex agglutination test, and counterimmunoelectrophoresis. The staphylococcal coaggultination and latex agglutination tests were more sensitive than counterimmunoelectrophoresis in identifying antigens of H. influenzae type b, S. pneumoniae, and N. meningitidis. None of the three tests successfully detected group B streptococcal antigens in body fluids. Nonspecific reactions noted with the staphylococcal coagglutination test could be usually eliminated after premixing test specimens with soluble protein A. PMID:34622

  3. Value of bacterial antigen detection in the diagnostic yield of transthoracic needle aspiration in severe community acquired pneumonia.

    PubMed Central

    Bella, F.; Tort, J.; Morera, M. A.; Espaulella, J.; Armengol, J.

    1993-01-01

    BACKGROUND--Transthoracic needle aspiration (TNA) with an ultrathin needle is a safe and highly specific procedure for obtaining a diagnosis in bacterial pneumonias, but its sensitivity is at best 70%. A study was performed to assess whether Streptococcus pneumoniae and Haemophilus influenzae type b antigen detection by latex agglutination from the TNA sample enhanced the diagnostic yield. METHODS--Blood cultures, TNA with an ultrathin needle (culture, Gram stain, and latex agglutination), serological tests, and pneumococcal antigen detection in the urine by counterimmunoelectrophoresis were performed in samples from 18 of 23 consecutive patients with severe community acquired pneumonia. RESULTS--The causative organism was identified in 16 cases (88%): S pneumoniae (10 cases), S pneumoniae plus H influenzae (two cases), Legionella pneumophila (three cases), and Mycoplasma pneumoniae (one case). The investigation of antigens by latex agglutination in the pulmonary aspirate increased the diagnostic yield of TNA from 50% to 78% and provided a rapid diagnosis (in less than two hours) with therapeutic implications in seven cases. Its effectiveness was not modified by prior antibiotic therapy. CONCLUSIONS--A latex agglutination test on the pulmonary aspirate enhances the diagnostic yield of TNA in severe community acquired pneumonia. PMID:8303628

  4. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue 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 methodology is chosen ...

  5. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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

  7. Optoacoustic sensing of ocular bacterial antigen using targeted gold nanorods

    NASA Astrophysics Data System (ADS)

    Maswadi, Saher; Page, Leland; Woodward, Lee; Glickman, Randolph D.; Barsalou, Norman

    2008-02-01

    Bacterial contamination can be detected using a minimally invasive optical method, based on laser-induced optoacoustic spectroscopy, to probe for specific antigens associated with a specific infectious agent. As a model system, we have used a surface antigen (Ag), isolated from Chlamydia trachomatis, and a complementary antibody (Ab). A preparation of 0.2 mg/ml of monoclonal Ab specific to the C. trachomatis surface Ag was conjugated to gold nanorods using standard commercial reagents, in order to produce a targeted contrast agent with a strong optoacoustic signal. The C. trachomatis Ag was absorbed in standard plastic microwells, and the binding of the complementary Ab-nanorod conjugate was tested in an immunoaffinity assay. Optoacoustic signals were elicited from the bound nanorods, using an optical parametric oscillator (OPO) laser system as the optical pump. The wavelength tuneability of the OPO optimized the spectroscopic measurement by exciting the nanorods at their optical absorption maxima. Optoacoustic responses were measured in the microwells using a probe beam deflection technique. Immunoaffinity assays were performed on several dilutions of purified C. trachomatis antigen ranging from 50 μg/ml to 1 pg/ml, in order to determine the detection limit for the optoacoustic-based assay. Only when the antigen was present, and the complementary Ab-NR reagent was introduced into the microwell, was an enhanced optoacoustic signal obtained, which indicated specific binding of the Ab-NR complex. The limit of detection with the current system design is between 1 and 5 pg/ml of bacterial Ag.

  8. Bacterial vectors for the delivery of tumor antigens.

    PubMed

    Wang, Yan; Toussaint, Bertrand; Le Gouëllec, Audrey

    2014-01-01

    The use of bacterial vectors, which offer ease of production and efficiency, has become an important mechanism for the delivery of protein antigens to antigen-presenting cells (APCs) in vivo. Proof of concept studies has been carried out utilizing different bacteria in various cancer models with some in clinical trials. Here we described the way to prepare Pseudomonas aeruginosa (P. aeruginosa) vaccines based on a virulence-attenuated strain to test the efficacy of different fragments of a well-known tumor antigen. This protocol could be applied to efficacy studies in murine models of human cancers. PMID:24619697

  9. Immunoproteomics: The Key to Discovery of New Vaccine Antigens Against Bacterial Respiratory Infections

    PubMed Central

    Dennehy, Ruth; McClean, Siobhán

    2012-01-01

    The increase in antibiotic resistance and the shortage of new antimicrobials to prevent difficult bacterial infections underlines the importance of prophylactic therapies to prevent infection by bacterial pathogens. Vaccination has reduced the incidence of many serious diseases, including respiratory bacterial infections. However, there are many pathogens for which no vaccine is available and some vaccines are not effective among all age groups or among immunocompromised individuals. Immunoproteomics is a powerful technique which has been used to identify potential vaccine candidates to protect against pathogenic bacteria. The combination of proteomics with the detection of immunoreactive antigens using serum highlights immunogenic proteins that are expressed during infection. This is particularly useful when patient serum is used as the antigens that promote a humoral response during human infection are identified. This review outlines examples of vaccine candidates that have been identified using immunoproteomics and have successfully protected animals against challenge when tested in immunisation studies. Many immunoreactive proteins are common to several unrelated pathogens, however some of these are not always protective in animal immunisation and challenge studies. Furthermore, examples of well-established immunogens, including Bordetella pertussis antigen FHA were not detected in immunoproteomics studies, indicating that this technology may underrepresent the immunoreactive proteins in a pathogen. Although only one step in the pathway towards an efficacious approved vaccine, immunoproteomics is an important technology in the identification of novel vaccine antigens. PMID:23305366

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

    PubMed

    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

  11. Bacterial detection: from microscope to smartphone.

    PubMed

    Gopinath, Subash C B; Tang, Thean-Hock; Chen, Yeng; Citartan, Marimuthu; Lakshmipriya, Thangavel

    2014-10-15

    The ubiquitous nature of bacteria enables them to survive in a wide variety of environments. Hence, the rise of various pathogenic species that are harmful to human health raises the need for the development of accurate sensing systems. Sensing systems are necessary for diagnosis and epidemiological control of pathogenic organism, especially in the food-borne pathogen and sanitary water treatment facility' bacterial populations. Bacterial sensing for the purpose of diagnosis can function in three ways: bacterial morphological visualization, specific detection of bacterial component and whole cell detection. This paper provides an overview of the currently available bacterial detection systems that ranges from microscopic observation to state-of-the-art smartphone-based detection. PMID:24836016

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

    PubMed

    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

  13. An O Antigen Capsule Modulates Bacterial Pathogenesis in Shigella sonnei

    PubMed Central

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

    2015-01-01

    Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment. PMID:25794007

  14. Detection of O antigens in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  15. [Towards bacterial detection in labile blood products].

    PubMed

    Morel, P

    2005-06-01

    Bacterial contamination of blood components represents today the highest infectious risk in blood transfusion, the risk is particularly high when it affects platelet concentrates. The prevention methods developed over the past ten years (donor selection, phlebotomy site preparation, first 30 ml diversion, systematic leuko-reduction...) which aimed at limiting the introduction of bacteria in donations and bacterial proliferation, has reduced the risk of transfusion reaction due to the bacterial contamination. Improving strategies for reducing the risks of bacterial contamination is one of the priorities of the French National Blood Transfusion Service (l'Etablissement français du sang - EFS). It is essential to improve existent prevention methods and develop the implication of all the actors (from donation to transfusion) involved in the prevention of this risk. Bacterial detection or pathogens inactivation are now available and are able to reduce (for detection) or prevent (for inactivation) the occurrence of reaction due to bacterial contamination of PC. Up to now, the choice is in favour of bacterial detection. Three methods (BacT/Alert, BioMerieux; eBDS, Pall; ScanSystem, Hemosystem) of detection of bacterial contamination in PC can be generalised now. Adaptations, need for their implementation are acceptable, especially concerning PC availability. PMID:15894499

  16. Biosensors for Whole-Cell Bacterial Detection

    PubMed Central

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

    2014-01-01

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

  17. Effect of Lewis blood group antigen expression on bacterial adherence to COS-1 cells.

    PubMed Central

    Gaffney, R A; Schaeffer, A J; Anderson, B E; Duncan, J L

    1994-01-01

    Epithelial cells from secretor individuals demonstrate decreased bacterial adherence compared with cells from nonsecretors. Lewis blood group antigen expression is one component of the secretor/nonsecretor phenotype and several epidemiologic studies have suggested a link between Lewis blood group antigen phenotype and susceptibility to urinary tract infections. In this study, we examined the possibility that the expression of the difucosylated Lewis blood group determinants, Leb and Ley (associated with the secretor phenotype), made cells less susceptible to Escherichia coli adherence by masking receptors for pili. COS-1 cells, which do not produce Lewis (Lea, Leb, Le(x), and Ley) blood group antigens, were used as target cells for bacterial adherence. The surface blood group antigen expression pattern of the cells was then modified by cotransfection with plasmids containing DNA inserts encoding alpha (1,2)-fucosyltransferase and alpha (1,3)- and alpha (1,4)-fucosyltransferases, resulting in the expression of Leb and Ley. E. coli HB101 expressing various adhesins (type 1, PapJ96, PapIA2, PapAD110, Prs, and S) from recombinant plasmids bound equally well to untransfected cells and transfected cells expressing Lea and Le(x) (nonsecretor phenotype) and Leb and Ley (secretor phenotype) antigens. We conclude that the presence of Leb and Ley antigens on cells from secretors does not alone mask receptors for E. coli pili or hinder bacterial adherence. PMID:8005692

  18. Point detection of bacterial and viral pathogens using oral samples

    NASA Astrophysics Data System (ADS)

    Malamud, Daniel

    2008-04-01

    Oral samples, including saliva, offer an attractive alternative to serum or urine for diagnostic testing. This is particularly true for point-of-use detection systems. The various types of oral samples that have been reported in the literature are presented here along with the wide variety of analytes that have been measured in saliva and other oral samples. The paper focuses on utilizing point-detection of infectious disease agents, and presents work from our group on a rapid test for multiple bacterial and viral pathogens by monitoring a series of targets. It is thus possible in a single oral sample to identify multiple pathogens based on specific antigens, nucleic acids, and host antibodies to those pathogens. The value of such a technology for detecting agents of bioterrorism at remote sites is discussed.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Quantum dot based fluorometric detection of cancer TF-antigen.

    PubMed

    Li, Nan; Chow, Ari M; Ganesh, Hashwin V S; Brown, Ian R; Kerman, Kagan

    2013-10-15

    Cancer is a major global health challenge that would benefit from advances in screening methods for early detection that are rapid and low cost. TF-antigen is a tumor-associated antigen displayed on cell surface proteins of a high percentage of human carcinomas. Here we present a fluorometric bioassay for TF-antigen (galactose-β-(1→3)-N-acetyl-d-galactosamine) that utilizes quantum dot (QD) technology coupled with magnetic beads for rapid detection of TF-antigen at high sensitivity (10(-7) M range). In the competitive bioassay, 4-aminophenyl β-d-galactopyranoside (4-APG) conjugated to QDs competes with TF-antigen for binding sites on peanut agglutinin (PNA) that is immobilized on magnetic beads. The bioassay is specific and ultrasensitive in the environment of complex protein mixtures, demonstrating its potential applicability for the screening of clinical samples. PMID:23980999

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

    PubMed Central

    Foley, Janet

    2015-01-01

    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. PMID:26288700

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

    PubMed Central

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

    2013-01-01

    CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32 phosphomycoketide (PM) as a previously unknown molecule and a CD1c-presented antigen. CD1c binding and presentation of mycoketide antigens absolutely required the unusual, mycobacteria-specific lipid branching patterns introduced by polyketide synthase 12 (pks12). Unexpectedly, one TCR responded to diversely glycosylated and unglycosylated forms of mycoketide when presented by DCs and B cells. Yet cell-free systems showed that recognition was mediated only by the deglycosylated phosphoantigen. These studies identify antigen processing of a natural bacterial antigen in the human CD1c system, indicating that cells act on glycolipids to generate a highly simplified neoepitope composed of a sugar-free phosphate anion. Using knowledge of this processed antigen, we generated human CD1c tetramers, and demonstrate that CD1c–PM complexes stain T cell receptors (TCRs), providing direct evidence for a ternary interaction among CD1c-lipid-TCR. Furthermore, PM-loaded CD1c tetramers detect fresh human T cells from peripheral blood, demonstrating a polyclonal response to PM antigens in humans ex vivo. PMID:23530121

  3. Real time viability detection of bacterial spores

    DOEpatents

    Vanderberg, Laura A.; Herdendorf, Timothy J.; Obiso, Richard J.

    2003-07-29

    This invention relates to a process for detecting the presence of viable bacterial spores in a sample and to a spore detection system, the process including placing a sample in a germination medium for a period of time sufficient for commitment of any present viable bacterial spores to occur, mixing the sample with a solution of a lanthanide capable of forming a fluorescent complex with dipicolinic acid, and, measuring the sample for the presence of dipicolinic acid, and the system including a germination chamber having inlets from a sample chamber, a germinant chamber and a bleach chamber, the germination chamber further including an outlet through a filtering means, the outlet connected to a detection chamber, the detection chamber having an inlet from a fluorescence promoting metal chamber and the detection chamber including a spectral excitation source and a means of measuring emission spectra from a sample, the detection chamber further connected to a waste chamber. A germination reaction mixture useful for promoting commitment of any viable bacterial spores in a sample including a combination of L-alanine, L-asparagine and D-glucose is also described.

  4. Detection of group B streptococcal antigens in amniotic fluid of rhesus monkeys.

    PubMed Central

    Hemming, V G; London, W T; Smith, L P; Curfman, B L; Fischer, G W; Sever, J L

    1983-01-01

    To simulate group B streptococci (GBS) amniotic fluid infections common in humans and to examine bacterial growth and the appearance of GBS antigens in vivo, GBS were injected into the amniotic cavity of 19 near-term rhesus monkeys. Transabdominal aspirates of amniotic fluid were obtained before bacterial challenge, after 2 and 6 h, and during cesarean section delivery (24 h). Each fluid was quantitatively cultured for GBS. Specimens of amniotic fluid and gastric aspirate from each infant were tested for the presence of GBS antigens with a commercial latex particle agglutination test (Wellcogen Strep B; Wellcome Diagnostics, Dartford, England). To eliminate nonspecific latex particle agglutination reactivity, presumably caused by proteins, a processing procedure was required. Despite active proliferation of bacteria, only 12% of the 2-h amniotic specimens were latex particle agglutination positive. In contrast, 94% of th3 6-h and 100% of the 24-h specimens had detectable antigens, as did 89% of the gastric fluid specimens aspirated from the 19 newborns. Latex particle agglutination tests, after proper processing, will readily detect GBS antigens in amniotic or gastric aspirate fluid from experimentally infected rhesus monkeys. PMID:6348078

  5. Bacterial surface antigens defined by monoclonal antibodies: the methanogens

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Magarinos, M.C.; Jovell, R.J.; Kandler, O.

    1982-01-01

    The methanogens (MB) are unique microbes of great evolutionary interest with applications in biotechnology-bioengineerings and are important in digestive processes. Their cell-wall composition is distinctively different from that of Eubacteria, e.g. the Methanobacteriaceae possess the peptidoglycan pseudomurein rather than murein. The range of cell-wall compositions among MB and their evolutionary and functional significance is not well known. The authors undertook a systematic study of the MB's surface structure using monoclonal antibodies through the following steps: (1) generation of hybridomas that produce antibody to several MB from 3 of their 4 families; (2) development of immunoenzymatic assays for MB's antigens and antibodies; (3) determination of the fine specificity of monoclonal antibodies by inhibition-blocking tests using cell-wall extracts and compounds of known structure; thus a set of monoclonal probes of predetermined specificity was assembled; and (4) resolution of surface determinants of MB representative of the Methanobacteriaceae using the monoclonal probes. Specific markers of MB strains were characterized. Two epitopes were identified within the pseudomurein molecule.

  6. Synthetic peptides with antigenic specificity for bacterial toxins.

    PubMed

    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

  7. [Detection and typing for swine leukocyte antigen].

    PubMed

    Li, Hua; Luo, Huai-Rong; Zhang, Ya-Ping; Qiu, Xiang-Pin; Ye, Chun

    2004-03-01

    Traditionally the cluster of swine leukocyte antigen (SLA) was typed by serological, cytological and biochemical methods. Many special molecular typing methods have been developed with the progress of molecular biological technology, such as PCR-RFLP, PCR-SSCP , MS and DNA sequencing. Here we discussed the advantages and disadvantages of each method based on the polymorphic and conservative (from the functional aspect, such as supertype and supermotif) characteristics of SLA, and illustrated the development of typing for SLA in the future. In addition, we pointed out the editorial mistakes about the serological haplotype of SLA in reference book and emphasized that the accurate polymorphism of SLA-DQB gene must be based on the cloning sequencing. PMID:15639990

  8. Common antigens prediction in bacterial bioweapons: a perspective for vaccine design.

    PubMed

    Jaiswal, Varun; Chauhan, Rajinder S; Rout, Chittaranjan

    2014-01-01

    Bioweapons (BWs) are a serious threat to mankind and the lack of efficient vaccines against bacterial bioweapons (BBWs) further worsens the situation in face of BW attack. Experts believe that difficulties in detection and ease in dissemination of deadly pathogens make BW a better option for attack compared to nuclear weapons. Molecular biology techniques facilitate the use of genetically modified BBWs thus creating uncertainty on which bacteria will be used for BW attack. In the present work, available resources such as proteomic sequences of BBWs, protective antigenic proteins (PAPs) reported in Protegen database and VaxiJen dataset, and immunogenic epitopes in immune epitope database (IEDB) were used to predict potential broad-specific vaccine candidates against BBWs. Comparison of proteomes sequences of BBWs and their analyses using in-house PERL scripts identified 44 conserved proteins and many of them were known to be immunogenic. Comparison of conserved proteins against PAPs identified six either as PAPs or their homologues with a potential of providing protection against multiple pathogens. Similarly, mapping of conserved proteins against experimentally known IEDB epitopes identified six epitopes which had exact epitope match in four proteins including three from earlier predicted six PAPs. These epitopes were also reported to provide protection against several pathogens. In the backdrop of conserved heat shock GroEL protein from Salmonella enterica providing protection against five diverse bacterial pathogens involved in different diseases, and synthetic proteins produced by combination of epitopes from Mycobacterium tuberculosis and 4 viruses providing protection against both bacterium and viruses, the identified putative immunogenic conserved proteins and immune-protective epitopes can further be explored for their potential as broad-specific vaccine candidates against BBWs. PMID:24300889

  9. Sensitive, Rapid Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    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.

  10. Immune response profile elicited by the model antigen ovalbumin expressed in fusion with the bacterial OprI lipoprotein.

    PubMed

    Basto, Afonso P; Badenes, Marina; Almeida, Sílvia C P; Martins, Carlos; Duarte, António; Santos, Dulce M; Leitão, Alexandre

    2015-03-01

    The use of immunogenic formulations targeting pattern recognition receptors towards modulation of immune responses is a promising strategy to develop better vaccines against infectious and malignant diseases. Molecules targeting TLR2 offer interesting properties that are relevant for vaccine development, including the possibility to covalently attach the lipidic ligands to the antigens. However, the type of immune response elicited by these formulations is still controversial. In this work, we used the model antigen ovalbumin (OVA) expressed in fusion with the bacterial lipoprotein OprI in order to characterize the immunomodulatory properties of this TLR ligand. Murine bone marrow-derived dendritic cells stimulated with OprI-OVA fusion lipoprotein produced high levels of the pro-inflammatory cytokines TNF-α and IL-6 and also IL-10, IL-12(p70) and IL-27, while TGF-β and IL-23 were not detected. Using OT-II and OT-I mice, an enhancement of MHC class II and class I antigen presentation was observed for the OVA antigen in fusion with OprI. Mice immunized by intraperitoneal route with this fusion lipoprotein in prime-boost protocols developed strong specific antibody responses including IgG1, IgG2c, IgG2b, IgG3 and IgE. These results, together with data obtained by restimulation of splenocytes from the immunized mice, point to an immune response profile that does not correspond to a strict Th1 or Th2 polarization. Finally, in a challenge experiment using a melanoma syngeneic mouse model (B16-OVA), prophylactic inoculation with OprI fused with the unrelated antigen eGFP increased the tumor growth, while the fusion with the tumor-associated antigen OVA delayed the tumor growth and increased mice survival. PMID:25467796

  11. Recombinant CHIK virus E1 coat protein of 11 KDa with antigenic domains for the detection of Chikungunya.

    PubMed

    Yathi, Krishna Kammara; Joseph, Julia Mary; Bhasker, Salini; Kumar, Ramesh; Chinnamma, Mohankumar

    2011-09-30

    Chikungunya is an acute febrile illness caused by an alpha virus technically called as CHIK virus. A smaller size of CHIK virus E1 coat protein -11 kDa was expressed in prokaryotic expression system. The recombinant protein was purified and confirmed by western blot analysis. The positions of the antigenic domain in the protein were identified and the immunoreactivity of recombinant protein with anti-CHIK IgM antibodies was ascertained. The antigen showed an 88% sensitivity and 100% specificity by Indirect ELISA. No cross reactivity of the antigen was observed with anti-Dengue virus serum samples. The results strongly support that the recombinant CHIK coat protein could be used as a diagnostic antigen for the detection of Chikungunya by Indirect ELISA. The relevance of a smaller size recombinant antigen highlights its large scale application in serodiagnosis of CHIK virus since bacterial expression is more simple and cost effective than eukaryotic system. PMID:21798263

  12. Human schistosomiasis: Schistosoma mansoni antigen detection in renal glomeruli.

    PubMed

    Hoshino-Shimizu, S; De Brito, T; Kanamura, H Y; Canto, A L; Silva, A O; Campos, A R; Penna, D O; Da Silva, L C

    1976-01-01

    Twelve kidney, five biopsy and seven necropsy specimens, all from schistosomiasis mansoni patients were studied by light and immunoflurescent microscopy in an attempt to detect antigen in the glomerular walls. Deposits of IgM, IgG,I gA, IgE, complement C3 and fibrinogen were observered in most cases. Antigen was successfully detected in two cases(one biopsy and one necropsy specimen), both exhibiting proliferative glomerulonephritis. The only clinical manifestation was a slight proteinuria. IgG antibodies eluted from the sutopsy kidney homogenates showed specific binding mostly to Schistosoma mansoni gut, thus spggesting that the fixed antibodies (eluates) are, at least partially, consituted by antibodies similar to the anti-circulating antigen. These data reinfroce the hypothesis that renal injury in schistosomiasis is mediated through an immune complex disease. PMID:65811

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

    PubMed

    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 (< 1.2 Mb), illustrate this variant generating capacity and its role in persistent infection. Borrelia burgdorferi VlsE diversity is encoded and expressed on a linear plasmid required for persistence and recent experiments have demonstrated that VlsE recombination is necessary for persistence in the immunocompetent host. In contrast, both 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

  14. Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections.

    PubMed Central

    Deitsch, K W; Moxon, E R; Wellems, T E

    1997-01-01

    Pathogenic microbes have evolved highly sophisticated mechanisms for colonizing host tissues and evading or deflecting assault by the immune response. The ability of these microbes to avoid clearance prolongs infection, thereby promoting their long-term survival within individual hosts and, through transmission, between hosts. Many pathogens are capable of extensive antigenic changes in the face of the multiple constitutive and dynamic components of host immune defenses. As a result, highly diverse populations that have widely different virulence properties can arise from a single infecting organism (clone). In this review, we consider the molecular and genetic features of antigenic variation and corresponding host-parasite interactions of different pathogenic bacterial, fungal, and protozoan microorganisms. The host and microbial molecules involved in these interactions often determine the adhesive, invasive, and antigenic properties of the infecting organisms and can dramatically affect the virulence and pathobiology of individual infections. Pathogens capable of such antigenic variation exhibit mechanisms of rapid mutability in confined chromosomal regions containing specialized genes designated contingency genes. The mechanisms of hypermutability of contingency genes are common to a variety of bacterial and eukaryotic pathogens and include promoter alterations, reading-frame shifts, gene conversion events, genomic rearrangements, and point mutations. PMID:9293182

  15. O Antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen Xylella fastidiosa

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    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

  17. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  18. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  19. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  20. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  1. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866.3402 Section 866.3402 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  2. Protein Stains to Detect Antigen on Membranes.

    PubMed

    Dsouza, Anil; Scofield, R Hal

    2015-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical. PMID:26139252

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

    PubMed

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

    NASA Astrophysics Data System (ADS)

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

    2006-03-01

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

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

    PubMed

    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

  6. Microbiology: Detection of Bacterial Pathogens and Their Occurrence.

    ERIC Educational Resources Information Center

    Reasoner, Donald J.

    1978-01-01

    Presents a literature review of bacterial pathogens that are related to water pollution, covering publications from 1976-77. This review includes: (1) bacterial pathogens in animals; and (2) detection and identification of waterborne bacterial pathogens. A list of 129 references is also presented. (HM)

  7. Benchtop Antigen Detection Technique using Nanofiltration and Fluorescent Dyes

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

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

    2016-10-15

    Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections. PMID:27590429

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

    PubMed

    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

  10. Serological versus antigen detection methods for Giardia duodenalis diagnosis.

    PubMed

    Bashir, M; Farid, A; Rabia, I; Mostafa, B; El Amir, A

    2014-12-01

    Giardiasis constitutes an important public health problem in the world. Contamination of the water with fecal materials including viruses and pathogenic protozoa still represents an environmental health hazard, especially in rural areas. The survey study evaluated the relation between seropositivity and some risk factors. Moreover, the study compared between the serological IgG and IgM level and antigen detection methods for the diagnosis of giardiasis. The results indicate that sex distribution and age were the mean risk factors for seroprevelence. In this study, sera samples were employed in sandwich ELISA assay, to detect circulating Giardia antigens. None of the negative control serum samples gave a positive reaction, but cross reaction was encountered with 3 case of Cryptosporidium. The specificity of the assay was 94.830/a. On the other hand, the sensitivity of the Giardia patient's sera was 94.12% which was higher than that of IgG (86.25%) and IgM (87.50%) secretion measurements. In conclusion, antigen detection methods give better and earlier diagnosis for giardiasis can be performed quickly and do not require an experienced and skilled morphologist. PMID:25643512

  11. Rapid detection of Yersinia pestis recombinant fraction 1 capsular antigen.

    PubMed

    Tsui, Pei-Yi; Tsai, Hui-Ping; Chiao, Der-Jiang; Liu, Cheng-Che; Shyu, Rong-Hwa

    2015-09-01

    Yersinia pestis, an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10(5) CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10(5) CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge. PMID:25994256

  12. Antigen

    MedlinePlus

    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 ...

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

    PubMed

    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

  14. Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein.

    PubMed

    Hug, Isabelle; Zheng, Blake; Reiz, Bela; Whittal, Randy M; Fentabil, Messele A; Klassen, John S; Feldman, Mario F

    2011-10-28

    Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications. PMID:21878645

  15. Staphylococcal Enterotoxin B Primes Cytokine Secretion and Lytic Activity in Response to Native Bacterial Antigens

    PubMed Central

    Mason, Kevin M.; Dryden, Tricia D.; Bigley, Nancy J.; Fink, Pamela S.

    1998-01-01

    Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-γ) and interleukin-12 than did sham-injected controls. The majority of IFN-γ production appeared to be CD8+ T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-γ. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to “bystander” fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-γ decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo. PMID:9784507

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

    PubMed

    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. Demonstration of cross-reactivity between bacterial antigens and class I human leukocyte antigens by using monoclonal antibodies to Shigella flexneri.

    PubMed Central

    Williams, K M; Raybourne, R B

    1990-01-01

    Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment. Images PMID:2187807

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

    PubMed Central

    2012-01-01

    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 <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. 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

  19. Optoacoustic detection of viral antigens using targeted gold nanorods

    NASA Astrophysics Data System (ADS)

    Maswadi, Saher; Woodward, Lee; Glickman, Randolph D.; Barsalou, Norman

    2009-02-01

    We are detecting antigens (Ag), isolated from infectious organisms, utilizing laser optoacoustic spectroscopy and antibody-coupled gold nanorod (NR) contrast agents specifically targeted to the antigen of interest. We have detected, in clinical ocular samples, both Herpes Simplex Virus Type 1 and 2 (HSV-1 and HSV-2) . A monoclonal antibody (Ab) specific to both HSV-1 and HSV-2 was conjugated to gold nanorods to produce a targeted contrast agent with a strong optoacoustic signal. Elutions obtained from patient corneal swabs were adsorbed in standard plastic micro-wells. An immunoaffinity reaction was then performed with the functionalized gold nanorods, and the results were probed with an OPO laser, emitting wavelengths at the peak absorptions of the nanorods. Positive optoacoustic responses were obtained from samples containing authentic (microbiologically confirmed) HSV-1 and HSV-2. To obtain an estimate of the sensitivity of the technique, serial dilutions from 1 mg/ml to 1 pg/ml of a C. trachomatis surface Ag were prepared, and were probed with a monoclonal Ab, specific to the C. trachomatis surface Ag, conjugated to gold nanorods. An optoacoustic response was obtained, proportional to the concentration of antigen, and with a limit of detection of about 5 pg/ml. The optoacoustic signals generated from micro-wells containing albumin or saline were similar to those from blank wells. The potential benefit of this method is identify viral agents more rapidly than with existing techniques. In addition, the sensitivity of the assay is comparable or superior to existing colorimetric- or fluorometric-linked immunoaffinity assays.

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

    PubMed

    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

  1. Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.

    PubMed

    Sata, T; Zuber, C; Rinderle, S J; Goldstein, I J; Roth, J

    1990-06-01

    The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them. PMID:2335739

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

    PubMed

    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

  3. Comparison of commercial kits for detection of cryptococcal antigen.

    PubMed Central

    Tanner, D C; Weinstein, M P; Fedorciw, B; Joho, K L; Thorpe, J J; Reller, L

    1994-01-01

    Although kits to detect cryptococcal antigen are used widely to diagnose cryptococcal infection, the comparative performance of commercially available assays has not been evaluated in the past decade. Therefore, we compared the sensitives and specificities of five commercially available kits for detecting cryptococcal antigen (four latex agglutination test kits--Calas [Meridian Diagnostics])--Crypto-LA [International Biological Labs], Myco-Immune [MicroScan], and Immy [Immunomycologics]--and an enzyme immunoassay kit, Premier [Meridian Diagnostics]) with culture for the diagnosis of cryptococcal meningitis and fungemia. Of 182 cerebrospinal fluid (CSF) and 90 serum samples submitted for cryptococcal antigen and fungal culture, 49 (19 and 30 samples, respectively) from 20 patients had a culture positive for Cryptococcus neoformans. For CSF specimens, the sensitivities and specificities of all kits were comparable (sensitivity, 93 to 100%; specificity, 93 to 98%). There was a significant difference in sensitivities of the kits when serum samples were tested with the International Biological Labs and MicroScan kits, which do not pretreat serum with pronase. These kits were less sensitive (sensitivity, 83%) than the Immy and Meridian latex kits (sensitivity, 97%), which do pretreat with pronase. The sensitivity of the Meridian enzyme immunoassay kit was comparable to that of the pronase-containing latex kits. These kits were of equivalent specificities (93 to 100%) when testing serum. Some of the currently available kits have limitations that need to be recognized for proper interpretation of results. Specifically, the use of pronase on serum samples reduces the number of false-positive results, and a titer of < or = 1:4 can be a false-positive result when CSF samples are being tested. PMID:7929757

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

    PubMed

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

    2014-08-27

    Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Laboratory diagnosis of bovine brucellosis mainly relies on serological diagnosis using serum and/or milk samples. Although there are several serological tests with different diagnostic performance and capacity to differentiate vaccinated from infected animals, there is still no standardized reference antigen for the disease. Here we validate the first recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of bovine brucellosis. This antigen can be produced in homogeneous batches without the need of culturing pathogenic brucellae; all characteristics that make it appropriate for standardization. An indirect immunoassay based on the detection of anti O-polysaccharide IgG antibodies in bovine samples was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept and to validate the antigen, we analyzed serum, whole blood and milk samples obtained from non-infected, experimentally infected and vaccinated animals included in a vaccination/infection trial performed in our laboratory as well as more than 1000 serum and milk samples obtained from naturally infected and S19-vaccinated animals from Argentina. Our results demonstrate that OAg-AcrA-based assays are highly accurate for diagnosis of bovine brucellosis, even in vaccinated herds, using different types of samples and in different platforms. We propose this novel recombinant glycoprotein as an antigen suitable for the development of new standard immunological tests for screening and confirmatory diagnosis of bovine brucellosis in regions or countries with brucellosis-control programs. PMID:24984948

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

    PubMed Central

    Geier, Christoph B.; Piller, Alexander; Linder, Angela; Sauerwein, Kai M. T.; Eibl, Martha M.; Wolf, Hermann M.

    2015-01-01

    Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency. PMID:26186701

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

    PubMed

    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

  7. Th2 Allergic Immune Response to Inhaled Fungal Antigens is Modulated By TLR-4-Independent Bacterial Products

    PubMed Central

    Allard, Jenna B.; Rinaldi, Lisa; Wargo, Matt; Allen, Gilman; Akira, Shizuo; Uematsu, Satoshi; Poynter, Matthew E.; Hogan, Deborah A.; Rincon, Mercedes; Whittaker, Laurie A.

    2009-01-01

    SUMMARY Allergic airway disease is characterized by eosinophilic inflammation, mucus hypersecretion and increased airway resistance. Fungal antigens are ubiquitous within the environment and are well know triggers of allergic disease. Bacterial products are also frequently encountered within the environment and may alter the immune response to certain antigens. The consequence of simultaneous exposure to bacterial and fungal products on the lung adaptive immune response has not been explored. Here we show that oropharyngeal aspiration of fungal lysates (Candida albicans, Aspergillus fumigatus) promotes airway eosinophilia, secretion of Th2 cytokines and mucus cell metaplasia. In contrast, oropharyngeal exposure to bacterial lysates (Pseudomonas aeruginosa) promotes airway inflammation characterized by neutrophils, Th1 cytokine secretion and no mucus production. More importantly, administration of bacterial lysates together with fungal lysates deviates the adaptive immune response to a Th1 type associated with neutrophilia and diminished mucus production. The immunomodulatory effect that bacterial lysates have on the response to fungi is TLR4-independent but MyD88 dependent. Thus, different types of microbial products within the airway can alter the host's adaptive immune response, and potentially impact the development of allergic airway disease to environmental fungal antigens. PMID:19224641

  8. Multiplexed BioCD for prostate specific antigen detection

    NASA Astrophysics Data System (ADS)

    Wang, Xuefeng; Zhao, Ming; Nolte, David D.

    2008-02-01

    Specific protein concentrations in human body fluid can serve as diagnostic markers for some diseases, and a quantitative and high-throughput technique for multiplexed protein detection would speed up diagnosis and facilitate medical research. For this purpose, our group developed the BioCD, a spinning-disc interferometric biosensor on which antibody is immobilized. The detection system adopts a common-path scheme making it ultra stable. The scaling mass sensitivity is below 10 pg/mm for protein surface density. A 25000-spot antibody BioCD was fabricated to measure the concentration of prostate specific antigen (PSA), a protein indicating prostate cancer if its level is high. Statistical analysis of our immunoassay results projects that the detection limit of PSA would reach 20 pg/ml in a 2 mg/ml background solution. For future prospects, a multiplexed BioCD can be produced for simultaneous diagnosis of diverse diseases. For instance, 100 markers above 200 pg/ml could be measured on a single disc given that the detection limit is inversely proportional to square root of the number of spots.

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

    PubMed Central

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

    2014-01-01

    Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity. PMID:25755893

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  12. [Partially automated antigen determination and antibody detection with microtiter plates].

    PubMed

    Rapp, C; Weisshaar, C

    1993-01-01

    In addition to several conventional methods for the detection of red cell antigens, the use of microplates has various advantages either as a solid-phase assay (enzyme immunoassay) or as native microplate. Microplates may also be used for the detection of red cell antibodies in 'pooled-cell solid-phase assays' of the second generation and for antibody screening. Blood donors and patients are the two main fields which are to be examined in immunohematology. There are various advantages in using the microplate in blood group serology: (i) if there is hardware already available, like sample processors and microplate readers, the use of microplates in blood group serology reduces the costs even if the equipment has to be purchased for this purpose only; (ii) low quantities of reagents are used in microplate assays; (iii) the application of bar codes on tubes and microplates guarantees the most security in sample identification; (iv) it is possible to investigate blood samples selectively depending on the available software if antibody detection is done as the sixth test beside anti-HIV, anti-HCV, HBsAG, lues antibodies and ALT, and (v) recording of data will be easy if electronic data processing is used. PMID:7693246

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

    PubMed

    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

  14. Detecting Bacterial Vaginosis Using Machine Learning

    PubMed Central

    Baker, Yolanda S.; Agrawal, Rajeev; Foster, James A.; Beck, Daniel; Dozier, Gerry

    2015-01-01

    Bacterial Vaginosis (BV) is the most common of vaginal infections diagnosed among women during the years where they can bear children. Yet, there is very little insight as to how it occurs. There are a vast number of criteria that can be taken into consideration to determine the presence of BV. The purpose of this paper is two-fold; first to discover the most significant features necessary to diagnose the infection, second is to apply various classification algorithms on the selected features. It is observed that certain feature selection algorithms provide only a few features; however, the classification results are as good as using a large number of features. PMID:26753179

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

    PubMed

    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

  16. Detection of Bacterial Spores with Lanthanide-Macrocycle Binary Complexes

    PubMed Central

    Cable, Morgan L.; Kirby, James P.; Levine, Dana J.; Manary, Micah J.; Gray, Harry B.; Ponce, Adrian

    2009-01-01

    The detection of bacterial spores via dipicolinate-triggered lanthanide luminescence has been improved in terms of detection limit, stability, and susceptibility to interferents by use of lanthanide-macrocycle binary complexes. Specifically, we compared the effectiveness of Sm, Eu, Tb and Dy complexes with the macrocycle 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) to the corresponding lanthanide aquo ions. The Ln(DO2A)+ binary complexes bind dipicolinic acid (DPA), a major constituent of bacterial spores, with greater affinity and demonstrate significant improvement in bacterial spore detection. Of the four luminescent lanthanides studied, the terbium complex exhibits the greatest dipicolinate binding affinity (100-fold greater than Tb3+ alone, and 10-fold greater than other Ln(DO2A)+ complexes) and highest quantum yield. Moreover, the inclusion of DO2A extends the pH range over which Tb-DPA coordination is stable, reduces the interference of calcium ions nearly 5-fold, and mitigates phosphate interference 1000-fold compared to free terbium alone. In addition, detection of Bacillus atrophaeus bacterial spores was improved by the use of Tb(DO2A)+, yielding a 3-fold increase in the signal-to-noise ratio over Tb3+. Out of the eight cases investigated, the Tb(DO2A)+ binary complex is best for the detection of bacterial spores. PMID:19537757

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2008-01-01

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

  19. Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

    PubMed Central

    Espino, A M; Finlay, C M

    1994-01-01

    A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive. PMID:8126178

  20. [Prevention of bacterial risk: pathogen inactivation/detection of bacteria].

    PubMed

    Morel, P; Naegelen, C; Deschaseaux, M; Bardiaux, L

    2013-05-01

    Bacterial contamination of blood products remains the most important infectious risk of blood transfusion in 2013. Platelet concentrates (PC) are in cause in the majority of the transfusion reaction due to bacterial contaminations. A lot of prevention methods have been developed over the last 10 years (pre-donation interview, skin decontamination, diversion of the first 30 mL of the donation, leuko-reduction...), they have focused on limiting the contamination of the donations and prevent the bacterial growth in donations and/or in the blood products. These measures were effective and led to significantly reducing the risk of adverse effects associated with bacterial growth. However, every year there are about six accidents (with a high level of imputability) and one death. The reduction of the bacterial risk remains a priority for the French Blood Establishment (EFS). The procedure for skin disinfection is going to be improved in order to further strengthen this crucial step to avoid the contamination of donation. Methods of pathogen inactivation applied to plasma and PC are available in France and their effectiveness is demonstrated on the bacterial risk. Methods for bacterial detection of PC are used in many countries now. Automated culture is the most common. Alternatives are now available in the form of rapid tests able to analyze the PC just before the delivery and avoid false negatives observed with automated culture. Assessments are under way to confirm these benefits in 2013. PMID:23622837

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

    PubMed

    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

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

    PubMed

    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

  3. Electrochemical immunosensor based on hyperbranched structure for carcinoembryonic antigen detection.

    PubMed

    Miao, Jingjing; Wang, Xiaobo; Lu, Liandi; Zhu, Peiyuan; Mao, Chun; Zhao, Haolin; Song, Youchao; Shen, Jian

    2014-08-15

    Sensitive determination of carcinoembryonic antigen (CEA) is very important in clinical research and diagnosis. Herein we report the design and synthesis of a new kind of immunosensor based on the benefits of hyperbranched structure. The hyperbranched polyester was grafted to the surface of indium tin oxides glass (ITO) electrode, and the grafting processes were characterized by attentuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). After CEA and horse radish peroxidase (HRP)-labeled antibody-conjugated AuNPs (HRP-Ab2-AuNPs) bioconjugates were immobilized on the surface of the hyperbranched structure-modified electrode, the optimized conditions of the above electrode were investigated. Moreover, the analytical performance of the proposed immunosensor showed a high sensitivity, a linear range from 0.01 to 80ng/mL with a low detection limit of 2.36pg/mL, and good selectivity for CEA. The designed immunoassay system holds great potential for ultrasensitive electrochemical biosensing of other analytes. PMID:24607616

  4. Bioluminescent enzyme immunoassay for the detection of norovirus capsid antigen.

    PubMed

    Sakamaki, Nozomi; Ohiro, Yoshiyuki; Ito, Mitsuki; Makinodan, Mitsuru; Ohta, Tsubasa; Suzuki, Wataru; Takayasu, Susumu; Tsuge, Harufumi

    2012-12-01

    An ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y = 0.66x - 3.21, r = 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 10(5) to 10(6) copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis. PMID:23081816

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

    PubMed

    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

  6. Fluorescence immunoassay for detecting periodontal bacterial pathogens in plaque.

    PubMed Central

    Wolff, L F; Anderson, L; Sandberg, G P; Aeppli, D M; Shelburne, C E

    1991-01-01

    A particle concentration fluorescence immunoassay has been modified into a bacterial concentration fluorescence immunoassay (BCFIA) to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilizes fluorescently tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected gram-negative plaque bacteria. Microorganisms closely associated with periodontal disease that can be identified in plaque with the BCFIA include Porphyromonas gingivalis, Bacteroides intermedius, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Eikenella corrodens. Briefly, the procedure involved mixing a patient's plaque sample or other bacterial preparation with a species-specific fluorescein isothiocyanate-labeled MAb in a specialized microtiter plate. This mixture was incubated to allow binding of the MAb to its homologous bacteria. The bound and unbound fluorescent tagged MAbs were separated by filtration in the modified microtiter plate, and the total bacterial bound fluorescence was determined with a fluorimeter. The number of a specific bacterial species in a given plaque sample or other bacterial suspension was estimated by reference to a primary standard carried through the BCFIA. The lower detection limit of the BCFIA was 10(3) to 10(4) bacterial cells from single cultures of bacteria or 10(4) bacterial cells in mixed cultures. The coefficient of variation within and between plates for each of the five bacterium-specific MAbs in screening plaque for the periodontal pathogens was less than 10%. These results demonstrate that microbes in plaque can be used as the solid phase in the BCFIA to detect and quantitate MAbs associated with specific bacteria quickly and reliably. PMID:1761686

  7. Detection Of Bacterial Endosymbionts In Clinical Acanthamoeba Isolates

    PubMed Central

    Iovieno, Alfonso; Ledee, Dolena R.; Miller, Darlene; Alfonso, Eduardo C.

    2009-01-01

    Purpose To determine the presence of four clinically relevant bacterial endosymbionts in Acanthamoeba isolates obtained from patients with Acanthamoeba keratitis (AK) and the possible contribution of endosymbionts to the pathogenesis of AK. Design Experimental study Participants Acanthamoeba isolates (N=37) recovered from cornea and contact lens paraphernalia of 23 patients with culture proven AK and 1 environmental isolate. Methods Acanthamoeba isolates were evaluated for the presence of microbial endosymbionts belonging to the bacterial genera Legionella, Pseudomonas, Mycobacteria and Chlamydia using molecular techniques (Polymerase chain reaction and sequence analysis, fluorescent in situ hybridization) and transmission electron microscopy. Corneal toxicity and virulence of Acanthamoeba isolates with and without endosymbionts were compared using a cytopathic effect (CPE) assay of human corneal epithelial cells in vitro. Initial visual acuity (VA), location and characteristics of the infiltrate, time to detection of the infection and symptoms duration at presentation were evaluated in all patients. Main Outcome Measures Prevalence and potential pathobiology of bacterial endosymbionts detected in Acanthamoeba isolates recovered from AK. Results Twenty-two of the 38 (59.4%) cultures examined contained at least one bacterial endosymbiont. One isolate contained two endosymbionts, Legionella and Chlamydia, confirmed by fluorescence in situ hybridization. Corneal toxicity (CPE) was significantly higher for Acanthamoebae hosting endosymbionts compared to isolates without endosymbionts (p<0.05). Corneal pathogenic endosymbionts such as Pseudomonas and Mycobacterium enhanced Acanthamoeba CPE significantly more than Legionella (p<0.05). In the presence of bacterial endosymbionts, there was a trend toward worse initial VA (p>0.05), central location (p<0.05), absence of radial perineuritis (p<0.05), delayed time to detection (p>0.05) and longer symptoms duration at

  8. Cross-Reactivity of the PLATELIA CANDIDA Antigen Detection Enzyme Immunoassay with Fungal Antigen Extracts

    PubMed Central

    Rimek, Dagmar; Singh, Jagpal; Kappe, Reinhard

    2003-01-01

    We studied the specificity of the PLATELIA CANDIDA Ag enzyme immunoassay by using 130 isolates of 63 clinically relevant fungal species. Antigen extracts of seven Candida spp. (Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis) repeatedly yielded positive reactions (>0.5 ng/ml). Geotrichum candidum and Fusarium verticillioides were found to yield borderline-positive reactions (0.25 to 0.50 ng/ml). Antigen preparations from the other 54 fungal species, including yeasts, molds, dermatophytes, and dimorphic fungi, did not cross-react in the assay. PMID:12843102

  9. Rapid diagnosis of typhoid fever by antigen detection.

    PubMed

    Sivadasan, K; Kurien, B; John, T J

    1984-01-21

    Salmonella typhi antigen was demonstrated in the blood of patients with typhoid fever by a passive staphylococcal agglutination test. This test was positive in 10 culture-proven typhoid patients and negative in 10 febrile patients without typhoid. PMID:6140444

  10. Development of Recombinant Antigen Array for Simultaneous Detection of Viral Antibodies

    PubMed Central

    Liu, Yi; Yu, Fengling; Huang, Haiyan; Han, Jinxiang

    2013-01-01

    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. PMID:24058498

  11. Detection of sugarcane yellow leaf virus by direct antigen coated enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The L9 (34) orthogonal diagram was applied to optimize detection conditions of direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) for Sugarcane yellow leaf virus (SCYLV) in sugarcane. Statistic analyses indicated that 150 µL of SCYLV in juice and leaf crude extract antigens was the ...

  12. Detection of Bacterial Endospores in Soil by Terbium Fluorescence

    PubMed Central

    Brandes Ammann, Andrea; Kölle, Linda; Brandl, Helmut

    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 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. PMID:21754939

  13. Serogroup-Specific Bacterial Engineered Glycoproteins as Novel Antigenic Targets for Diagnosis of Shiga Toxin-Producing-Escherichia coli-Associated Hemolytic-Uremic Syndrome

    PubMed Central

    Melli, Luciano J.; Ciocchini, Andrés E.; Caillava, Ana J.; Vozza, Nicolás; Chinen, Isabel; Rivas, Marta; Feldman, Mario F.

    2014-01-01

    Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis. PMID:25472487

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

    PubMed Central

    2012-01-01

    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. PMID:23126230

  15. Genetic Diversity of O-Antigens in Hafnia alvei and the Development of a Suspension Array for Serotype Detection

    PubMed Central

    Duan, Zhifeng; Niedziela, Tomasz; Lugowski, Czeslaw; Cao, Boyang; Wang, Tianwei; Xu, Lingling; Yang, Baopeng; Liu, Bin; Wang, Lei

    2016-01-01

    Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes. PMID:27171009

  16. Genetic Diversity of O-Antigens in Hafnia alvei and the Development of a Suspension Array for Serotype Detection.

    PubMed

    Duan, Zhifeng; Niedziela, Tomasz; Lugowski, Czeslaw; Cao, Boyang; Wang, Tianwei; Xu, Lingling; Yang, Baopeng; Liu, Bin; Wang, Lei

    2016-01-01

    Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes. PMID:27171009

  17. Reaction of goat's milk with stained antigens in the detection of brucellosis

    PubMed Central

    Alivisatos, G. P.; Edipides, T.

    1953-01-01

    Testing for brucellosis in goats by the reaction of milk with stained antigens was carried out on 206 milk-samples and the results compared with those of sero-agglutination tests on the same animals. The authors find that in most cases the milk reactions correspond to the sero-agglutination results, and that it is the agglutination of the stained antigen which is the principal sign of a positive reaction rather than ring formation, which is very irregular. Haematoxylin antigen is generally more reliable than Bendtsen antigen, but, with either, the results should be read after 1-2 hours' incubation at 37°C. The authors conclude that the stained antigen test can be used for the detection of infection in herds of goats, but that it is not yet possible to say whether it is also valuable for detecting individual infection. PMID:13141139

  18. Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay.

    PubMed Central

    Evans, D G; Evans, D J; Clegg, S

    1980-01-01

    An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E. coli was the known causative agent. The inhibition method, or blocking technique, was used. In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin. CFA/I purified from E. coli strain H-10407 (O78:H11) was used. Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen. Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E. coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures. Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar. These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E. coli. PMID:7031075

  19. Liquid crystals as optical amplifiers for bacterial detection.

    PubMed

    Zafiu, C; Hussain, Z; Küpcü, S; Masutani, A; Kilickiran, P; Sinner, E-K

    2016-06-15

    Interactions of bacteria with target molecules (e.g. antibiotics) or other microorganisms are of growing interest. The first barrier for targeting gram-negative bacteria is layer of a Lipopolysaccharides (LPS). Liquid crystal (LC) based sensors covered with LPS monolayers, as presented in this study, offer a simple model to study and make use of this type of interface for detection and screening. This work describes in detail the production and application of such sensors based on three different LPS that have been investigated regarding their potential to serve as sensing layer to detect bacteria. The LPS O127:B8 in combination with a LC based sensor was identified to be most useful as biomimetic sensing surface. This LPS/LC combination interacts with three different bacteria species, one gram-positive and two gram-negative species, allowing the detection of bacterial presence regardless from their viability. It could be shown that even very low bacterial cell numbers (minimum 500 cell ml(-1)) could be detected within minutes (maximum 15 min). The readout mechanism is the adsorption of bacterial entities on surface bond LPS molecules with the LC serving as an optical amplifier. PMID:26827146

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

    PubMed

    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

  1. A Cronobacter turicensis O1 Antigen-Specific Monoclonal Antibody Inhibits Bacterial Motility and Entry into Epithelial Cells

    PubMed Central

    Lehner, Angelika; Dietrich, Richard; Kleinsteuber, Ina; Canals, Rocío; Zurfluh, Katrin; Weiner, Kerstin; Märtlbauer, Erwin

    2014-01-01

    Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited. PMID:25534937

  2. Sensitive immunoradiometric assay for the detection of Paracoccidioides brasiliensis antigens in human sera.

    PubMed Central

    Ferreira-da-Cruz, M F; Galvão-Castro, B; Daniel-Ribeiro, C T

    1991-01-01

    In the present study we report the standardization of an immunoradiometric assay (IRMA) for detection of Paracoccidioides brasiliensis circulating antigens that could be useful in the diagnosis and prognosis of paracoccidioidomycosis. For this purpose we studied the reactivities of P. brasiliensis and other mycotic antigens with rabbit polyclonal anti-P. brasiliensis antibodies (immunoglobulin G) in order to evaluate the sensitivity and specificity of an IRMA for detecting P. brasiliensis antigens. The results were compared with those obtained by the double immunodiffusion test, the standard technique for the serodiagnosis of paracoccidioidomycosis. By using the immunoglobulin G fraction of rabbit antisera (900 ng per well), it was possible to detect up to 3.6 ng (0.12 micrograms/ml) of cellular antigen and 360 ng (12 micrograms/ml) of metabolic antigen in contrast to the double immunodiffusion test that could detect only 12 micrograms (1.2 mg/ml) of both antigens. IRMA was shown to be feasible and very sensitive and may therefore help, together with clinical data, in establishing early diagnosis and assessing disease activity. It could also allow the study of relationships between P. brasiliensis circulating antigens and host defense mechanisms during the disease. PMID:1907608

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

    PubMed Central

    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 lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition. PMID:15731032

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

    USGS Publications Warehouse

    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.

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed Central

    Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; de Souza, Wayner Vieira; 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

  9. Antigenic Crossreactivity between Bacterial and Plant Cytochrome P-450 Monoxygenases 1

    PubMed Central

    Stewart, Cassie B.; Schuler, Mary A.

    1989-01-01

    Although cytochrome P-450 monoxygenases mediate critical reactions in plant microsomes, characterization of their activities has been difficult due to their inherent instability and the lack of a crossreacting P-450 antibody. We have surveyed the effects of protein stabilizing agents on t-cinnamic acid hydroxylase (t-CAH), a prominent microsomal P-450, and on total P-450 monoxygenase content. Trans-cinnamic acid is the most effective protecting agent for t-CAH activity. Leupeptin, a broad spectrum protease inhibitor, stabilizes t-CAH activity and increases the apparent P-450 content more than serine protease inhibitors such as phenylmethylsulfonyl fluoride. The combination of t-cinnamic acid and protease inhibitors increase the level of detectable t-CAH activity 4- to 14-fold over the levels detected by previously published procedures. In order to estimate the molecular weights and diversity of the plant P-450 monoxygenases in wounded pea epicotyls, we have prepared two polyclonal antibodies against the Pseudomonas putida camphor hydroxylase (P-450cam). One of the heterologous antibodies cross-reacts with constitutive microsomal polypeptides between 52 and 54 kilodaltons and several pea (Pisum sativum L.) mitochondrial proteins between 47 and 48 kilodaltons. The other polyclonal antibody cross-reacts strongly with two wound-induced polypeptides (65 and 47 kilodaltons) and weakly with one constitutive polypeptide (58 kilodaltons). We conclude that at least two subclasses of plant P-450 monoxygenases share common epitopes with the bacterial P-450 enzyme. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:16666804

  10. Bovine viral diarrhea virus antigen detection across whole cattle hides using two antigen-capture enzyme-linked immunosorbent assays.

    PubMed

    Vander Ley, Brian L; Ridpath, Julia F; Sweiger, Shaun H

    2012-05-01

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle requires substantial quantities of known positive- and negative-sample material. The objective of this study was to determine what sections of bovine skin contained Bovine viral diarrhea virus antigen. Two commercially available antigen-capture enzyme-linked immunoassays were used to test subsamples representing the entire skin of 3 persistently infected calves. Both assays detected Bovine viral diarrhea virus antigen in the samples indicated for use by assay protocol. However, one assay identified all subsamples as positive, while the second assay identified 64.4% of subsamples as positive. These results show that use of samples other than those specified by the assay protocol must be validated for each individual assay. In this study, alternative sample sites and use of the entire hide for proficiency testing would be acceptable for only one of the assays tested. PMID:22529122

  11. Rapid Bacterial Detection via an All-Electronic CMOS Biosensor.

    PubMed

    Nikkhoo, Nasim; Cumby, Nichole; Gulak, P Glenn; Maxwell, Karen L

    2016-01-01

    The timely and accurate diagnosis of infectious diseases is one of the greatest challenges currently facing modern medicine. The development of innovative techniques for the rapid and accurate identification of bacterial pathogens in point-of-care facilities using low-cost, portable instruments is essential. We have developed a novel all-electronic biosensor that is able to identify bacteria in less than ten minutes. This technology exploits bacteriocins, protein toxins naturally produced by bacteria, as the selective biological detection element. The bacteriocins are integrated with an array of potassium-selective sensors in Complementary Metal Oxide Semiconductor technology to provide an inexpensive bacterial biosensor. An electronic platform connects the CMOS sensor to a computer for processing and real-time visualization. We have used this technology to successfully identify both Gram-positive and Gram-negative bacteria commonly found in human infections. PMID:27618185

  12. Sandwich-ELISA detection of venom antigens in envenoming by Phoneutria nigriventer spider.

    PubMed

    Chávez-Olórtegui, C; Bohórquez, K; Alvarenga, L M; Kalapothakis, E; Campolina, D; Maria, W S; Diniz, C R

    2001-06-01

    Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Phoneutria nigriventer spider venom. Horse anti-P. nigriventer immunoglobulins were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with P. nigriventer venom from those in mice inoculated with Lycosa sp. and Loxosceles intermedia spider venoms, Tityus serrulatus scorpion venom and Apis mellifera bee venom. Measurable absorbance signals were obtained with 0.8ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice and to detect antigens in the sera of patients envenomed by P. nigriventer. PMID:11137553

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

    PubMed

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

    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 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. PMID:26270053

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

    PubMed

    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

  15. Detection of Aspergillus antigens associated with invasive infection.

    PubMed Central

    Haynes, K A; Latge, J P; Rogers, T R

    1990-01-01

    Serial urine samples were collected from 33 neutropenic patients, 10 of whom developed invasive aspergillosis (IA) while undergoing bone marrow transplantation or remission induction therapy for leukemia. Concentrated urine samples from the infected patients were subjected to polyacrylamide gel electrophoresis, blotted, and then incubated with antiserum raised to a cell wall extract of Aspergillus fumigatus (anti-CW) or an immunoglobulin G monoclonal antibody to A. fumigatus galactomannan (EBA1). When IA patient urine blots were probed with anti-CW, major bands at 11 and 18 kilodaltons (kDa); intermediate bands at 13, 14, and 29 kDa; and minor bands at 38 and 44 kDa were seen. In contrast, EBA1 showed diffuse staining at molecular masses larger than 45 kDa and a single weak band at 21 kDa. Urine samples from the 23 patients with no evidence of IA were unreactive with both anti-CW and EBA1. These antigen bands are likely to represent immunodominant antigens which are excreted during IA and should play a valuable role in the development of rapid diagnostic tests for aspergillosis. Images PMID:2229387

  16. Selective detection of bacterial layers with terahertz plasmonic antennas.

    PubMed

    Berrier, Audrey; Schaafsma, Martijn C; Nonglaton, Guillaume; Bergquist, Jonas; Rivas, Jaime Gómez

    2012-11-01

    Current detection and identification of micro-organisms is based on either rather unspecific rapid microscopy or on more accurate but complex and 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. PMID:23162730

  17. Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates

    SciTech Connect

    Sano, T.; Smith, C.L.; Cantor, C.R. )

    1992-10-02

    An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.

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

    PubMed

    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

  19. IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens.

    PubMed

    Maglione, Paul J; Simchoni, Noa; Black, Samuel; Radigan, Lin; Overbey, Jessica R; Bagiella, Emilia; Bussel, James B; Bossuyt, Xavier; Casanova, Jean-Laurent; Meyts, Isabelle; Cerutti, Andrea; Picard, Capucine; Cunningham-Rundles, Charlotte

    2014-12-01

    IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans. PMID:25320238

  20. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  1. Diagnostic markers of ovarian cancer by high-throughput antigen cloning and detection on arrays.

    PubMed

    Chatterjee, Madhumita; Mohapatra, Saroj; Ionan, Alexei; Bawa, Gagandeep; Ali-Fehmi, Rouba; Wang, Xiaoju; Nowak, James; Ye, Bin; Nahhas, Fatimah A; Lu, Karen; Witkin, Steven S; Fishman, David; Munkarah, Adnan; Morris, Robert; Levin, Nancy K; Shirley, Natalie N; Tromp, Gerard; Abrams, Judith; Draghici, Sorin; Tainsky, Michael A

    2006-01-15

    A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations. PMID:16424057

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

    PubMed

    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

  3. Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

    NASA Astrophysics Data System (ADS)

    Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

    2005-11-01

    Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

  4. Immunofluorescence versus ELISA for the detection of antinuclear antigens.

    PubMed

    Rondeel, Jan M M

    2002-05-01

    Determining the presence and specificity of antinuclear antigens (ANA) is a challenge to a laboratory involved in the diagnosis of connective tissue disease (CTD). The immunofluorescent technique (IF), once considered the gold standard, is more and more displaced by ELISA. ELISA can be fully automated and the interpretation does not require the extensive experience needed in IF. However, literature in which both techniques are compared does not give unequivocal conclusions that ELISA indeed performs better. The clue as to which technique is best in the cascade testing of ANA, is given by its clinical value, not only by its technical and logistic performance. Selective test ordering is strongly recommended to increase the predictive value of these tests. The pros and cons of both techniques are discussed. PMID:12050861

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

    SciTech Connect

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

    1980-12-01

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

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

    PubMed

    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

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

    PubMed

    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

  8. Clinical comparison of two assays for rapid detection of cytomegalovirus early nuclear antigen.

    PubMed

    Woods, G L; Johnson, A M; Thiele, G M

    1990-03-01

    Three methods for detection of cytomegalovirus (CMV) in 218 clinical specimens were compared: (1) shell vial assay to detect the early nuclear antigen after incubation for 16 hours and 40 hours (Syva Company); (2) 24-well plate assay to detect the early nuclear antigen after incubation for 16 hours (DuPont); and (3) convention tissue cell culture. CMV was detected in 26 specimens (12%) by one or more of these methods. With the shell vial assay, 12 (46%) and 15 (58%) specimens were positive after incubation for 16 hours and 40 hours, respectively. CMV was detected in 17 specimens (65%) by the 24-well plate assay. There was no significant difference in the detection of CMV between these assays. CMV was identified by conventional tissue culture in 15 of 22 (68%) evaluable cultures after an average of 14.2 days. More specimens were positive by conventional culture than by the 16-hour shell vial assay (P = 0.035). For optimal detection of CMV in clinical specimens, both conventional tissue cell culture and an early antigen assay should be performed. The two early antigen assays evaluated in this study yielded comparable results. However, the 24-well plates are more easily manipulated, and the 24-well plate assay, as performed, was easier to interpret and more cost efficient. PMID:2155527

  9. Detection with monoclonal antibody of Salmonella typhi antigen 9 in specimens from patients.

    PubMed Central

    Chaicumpa, W; Thin-Inta, W; Khusmith, S; Tapchaisri, P; Echeverria, P; Kalambaheti, T; Chongsa-Nguan, M

    1988-01-01

    Monoclonal antibodies were raised against Barber antigen (Ba) of Salmonella typhi 0901. Antibodies produced to antigen 9 of group D salmonellae were used in double- and triple-sandwich antibody enzyme-linked immunosorbent assays (ELISAs) for detecting antigen 9 in urine and plasma specimens from three groups of patients and 49 controls. The triple-antibody ELISA detected the antigen in urine samples from 11 of 18 (65%) patients with hemoculture-proven typhoid (group 1) and 12 of 39 (31%) patients with clinical features compatible with typhoid but whose hemocultures were negative (group 2). This ELISA was negative in three patients from whom Salmonella paratyphi A, Escherichia coli, and Klebsiella pneumoniae (group 3) were isolated by hemoculture and in all healthy controls. The double-antibody sandwich ELISA was positive in 41 and 15% of urine samples from patients in groups 1 and 2, respectively, and was negative with samples from two patients from group 3 and all controls. The sensitivity and specificity compared with those for healthy controls were 65 and 100%, respectively, for the triple-antibody ELISA. Although as little as 7.8 ng of homologous lipopolysaccharide could be detected, background in clinical specimens prevented accurate interpretation of the detection of this antigen in serum. Results were best with urine specimens. Images PMID:3183027

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

    PubMed

    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

  11. Novel use of a radiolabelled antibody against stage specific embryonic antigen for the detection of occult abscesses in mammals

    DOEpatents

    Thakur, Madhukar L.

    1990-01-01

    The invention discloses improved reagents containing antibodies against stage specific embryonic antigen-1 antibodies and improved methods for detection of occult abscess and inflammation using the improved reagents.

  12. Investigation of magnetic microdiscs for bacterial pathogen detection

    NASA Astrophysics Data System (ADS)

    Castillo-Torres, Keisha Y.; Garraud, Nicolas; Arnold, David P.; McLamore, Eric S.

    2016-05-01

    Despite strict regulations to control the presence of human pathogens in our food supply, recent foodborne outbreaks have heightened public concern about food safety and created urgency to improve methods for pathogen detection. Herein we explore a potentially portable, low-cost system that uses magnetic microdiscs for the detection of bacterial pathogens in liquid samples. The system operates by optically measuring the rotational dynamics of suspended magnetic microdiscs functionalized with pathogen-binding aptamers. The soft ferromagnetic (Ni80Fe20) microdiscs exhibit a closed magnetic spin arrangement (i.e. spin vortex) with zero magnetic stray field, leading to no disc agglomeration when in free suspension. With very high surface area for functionalization and volumes 10,000x larger than commonly used superparamagnetic nanoparticles, these 1.5-μm-diameter microdiscs are well suited for tagging, trapping, actuating, or interrogating bacterial targets. This work reports a wafer-level microfabrication process for fabrication of 600 million magnetic microdiscs per substrate and measurement of their rotational dynamics response. Additionally, the biofunctionalization of the microdiscs with DNA aptamers, subsequent binding to E. coli bacteria, and their magnetic manipulation is reported.

  13. Ultrasensitive immunoassay for prostate specific antigen using scanning tunneling microscopy-based electrical detection

    NASA Astrophysics Data System (ADS)

    Choi, Jeong-Woo; Oh, Byung-Keun; Jang, Yong-Hark; Kang, Da-Yeon

    2008-07-01

    We characterized a vertically configured electrical detection system that used scanning tunneling microscopy (STM) to detect antigen-antibody binding. This technique could be used to easily construct a multiple measurement system in a protein chip. We utilized immunocomplexes comprised of our model protein, prostate specific antigen (PSA), corresponding antibody fragments, and gold nanoparticle-antibody conjugates. The electrical tunneling current between the STM tip and these complexes exhibited a peaklike pulse, the frequency of which depended on the surface density of the bound complexes. We could therefore quantitatively measure PSA concentrations as low as 10fg/mL using periodogram analysis of this peak frequency.

  14. Detection Of Viral And Bacterial Pathogens In Acute Respiratory Infections

    PubMed Central

    Obasi, Chidi N.; Barrett, Bruce; Brown, Roger; Vrtis, Rose; Barlow, Shari; Muller, Daniel; Gern, James

    2013-01-01

    Objectives The role of bacteria in acute respiratory illnesses (ARI) of adults and interactions with viral infections is incompletely understood. This study tested the hypothesis that bacterial co-infection during ARI adds to airway inflammation and illness severity. Methods Two groups of 97 specimens each were randomly selected from multiplex-PCR identified virus-positive and virus-negative nasal specimens obtained from adults with new onset ARI, and 40 control specimens were collected from healthy adults. All specimens were analyzed for Haemophilus influenza(HI), Moraxella catarrhalis(MC) and Streptococcus pneumonia(SP) by quantitative-PCR. General linear models tested for relationships between respiratory pathogens, biomarkers (nasal wash neutrophils and CXCL8), and ARI-severity. Results Nasal specimens from adults with ARIs were more likely to contain bacteria (37% overall; HI=28%, MC=14%, SP=7%) compared to specimens from healthy adults (5% overall; HI=0%, MC=2.5%, SP=2.5%;p<0.001). Among ARI specimens, bacteria were more likely to be detected among virus-negative specimens compared to virus-positive specimens (46% vs. 27%;p=0.0046). The presence of bacteria was significantly associated with increased CXCL8 and neutrophils, but not increased symptoms. Conclusion Pathogenic bacteria were more often detected in virus-negative ARI, and also associated with increased inflammatory biomarkers. These findings suggest the possibility that bacteria may augment virus-induced ARI and contribute to airway inflammation. Summary We tested whether bacterial pathogens were associated with ARI illness and inflammation. Bacteria were detected more often in nasal secretions during ARI, especially in samples without detectable viruses, and were associated with increased airway inflammation, but not increased symptoms. PMID:24211414

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

    PubMed

    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

  16. Capsular polysaccharide antigens for detection of serotype-specific antibodies to Actinobacillus pleuropneumoniae.

    PubMed Central

    Bossé, J T; Johnson, R P; Rosendal, S

    1990-01-01

    Capsular polysaccharides (CPS) of serotypes 1, 2, 5 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae were obtained from 18 h culture supernatants by precipitation with hexadecyltrimethyl-ammonium bromide (Cetavlon) followed by extraction with sodium chloride and reprecipitation in ethanol. These crude extracts, and portions purified further by phenol extraction to remove contaminating proteins, were evaluated as antigens for the detection of serotype-specific antibodies to A. pleuropneumoniae in sera from immunized rabbits and swine by an enzyme-linked immunosorbent assay. The crude extracts reacted strongly with homologous antisera, but except for serotype 1 showed considerable cross-reactivity with antisera to other serotypes. Phenol extraction greatly improved the serospecificity of the antigens from serotypes 1, 7 and, to a lesser extent, 5. The serotype 2 CPS antigen showed poor reactivity following phenol extraction, and did not appear as useful for detection of serotype-specific antibodies. PMID:2379111

  17. Trichinella spiralis: monoclonal antibody against the muscular larvae for the detection of circulating and fecal antigens in experimentally infected rats.

    PubMed

    Zumaquero-Ríos, José-Lino; García-Juarez, Jazmín; de-la-Rosa-Arana, Jorge-Luis; Marcet, Ricardo; Sarracent-Pérez, Jorge

    2012-12-01

    In this work we search for antigens of Trichinella spiralis in sera and stool of rats experimentally infected. The kinetic of antibodies to excretory and secretory (ES) antigens of muscle larvae (ML) was also determined. Wistar rats were infected with 15 ML per gram of body weight and blood samples were collected weekly for 10 weeks. Antibodies were studied using an indirect ELISA. For detection of circulating antigens and coproantigens, a sandwich ELISA was developed with the use of polyclonal rabbit antibodies obtained against the total extract of ML and an IgM monoclonal antibody (Mab) against ES antigens of ML. No reactivity was observed between Mab and the total worm antigens of Angiostrongylus cantonensis, Ascaris suum, Echinococcus granulosus, Fasciola hepatica, Strongyloides stercoralis, Taenia solium, Toxocara canis and Trichuris trichiura. The IgM Mab recognized antigens of 45, 49, and 55 kDa in ES antigens and was unable to bind ES antigens deglycosylated with trifluoromethanesulphonic acid (TFMS) indicating that a glycan structure is present in the epitope recognized by this Mab. The sensitivity of sandwich ELISA was 1 ng/mL. Circulating antigens were detected in all infected rats between 3 and 8 weeks post infection and coproantigens were found during the first two days post infection. Antibodies were detected since the third week post infection through the end of experiment. These results suggested that antigen detection by our sandwich ELISA could be a useful complementary laboratory test for antibody detection. PMID:23026455

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Biochemical principle of Limulus test for detecting bacterial endotoxins

    PubMed Central

    Iwanaga, Sadaaki

    2007-01-01

    A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1,3)-β-D-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, and factor G, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. The molecular structures of these proteins have also been elucidated. Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel. Here, I will focus on the biochemical principle of Limulus test for detecting bacterial endotoxins, and its activation and regulation mechanism on the LPS-mediated coagulation cascade. PMID:24019589

  20. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria. PMID:27088225

  1. Detection of influenza antigenic variants directly from clinical samples using polyclonal antibody based proximity ligation assays

    PubMed Central

    Martin, Brigitte E.; Jia, Kun; Sun, Hailiang; Ye, Jianqiang; Hall, Crystal; Ware, Daphne; Wan, Xiu-Feng

    2016-01-01

    Identification of antigenic variants is the key to a successful influenza vaccination program. The empirical serological methods to determine influenza antigenic properties require viral propagation. Here a novel quantitative PCR-based antigenic characterization method using polyclonal antibody and proximity ligation assays, or so-called polyPLA, was developed and validated. This method can detect a viral titer that is less than 1000 TCID50/mL. Not only can this method differentiate between different HA subtypes of influenza viruses but also effectively identify antigenic drift events within the same HA subtype of influenza viruses. Applications in H3N2 seasonal influenza data showed that the results from this novel method are consistent with those from the conventional serological assays. This method is not limited to the detection of antigenic variants in influenza but also other pathogens. It has the potential to be applied through a large-scale platform in disease surveillance requiring minimal biosafety and directly using clinical samples. PMID:25546251

  2. Biomarker Detection Using NAPPA Tumor Antigen Arrays — EDRN Public Portal

    Cancer.gov

    Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p 91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.

  3. Detection of Schistosoma mansoni circulating cathodic antigen for evaluation of resistance induced by irradiated cercariae.

    PubMed

    Barsoum, I S; Bogitsh, B J; Colley, D G

    1992-08-01

    The appearance of serum levels of circulating cathodic antigen (CCA) detectable by a monoclonal antibody (mAb) (5H11) antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) system was evaluated during acute Schistosoma mansoni infections in female CF1 mice exposed to either 100 or 25 cercariae. Measurable CCA levels occurred in these groups at 5 and 7 wk after infection, respectively. The kinetics of appearance of CCA were thus related to the intensity of infection. The level of resistance developed by female C57BL/6 mice upon immunization with irradiated cercariae, as expressed by both worm burden and CCA levels after cercarial challenge was evaluated. Immunization conferred 44% protection against the challenge infection, and the level of CCA detected in the sera of the control group was significantly (P less than 0.02) higher than that found in the sera of the immunized group, 6 wk after challenge. These results demonstrate that CCA detection by the 5H11 mAb antigen-capture sandwich ELISA can reflect vaccine-induced resistance against S. mansoni. Localization studies showed that 5H11 reacts with a CCA epitope in the adult worm gut and to a lesser extent with the male tegument. Adaptations of this and other antigen detection systems may prove useful in monitoring the efficacy of developmental vaccines, an ability that may be essential for the extension of such studies to humans. PMID:1635027

  4. Production of a novel monoclonal antibody, JT-95, which can detect antigen of thyroid carcinoma.

    PubMed

    Takeyama, H; Hosoya, T; Sakurai, K; Mori, Y; Watanabe, M; Kisaki, H; Ohno, T

    1996-04-15

    Monoclonal antibody (MAb) JT-95 was produced by immunization of mice with membrane fractions of a human thyroid carcinoma. Immuno-histochemical staining has demonstrated that the antigen recognized by JT-95 is strongly expressed in 95 (95%) of 100 cases of papillary carcinomas and in 3 (75%) of 4 cases of follicular carcinomas. In benign diseases of the thyroid gland, MAb JT-95 reacted with 0 (0%) of 39 adenomas, 1 (4%) of 21 adenomatous goiters, 0 (0%) of 8 hyperthyroidism specimens, and 3 (38%) of 8 chronic thyroiditis specimens. The antigen detected by MAb JT-95 has an apparent Mr 250,000 in thyroid carcinomas. Moreover, circulating antigen in thyroid carcinoma patients was detected by MAb JT-95 in an ELISA and in Western blotting. The circulating antigen has a Mr 105,000. MAb JT-95 conjugated with (131) I was administrated to nude mice bearing a human thyroid carcinoma. JT-95 131I accumulation at the transplanted tumor was visualized by autoradiography with 2.68-14.75-fold higher levels detected at the xenograft compared to that for normal organs. Based on these data, MAb JT-95 may be useful in the diagnosis detection and therapy of thyroid carcinoma. PMID:8620498

  5. Salmonella typhimurium-induced M1 macrophage polarization is dependent on the bacterial O antigen.

    PubMed

    Luo, Fengling; Sun, Xiaoming; Qu, Zhen; Zhang, Xiaolian

    2016-02-01

    Recently, macrophages were shown to be capable of differentiating toward two phenotypes after antigen stimulation: a classically activated (M1) or an alternatively activated phenotype (M2). To investigate the effect of Salmonella enteric serovar typhimurium (S. typhimurium) on macrophage differentiation, we compared macrophage phenotypes after infection of murine bone marrow-derived macrophages with wild-type S. typhimurium and its isogenic rfc mutant. S. typhimurium C5 induced M1 macrophage polarization and enhanced inducible nitric oxide synthase expression by macrophages; this induction was dependent on Toll-like receptor 4. In contrast, the Δrfc mutant (S. typhimurium C5 rfc::Km(r)) lost this function and induced an M2 response in the macrophages. Here, we propose that S. typhimurium C5 is capable of polarizing macrophages towards the M1 phenotype and that this polarization is dependent on the O antigen encoded by rfc. Our finding indicates that M1 macrophage polarization induced by S. typhimurium may be related to the ability of this intracellular bacterium to survive and replicate within macrophages, which is essential for systemic disease. PMID:26745982

  6. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

    2009-01-01

    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  7. Detecting rare gene transfer events in bacterial populations.

    PubMed

    Nielsen, Kaare M; Bøhn, Thomas; Townsend, Jeffrey P

    2014-01-01

    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. PMID:24432015

  8. Detecting rare gene transfer events in bacterial populations

    PubMed Central

    Nielsen, Kaare M.; Bøhn, Thomas; Townsend, Jeffrey P.

    2014-01-01

    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. PMID:24432015

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

    PubMed Central

    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

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

    PubMed

    Bilgic, Huseyin Bilgin; Karagenc, Tulin; Bakırcı, Serkan; Shiels, Brian; Tait, Andrew; Kinnaird, Jane; Eren, Hasan; Weir, William

    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

  11. A valuable antigen detection method for diagnosis of acute hepatitis E.

    PubMed

    Wen, Gui-Ping; Tang, Zi-Min; Yang, Fan; Zhang, Ke; Ji, Wen-Fang; Cai, Wei; Huang, Shou-Jie; Wu, Ting; Zhang, Jun; Zheng, Zi-Zheng; Xia, Ning-Shao

    2015-03-01

    Hepatitis E virus (HEV) is a serious public health problem. The commonly used tests that are specific for current HEV infection diagnosis include the detection of anti-HEV IgM and HEV RNA. Here, we report an improved enzyme-linked immunosorbent assay (ELISA) method for HEV antigen detection with a linear range equivalent to 6.3 × 10(3) to 9.2 × 10(5) RNA copies per ml. The monoclonal antibody (MAb) 12F12, a high-ability MAb that binds HEV virus, was selected as the capture antibody from a panel of 95 MAbs. The positive period of HEV antigenemia in infected monkeys using this test was, on average, 3 weeks longer than previously reported and covered the majority of the acute phase. The positive detection rates of IgM, RNA, and new antigen from the first serum samples collected from 16 confirmed acute hepatitis E patients were 81% (13/16), 81% (13/16), and 100% (16/16), respectively. In three patients, the initial serum specimens that tested negative for IgM, despite the presence of symptoms of acute hepatitis and elevated alanine aminotransferase (ALT) levels, were positive for HEV antigen and HEV RNA. In contrast, the serum samples of the three RNA-negative patients were antigen positive (and IgM positive), possibly due to the degradation of HEV nucleic acids. Our results suggest that this new antigen detection method has acceptable concordance with RNA detection and could serve as an important tool for diagnosing acute hepatitis E. PMID:25540394

  12. Detection of Salivary IgA Antibodies Against the HlyE Antigen as a Diagnosis of Typhoid Fever

    PubMed Central

    Chin, Kai Ling; Redhuan, Nur Eliyana Mohd; Balaram, Prabha; Phua, Kia Kien

    2016-01-01

    Introduction The Salmonella typhi (S. typhi) haemolysin E protein (HlyE) has been shown to be a sensitive and specific antigen for the detection of typhoid fever through the detection of anti-HlyE antibodies in sera. Saliva can also be a useful diagnostic fluid as it also contains antibodies against bacterial pathogens. Aim This study aims to evaluate the potential detection of salivary anti-HlyE antibodies as a diagnosis of typhoid fever. Materials and Methods Saliva was collected from acute typhoid patients (n=16) who presented at Hospital Universiti Sains Malaysia with prolonged fever of more than five days and were positive for S. Typhi blood culture. Saliva was also collected from convalescent typhoid patients (n=11), patients with other febrile fevers (n=15), and from healthy individuals (n=25). An ELISA was developed to detect the presence of IgA antibodies against HlyE in the saliva of typhoid patients. Results The acute typhoid group had a higher mean absorbance value of 1.496 compared to the convalescent typhoid (0.538), other febrile fevers (0.678), and healthy individuals (0.457) group. Conclusion This study demonstrated the utility of salivary anti-HlyE IgA antibody as a biomarker for the diagnosis of typhoid fever. Follow-up studies with a larger sample size will allow the optimization of the sensitivity and specificity of the assay. This non-invasive method can be useful for mass screening programs. PMID:27504289

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

    PubMed Central

    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. PMID:18716008

  14. A novel agglutination test for antigen-specific detection of platelet antibodies.

    PubMed

    Meyer, Oliver; Agaylan, Ashraf; Borchert, Hans-Hubert; Aslan, Tunay; Bombard, Stéphane; Kiesewetter, Holger; Salama, Abdulgabar

    2006-10-15

    A simple and rapid antigen-specific assay for the identification antibodies to platelets is lacking, yet. Red-dyed polystyrene microbeads were coated with monoclonal antibodies to various platelet glycoprotein complexes, and used for the detection of platelet autoantibodies and alloantibodies. The results were largely identical with those obtained by monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). The new test is reliable yet less complex and time-consuming than the currently available assays, and it can be implemented in any routine laboratory. PMID:16933262

  15. Procedures for Sxs antigen detection by antibody-mediated cytotoxicity tests. A comparative analysis.

    PubMed

    Sánchez, A; Jiménez, R; Burgos, M; Díaz de la Guardia, R

    1994-11-01

    Biological reagents used in the serological detection of Sxs antigen by antibody-mediated cytotoxicity tests were compared in order to optimize the method. Our analyses showed that: (a) red cell-free spleen cells are the best target cells, (b) rabbit serum used as the complement source should be obtained from females, and absorbed with female spleen cells before use, (c) antiserum obtained by immunizing females with repeated injections of syngenic male spleen cells provides the highest anti-Sxs antibody titer, and (d) of the different biological fluids investigated, testis supernatant has highest concentration of Sxs antigen. PMID:7836542

  16. Modulation of cytokine release from colonic explants by bacterial antigens in inflammatory bowel disease.

    PubMed

    Dionne, S; Laberge, S; Deslandres, C; Seidman, E G

    2003-07-01

    The intestinal flora play an important role in experimental colitis and inflammatory bowel disease (IBD). Using colonic explant cultures from 132 IBD and control subjects, we examined tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 and interleukin-1 receptor antagonist (IL-1RA) production in vitro in response to bacterial activators. Unstimulated TNF-alpha release was increased significantly in rectal biopsies from involved IBD tissue, correlating with inflammation severity. Whereas lipopolysaccharide (LPS) only moderately stimulated TNF-alpha production from inflamed tissue, pokeweed mitogen (PWM) induced its release in all groups, with a stronger response in involved IBD tissue. Superantigen staphylococcal enterotoxin A (SEA) had a similar, but weaker effect. SEB was observed to be the strongest inducer of TNF-alpha for all groups, again with a more marked response in inflamed tissue. Stimulated release of IL-1 was considerably less than for TNF-alpha. The superantigens' superior potency over LPS was not as marked for IL-1 as it was for TNF-alpha. In addition to IL-1, IL-1RA release was also triggered by the bacterial products. The net effect of activation on the IL-1RA/IL-1 ratio was relatively modest. Release of the proinflammatory cytokines TNF-alpha and IL-1, as well as that of the anti-inflammatory cytokine IL-1RA was increased by incubation of colonic tissue with bacterial factors. TNF-alpha production and release was increased significantly in involved colonic explants from IBD. SEB was even capable of inducing TNF-alpha release from uninvolved colonic tissue. PMID:12823284

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

    USGS Publications Warehouse

    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.

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

    PubMed Central

    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 microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections. PMID:3021807

  19. Infection with Bartonella weissii and Detection of Nanobacterium Antigens in a North Carolina Beef Herd

    PubMed Central

    Breitschwerdt, Edward B.; Sontakke, Sushama; Cannedy, Allen; Hancock, Susan I.; Bradley, Julie M.

    2001-01-01

    Very recently, Bartonella organisms have been isolated from large ruminants (deer, elk, and dairy and beef cattle) located in the United States and in France. In this study, we report the serologic, microbiologic, and molecular findings related to the isolation of a Bartonella species in North Carolina beef cattle and the detection of nanobacterial antigen using a commercially available enzyme-linked immunosorbent assay. Between August 1998 and September 1999, blood was collected from 38 cattle ranging in age from 1 month to 6.5 years. After a 1-month incubation period, a Bartonella sp. was isolated on a 5% rabbit blood agar plate from three of six EDTA blood samples. PCR amplification of the 16S rRNA gene from all three isolates resulted in a DNA sequence that was 100% identical to that of B. weissii 16S rRNA (GenBank no. AF199502). By IFA testing, 36 of 38 cattle had antibodies (≥1:64) to Bartonella weissii (bovine origin) antigens. Nanobacterial antigen was detected in 22 of 22 serum samples. We conclude that infection with an organism similar or closely related to B. weissii can occur in North Carolina cattle and that although their actual existence is still controversial Nanobacterium antigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration. PMID:11230398

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

    PubMed

    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

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  3. Evaluation of enzyme-linked immunosorbent assay for detection of Salmonella typhi antigen and antibodies.

    PubMed

    el Fattah, M M; Hassan, E M; Shaheen, H; Awad, R A; Girgis, N

    1991-01-01

    Enteric fever is considered a major health problem in developing countries. The need for a rapid, accurate and conclusive method for diagnosis is important for adequate and proper treatment. The usefulness and reliability of the ELISA test in detection of S. typhi O antigen and specific IgG and IgM antibodies were assessed using sera obtained from 63 subjects clinically suspected to have enteric fever, 22 febrilenon-enteric subjects and 20 normal subjects. ELISA detection of S. typhi somatic antigen was positive in 75% of subjects with positive clot cultures. IgG and IgM antibodies to S. typhi lipopolysaccharide (LPS) were detected in sera from 83% and 88% of enteric fever subjects, respectively. While anti-LPS IgM was negative in all sera from febrile non-enteric subjects, 9% were positive for anti-LPS-IgG. The use of an ELISA for detection of anti-S. typhi LPS antibody in combination with the Widal test and/or the O antigen detection ELISA would provide a useful (95.8% sensitivity) adjunct to standard culture methods and allow for an earlier and more rapid diagnosis of enteric fever. PMID:1724780

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    PubMed Central

    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

  7. Detection of Antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen.

    PubMed Central

    Peterson, R R; Deeb, B J; DiGiacomo, R F

    1997-01-01

    As infection with Pasteurella multocida is common in rabbits, an enzyme immunoassay (EIA) was developed for its detection. A murine immunoglobulin G monoclonal antibody was used to capture a 37-kDa polypeptide of P. multocida serotype A:12 in an EIA to detect antibodies to P. multocida. The 37-kDa antigen was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits. The sensitivity of the P37 EIA, determined with sera from 56 rabbits infected with P. multocida, was 98%. Specificity, evaluated with sera from 62 rabbits from colonies free of P. multocida, was 92%. Titration curves of sera from rabbits immunized with P. multocida serotype A:3 or A:12 coincided, indicating that the P37 EIA was equally efficient in detecting antibodies to the two major serotypes of the organism. Comparison of the P37 EIA with the current serodiagnostic test, a bacterial lysate EIA, revealed relatively good correlation (r = 0.68). However, specificity was greatly improved, as 34% of uninfected rabbits were falsely positive by the lysate EIA whereas only 3% of uninfected rabbits were falsely positive by the P37 EIA. The coefficient of variation for same-day tests was 10%, and that for interday tests was 15%, indicating good reproducibility. The greater sensitivity and specificity of the P37 EIA should significantly enhance diagnostic capability to identify rabbits infected with P. multocida. PMID:8968909

  8. An antibody profile of systemic lupus erythematosus detected by antigen microarray

    PubMed Central

    Fattal, Ittai; Shental, Noam; Mevorach, Dror; Anaya, Juan-Manuel; Livneh, Avi; Langevitz, Pnina; Zandman-Goddard, Gisele; Pauzner, Rachel; Lerner, Miriam; Blank, Miri; Hincapie, Maria-Eugenia; Gafter, Uzi; Naparstek, Yaakov; Shoenfeld, Yehuda; Domany, Eytan; Cohen, Irun R

    2010-01-01

    Patients with systemic lupus erythematosus (SLE) produce antibodies to many different self-antigens. Here, we investigated antibodies in SLE sera using an antigen microarray containing many hundreds of antigens, mostly self-antigens. The aim was to detect sets of antibody reactivities characteristic of SLE patients in each of various clinical states – SLE patients with acute lupus nephritis, SLE patients in renal remission, and SLE patients who had never had renal involvement. The analysis produced two novel findings: (i) an SLE antibody profile persists independently of disease activity and despite long-term clinical remission, and (ii) this SLE antibody profile includes increases in four specific immunoglobulin G (IgG) reactivities to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), Epstein–Barr virus (EBV) and hyaluronic acid; the profile also includes decreases in specific IgM reactivities to myeloperoxidase (MPO), CD99, collagen III, insulin-like growth factor binding protein 1 (IGFBP1) and cardiolipin. The reactivities together showed high sensitivity (> 93%) and high specificity for SLE (> 88%). A healthy control subject who had the SLE antibody profile was later found to develop clinical SLE. The present study did not detect antibody reactivities that differentiated among the various subgroups of SLE subjects with statistical significance. Thus, SLE is characterized by an enduring antibody profile irrespective of clinical state. The association of SLE with decreased IgM natural autoantibodies suggests that these autoantibodies might enhance resistance to SLE. PMID:20201986

  9. Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria

    PubMed Central

    Karan, Ram; Barnes, Susan; Ekulona, Folasade; Chakravortty, Dipshikha; DasSarma, Shiladitya

    2014-01-01

    Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3’-coding region, named sopB4 and B5, were each fused to the C-terminal coding region of the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases. PMID

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

    PubMed

    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

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

    SciTech Connect

    Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.; Murrell, K.D.

    1989-02-01

    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.

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Novel fusion antigen displayed-bacterial ghosts vaccine candidate against infection of Escherichia coli O157:H7

    PubMed Central

    Cai, Kun; Tu, Wei; Liu, Yuenan; Li, Tao; Wang, Hui

    2015-01-01

    Infection with Escherichia coli O157:H7 may develop into hemorrhagic colitis, or hemolytic uremic syndrome (HUS), which usually causes kidney failure or even death. The adhesion and toxins are the important virulent factors. In this study, a novel vaccine candidate rSOBGs was constructed based on the bacterial ghost (BG). rSOBGs maintained the integrity of cellular morphology and displayed the linear Stx2Am-Stx1B antigen on the surface of outer membrane. rSOBGs induced Stxs-specific IgA/IgG antibodies and stronger intimin-specific IgA/IgG antibodies effectively in sera in this study. In vivo, the rSOBGs provided the higher protection rate (52%) than native bacterial ghost-OBGs (12%) when challenged intragastricly with high dose (500 LD50) viable E. coli O157:H7. Meanwhile, the rSOBGs provided higher protection rate (73.33%) than OBGs when challenged with 2 LD50 even to 5 LD50 lysed E. coli O157:H7. In vitro, the rSOBGs-immunized sera possessed neutralizing activity to lysed pathogenic bacteria. Furthermore, the results of histopathology also displayed that the administration of rSOBGs have the ability to reduce or inhibit the adhesion lesions and toxins damages of organs. The novel vaccine candidate rSOBGs induced both anti-toxin and anti-adhesion immune protection, suggesting the possibility to prevent the infectious diseases caused by Escherichia coli O157:H7. PMID:26626573

  15. Novel fusion antigen displayed-bacterial ghosts vaccine candidate against infection of Escherichia coli O157:H7.

    PubMed

    Cai, Kun; Tu, Wei; Liu, Yuenan; Li, Tao; Wang, Hui

    2015-01-01

    Infection with Escherichia coli O157:H7 may develop into hemorrhagic colitis, or hemolytic uremic syndrome (HUS), which usually causes kidney failure or even death. The adhesion and toxins are the important virulent factors. In this study, a novel vaccine candidate rSOBGs was constructed based on the bacterial ghost (BG). rSOBGs maintained the integrity of cellular morphology and displayed the linear Stx2Am-Stx1B antigen on the surface of outer membrane. rSOBGs induced Stxs-specific IgA/IgG antibodies and stronger intimin-specific IgA/IgG antibodies effectively in sera in this study. In vivo, the rSOBGs provided the higher protection rate (52%) than native bacterial ghost-OBGs (12%) when challenged intragastricly with high dose (500 LD50) viable E. coli O157:H7. Meanwhile, the rSOBGs provided higher protection rate (73.33%) than OBGs when challenged with 2 LD50 even to 5 LD50 lysed E. coli O157:H7. In vitro, the rSOBGs-immunized sera possessed neutralizing activity to lysed pathogenic bacteria. Furthermore, the results of histopathology also displayed that the administration of rSOBGs have the ability to reduce or inhibit the adhesion lesions and toxins damages of organs. The novel vaccine candidate rSOBGs induced both anti-toxin and anti-adhesion immune protection, suggesting the possibility to prevent the infectious diseases caused by Escherichia coli O157:H7. PMID:26626573

  16. Osteosarcoma Microenvironment: Whole-Slide Imaging and Optimized Antigen Detection Overcome Major Limitations in Immunohistochemical Quantification

    PubMed Central

    Kunz, Pierre; Fellenberg, Jörg; Moskovszky, Linda; Sápi, Zoltan; Krenacs, Tibor; Poeschl, Johannes; Lehner, Burkhard; Szendrõi, Miklos; Ewerbeck, Volker; Kinscherf, Ralf; Fritzsching, Benedikt

    2014-01-01

    Background In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. Methods Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. Results More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35–65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. Conclusion Standard staining and quantification procedures are not

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

    NASA Astrophysics Data System (ADS)

    Yu, An; Geng, Tingting; Fu, Qiang; Chen, Chao; Cui, Yali

    2007-04-01

    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 5 mol 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).

  18. Biomimetic/Optical Sensors for Detecting Bacterial Species

    NASA Technical Reports Server (NTRS)

    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

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

    PubMed Central

    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 <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors. PMID:15663862

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

    PubMed

    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

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

    PubMed Central

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

    2000-01-01

    A combination cassette format nonenzymatic rapid immunoassay for detection of Giardia and Cryptosporidium antigens was evaluated by using 556 patient stool specimens from three clinical laboratories. This assay (Genzyme Diagnostics Contrast Giardia/Cryptosporidium), which can be used with fresh or formalin-fixed specimens, had unadjusted sensitivities and specificities of 96.1 and 98.5% for Giardia and 100 and 98.7% for Cryptosporidium, respectively, in this study. PMID:10618122

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

    PubMed

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

    2016-04-01

    We performed a meta-analysis of diagnostic data to evaluate the performance of Histoplasma antigen detection tests for diagnosing histoplasmosis. We included all studies involving human subjects that assessed the performance of any antigen detection test for histoplasmosis in urine or serum by carrying out an exhaustive and reproducible search of the literature between 1980 and 2014 from four databases. Quality of the articles was assessed, and meta-analysis was performed under the random effects model, calculating sensitivity, specificity, likelihood and odds ratios, and ROC curve using Meta-DiSc(es). Nine out of a total of 23 studies met strict quality criteria and were therefore included. The overall sensitivity for antigen detection in serum and urine was 81% (95% CI 78-83%), while specificity was 99% (95% CI 98-99%). Sensitivity for antigenuria and antigenemia was 79% (95% CI 76-82%) and 82% (95% CI 79-85%), respectively; specificity values were 99% (95% CI 98-100%) in urine and 97% (95% CI 96-98%) in serum. The positive and negative likelihood ratios were 49.5 (95% CI 20.7-118.7) and 0.19 (95% CI 0.14-0.26), respectively, while the diagnostic OR was 362 (95% CI 121.2-1080.3) and area under the curve was 0.99. In conclusion, the performance of Histoplasma antigen detection assay of urine was not significantly different from that of blood, indicating that antigenuria and antigenemia have equal diagnostic value in histoplasmosis. PMID:26559429

  3. Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

    PubMed Central

    Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

    2012-01-01

    There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

  4. Method and apparatus for detecting and quantifying bacterial spores on a surface

    NASA Technical Reports Server (NTRS)

    Ponce, Adrian (Inventor)

    2009-01-01

    A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: a matrix including lanthanide ions is provided on the surface containing the bacterial spores; functionalized aromatic molecules are released from the bacterial spores on the surface; a complex of the lanthanide ion and the aromatic molecule is formed on the surface; the complex of the lanthanide ion and the aromatic molecule is excited to generate a characteristic luminescence of the complex on the surface; and the bacterial spores exhibiting the luminescence of the complex on the surface are detected and quantified.

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

    PubMed Central

    Kumar, Jyoti; Dixit, Shivendra Kumar; Kumar, Rajiv

    2015-01-01

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

  6. Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens.

    PubMed Central

    Gómez, B L; Figueroa, J I; Hamilton, A J; Ortiz, B; Robledo, M A; Hay, R J; Restrepo, A

    1997-01-01

    The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses. PMID:9399534

  7. Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens.

    PubMed

    Gómez, B L; Figueroa, J I; Hamilton, A J; Ortiz, B; Robledo, M A; Hay, R J; Restrepo, A

    1997-12-01

    The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses. PMID:9399534

  8. Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology.

    PubMed

    Zijlmans, H J; Bonnet, J; Burton, J; Kardos, K; Vail, T; Niedbala, R S; Tanke, H J

    1999-02-01

    A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2-0.4 microm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology. PMID:9918652

  9. Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

    PubMed Central

    Cao, Boyang; Yao, Fangfang; Liu, Xiangqian; Feng, Lu

    2013-01-01

    Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila. PMID:23974134

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

    PubMed Central

    Bruning, A.H.L.; Susi, P.; Toivola, H.; Christensen, A.; Söderlund-Venermo, M.; Hedman, K.; Aatola, H.; Zvirbliene, A.; Koskinen, J.O.

    2016-01-01

    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. PMID:27014463

  11. Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

    PubMed

    Hunt, Ann R; Hall, Roy A; Kerst, Amy J; Nasci, Roger S; Savage, Harry M; Panella, Nicholas A; Gottfried, Kristy L; Burkhalter, Kristen L; Roehrig, John T

    2002-06-01

    An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay. PMID:12037058

  12. Bacterial membranes enhance the immunogenicity and protective capacity of the surface exposed tick Subolesin-Anaplasma marginale MSP1a chimeric antigen.

    PubMed

    Contreras, Marinela; Moreno-Cid, Juan A; Domingos, Ana; Canales, Mario; Díez-Delgado, Iratxe; Pérez de la Lastra, José M; Sánchez, Emilio; Merino, Octávio; Zavala, Rigoberto López; Ayllón, Nieves; Boadella, Mariana; Villar, Margarita; Gortázar, Christian; de la Fuente, José

    2015-09-01

    Ticks are vectors of diseases that affect humans and animals worldwide. Tick vaccines have been proposed as a cost-effective and environmentally sound alternative for tick control. Recently, the Rhipicephalus microplus Subolesin (SUB)-Anaplasma marginale MSP1a chimeric antigen was produced in Escherichia coli as membrane-bound and exposed protein and used to protect vaccinated cattle against tick infestations. In this research, lipidomics and proteomics characterization of the E. coli membrane-bound SUB-MSP1a antigen showed the presence of components with potential adjuvant effect. Furthermore, vaccination with membrane-free SUB-MSP1a and bacterial membranes containing SUB-MSP1a showed that bacterial membranes enhance the immunogenicity of the SUB-MSP1a antigen in animal models. R. microplus female ticks were capillary-fed with sera from pigs orally immunized with membrane-free SUB, membrane bound SUB-MSP1a and saline control. Ticks ingested antibodies added to the blood meal and the effect of these antibodies on reduction of tick weight was shown for membrane bound SUB-MSP1a but not SUB when compared to control. Using the simple and cost-effective process developed for the purification of membrane-bound SUB-MSP1a, endotoxin levels were within limits accepted for recombinant vaccines. These results provide further support for the development of tick vaccines using E. coli membranes exposing chimeric antigens such as SUB-MSP1a. PMID:26219233

  13. Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

    PubMed Central

    Stibbs, H H; Samadpour, M; Manning, J F

    1988-01-01

    An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy. PMID:3183015

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

    PubMed

    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

  15. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

    PubMed Central

    Tang, Jonathan CY; Drokhlyansky, Eugene; Etemad, Behzad; Rudolph, Stephanie; Guo, Binggege; Wang, Sui; Ellis, Emily G; Li, Jonathan Z; Cepko, Constance L

    2016-01-01

    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001 PMID:27205882

  16. Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water and juice samples.

    PubMed

    Mutreja, Ruchi; Jariyal, Monu; Pathania, Preeti; Sharma, Arunima; Sahoo, D K; Suri, C Raman

    2016-11-15

    A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water and juice samples with a sensitivity upto 10(1)CFUmL(-1), with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection of Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view. PMID:27261886

  17. Improved Detection of Hepatitis B Virus Surface Antigen by a New Rapid Automated Assay

    PubMed Central

    Weber, Bernard; Bayer, Anja; Kirch, Peter; Schlüter, Volker; Schlieper, Dietmar; Melchior, Walter

    1999-01-01

    The performance of hepatitis B virus (HBV) surface antigen (HBsAg) screening assays is continuously improved in order to reduce the residual risk of transfusion-associated hepatitis B. In a multicenter study, a new automated rapid screening assay, Elecsys HBsAg (Roche Diagnostics), was compared to well-established tests (Auszyme Monoclonal [overnight incubation] version B and IMx HBsAg [Abbott]). Included in the evaluation were 23 seroconversion panels; sera from the acute and chronic phases of infection; dilution series of various HBsAg standards, HBV subtypes, and S gene mutants; and isolated anti-HBV core antigen-positive samples. To challenge the specificity of the new assay, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. Elecsys HBsAg showed a higher sensitivity for HBsAg subtypes ad, ay, adw2, adw4, ayw1, ayw2, ayw4, and adr detection in dilution series of different standards or sera than Auszyme Monoclonal version B and/or IMx HBsAg. Acute hepatitis B was detected in 11 to 16 of 23 seroconversion panels between 2 and 16 days earlier with Elecsys HBsAg than with the alternative assays. Elecsys HBsAg and Auszyme Monoclonal version B detected HBsAg surface mutants with equal sensitivity. The sensitivity and specificity of Elecsys HBsAg were 100%. Auszyme Monoclonal version B had a 99.9% specificity, and its sensitivity was 96.6%. IMx HBsAg showed a poorer sensitivity and specificity than the other assays. In conclusion, Elecsys HBsAg permits earlier detection of acute hepatitis B and different HBV subtypes than the alternative assays. By using highly sensitive HBsAg screening assays, low-level HBsAg carriers among isolated anti-HBV core antigen-positive individuals can be detected. PMID:10405414

  18. Reverse phase passive haemagglutination and single radial immunodiffusion to detect epsilon antigen of Clostridium perfringens type D.

    PubMed

    Beh, K J; Buttery, S H

    1978-11-01

    Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl. perfringens type D. It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml. When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen. However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult. No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep. The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem. PMID:223537

  19. Novel use of a radiolabelled antibody against stage specific embryonic antigen for the detection of occult abscesses in mammals

    DOEpatents

    Thakur, M.L.

    1990-04-17

    The invention discloses improved reagents containing antibodies against stage specific embryonic antigen-1 antibodies and improved methods for detection of occult abscess and inflammation using the improved reagents. No Drawings

  20. Production of monoclonal antibodies to hepatitis B surface and core antigens, and use in the detection of viral antigens in liver biopsies.

    PubMed Central

    Tedder, R. S.; Guarascio, P.; Yao, J. L.; Lord, R. B.; Eddleston, A. L.

    1983-01-01

    Hybridomas secreting monoclonal antibodies to HBsAg and HBcAg were prepared from immunized mice. An antibody capture radioimmunoassay was used to detect and select appropriate hybrids for propagation and cloning. The advantages of this assay were discussed. The resulting monoclonal antibodies were compared with conventional polyclonal antisera for the detection of virus antigens in liver tissue and found to give excellent results. Images Plate 2 Plate 1 PMID:6337208

  1. Detection of antibodies to Salmonella "O" antigens in typhoid fever by counterimmunoelectrophoresis. I. Description of technique.

    PubMed

    Hernández-Velarde, R; Muñoz, O; Garduño-Rodríguez, G; Gonzáles-Arroyo, S; Gutiérrez, G

    1979-01-01

    In the serum of patients with typhoid fever counterimmunoelectrophoretic techniques were used for the detection of antibodies to Salmonella "O" antigen. Lipopolysacharides (LPS) obtained with phenol and water from Salmonella typhi (antigens 0, 9 and 12) were used. Positive results were obtained in those patients with typhoid fever (20). The lower and higher titration levels were 1:8 and 1:32 respectively; the geometric mean was 1:16. The variation coefficient during the intra assay tests was 0.19, and remained stable throughout the inter-assay tests. Reproducibility, as well as a rapid technique, make this test a valuable tool for the serologic diagnosis of typhoid fever. PMID:420534

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

    PubMed Central

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

    2016-01-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis. PMID:27417097

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

    PubMed

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

    2016-06-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis. PMID:27417097

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

    PubMed Central

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

    1977-01-01

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

  5. Prostate specific antigen detection using AlGaN /GaN high electron mobility transistors

    NASA Astrophysics Data System (ADS)

    Kang, B. S.; Wang, H. T.; Lele, T. P.; Tseng, Y.; Ren, F.; Pearton, S. J.; Johnson, J. W.; Rajagopal, P.; Roberts, J. C.; Piner, E. L.; Linthicum, K. J.

    2007-09-01

    Antibody-functionalized Au-gated AlGaN /GaN high electron mobility transistors (HEMTs) were used to detect prostate specific antigen (PSA). The PSA antibody was anchored to the gate area through the formation of carboxylate succinimdyl ester bonds with immobilized thioglycolic acid. The AlGaN /GaN HEMT drain-source current showed a rapid response of less than 5s when target PSA in a buffer at clinical concentrations was added to the antibody-immobilized surface. The authors could detect a wide range of concentrations from 10pg/mlto1μg/ml. The lowest detectable concentration was two orders of magnitude lower than the cutoff value of PSA measurements for clinical detection of prostate cancer. These results clearly demonstrate the promise of portable electronic biological sensors based on AlGaN /GaN HEMTs for PSA screening.

  6. Detection of a Pancreatic Cancer-Associated Antigen (DU-PAN-2 Antigen) in Serum and Ascites of Patients with Adenocarcinoma

    NASA Astrophysics Data System (ADS)

    Metzgar, Richard S.; Rodriguez, Ned; Finn, Olivera J.; Lan, Michael S.; Daasch, Vicki N.; Fernsten, Philip D.; Meyers, William C.; Sindelar, William F.; Sandler, Robert S.; Seigler, H. F.

    1984-08-01

    A competition radioimmunoassay was developed, utilizing a murine monoclonal antibody to human pancreatic adenocarcinoma cells. Immunoblotting of a standard antigen preparation from either serum or ascites fluid after electrophoresis in 1% agarose showed that the specific DUPAN-2 activity resided in two major high molecular weight bands. DU-PAN-2 antigen levels were expressed as arbitrary units based on a standard partially purified antigen preparation. The inhibition curve with standard antigen was reproducible (SD < 10%) and essentially linear from 25 to 200 units/ml. The mean DU-PAN-2 antigen concentration for the sera from 126 normal individuals was 81 units/ml. Sera from pediatric patients with malignancy had a mean of 127 units/ml, while nasopharyngeal, stage III melanoma, and ovarian carcinoma patients had means of 89, 92, and 119 units/ml, respectively. All values in normal subjects as well as the melanoma, nasopharyngeal, ovarian, and pediatric cancer patients were less than 400 units/ml. Intermediate antigen levels were detected in patients with alimentary tract malignancies. Eight of 20 gastric cancer and 8 of 76 colorectal carcinoma patients and 3 patients with benign or nonmalig nant gastrointestinal tract disease had DU-PAN-2 values exceeding 400 units/ml. Ascites fluids from 6/6 and pancreatic juice from 2/2 pancreatic cancer patients had values greater than 750 units/ml. Serum from 68% of the 89 pancreatic cancer patients tested had DU-PAN-2 antigen levels greater than 400 units/ml. The mean serum value in this patient population was 4888 units/ml.

  7. A novel nanostructured biosensor for the detection of the dust mite antigen Der p2

    PubMed Central

    Tsai, Jaw-Ji; Bau, I-Jiuan; Chen, He-Tsing; Lin, Yu-Ta; Wang, Gou-Jen

    2011-01-01

    The group 2 allergen, Der p2, has been reported to activate innate toll-like receptors (TLRs) on respiratory epithelial cells and thus aggravate respiratory diseases. In this study, a highly sensitive nanostructured biosensor based on a 3D sensing element with uniformly deposited gold nanoparticles is proposed for the detection of the dust mite antigen Der p2. The barrier layer comprises an anodic aluminum oxide (AAO) film which is used as the template in this highly sensitive nanostructured biosensor. Simple electrochemical deposition without reducing agent and stabilizer was enough to uniformly synthesize gold nanoparticles on the surface of the barrier layer. The size and the distribution density of the nanoparticles can be well controlled by the applied potential during electrochemical deposition. Following this procedure, the dust mite monoclonal antibodies (IgG) were then immobilized through the 11-MUA (11-mercaptoundecanoic acid), (1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide)/(N-hydroxysuccinimide) self-assembled monolayer approach for the dust mite antigen Der p2 detection. The detection limit of the proposed 3D gold nanoparticle-based nanostructured biosensor was examined using electrochemical impedance spectroscopy analysis and found to be 1 pg/mL. The dynamic range was found to be 5 μg/mL. The proposed nanostructured biosensor would be useful for fast detection of rare molecules in a solution. PMID:21822382

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

    PubMed

    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

  9. Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype typhi antigens in urine.

    PubMed

    Fadeel, Moustafa Abdel; Crump, John A; Mahoney, Frank J; Nakhla, Isabelle A; Mansour, Adel M; Reyad, Baheia; El Melegi, Dawlat; Sultan, Yehia; Mintz, Eric D; Bibb, William F

    2004-03-01

    We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies to capture somatic antigen 9 (O9), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) from the urine of persons with and without typhoid fever. Sequential urine samples were collected from 44 patients with blood culture-confirmed typhoid fever and from two control groups. The first control group included patients with brucellosis (n = 12) and those with clinically diagnosed, non-typhoid, acute, febrile illness (n = 27). The second control group was a sample of healthy volunteer laboratory workers (n = 11). When assessed relative to date of fever onset, sensitivity was highest during the first week for all three antigens: Vi was detected in the urine of nine (100%) patients, O9 in 4 (44%) patients, and Hd in 4 (44%) patients. Sequential testing of two urine samples from the same patient improved test sensitivity. Combined testing for Vi with O9 and Hd produced a trend towards increased sensitivity without compromising specificity. The specificity for Vi exceeded 90% when assessed among both febrile and healthy control subjects, but was only 25% when assessed among patients with brucellosis. Detection of urinary Vi antigen with this ELISA shows promise for the diagnosis of typhoid fever, particularly when used within the first week after fever onset. However, positive reactions for Vi antigen in patients with brucellosis must be understood before urinary Vi antigen detection can be developed further as a useful rapid diagnostic test. PMID:15031525

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay.

    PubMed

    Gonçalves, Marcelo Luiz Carvalho; Araújo, Adauto; Duarte, Rosemere; da Silva, Joaquim Pereira; Reinhard, Karl; Bouchet, Françoise; Ferreira, Luiz Fernando

    2002-01-01

    The objective of this experiment was to assess the utility of a commercially available enzyme-linked immunosorbent assay (ELISA) kit for diagnosis of giardiasis in archaeological human remains. The kit, a monoclonal antibody assay, is used to detect the presence of Giardia-specific antigen 65 (GSA65) in human faeces. We utilized the assay in ancient faecal material. The material included desiccated faeces found in mummies or in archaeological sites, and sediments from latrines. A total of 83 specimens, previously examined microscopically for parasites, were examined. The ELISA detected 3 positive samples, dated to about 1200 AD, 1600 AD and 1700 AD. The ELISA was superior to direct observation. It was possible to identify G. duodenalis cysts by direct microscopy in only one of these samples. The results did not show cross-reactivity between this protozoan and helminths. The use of ELISA to detect G. duodenalis coproantigen could help the diagnosis of giardiasis in ancient human remains. PMID:12625140

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

    PubMed

    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

  14. Enhanced detection of bladder cancer using the epithelial surface marker epithelial membrane antigen: a preliminary report.

    PubMed

    Ring, K S; Karp, F; Benson, M C

    1990-09-01

    The flow cytometry (FCM) technique allows for the rapid quantitative analysis of the DNA content of individual cells. In a variety of genitourinary tumors, DNA ploidy has a significant impact upon prognosis and ultimate patient survival. In patients having transitional cell cancer (TCC) of the bladder, FCM of voided urine and bladder barbotage specimens is highly correlated with cytologic analysis in the detection of malignant cells. One problem with this technique has been decreased sensitivity in samples containing large numbers of inflammatory cells. To improve FCM detection of TCC in bladder wash specimens, we developed a technique using a monoclonal antibody (Mab) specific to human, epithelial membrane antigen (EMA). The EMA cell-surface marker enabled us to differentiate bladder epithelial cells from lymphocytes and cellular debris. In combination with DNA analysis using propidium iodide, the EMA Mab increased the sensitivity and specificity of FCM compared to conventional analysis using propidium iodide alone. We conclude that epithelial cell-surface antigen staining using both EMA Mab and DNA staining can increase the FCM detection of TCC in bladder wash specimens. PMID:2074517

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

    NASA Technical Reports Server (NTRS)

    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.

  16. Comparison of techniques for detecting antigens of Giardia lamblia and Cryptosporidium parvum in faeces.

    PubMed Central

    Tee, G H; Moody, A H; Cooke, A H; Chiodini, P L

    1993-01-01

    AIM--To compare the use of commercial monoclonal antibody test systems--the Giardia CEL IF test and the Crypto CEL IF test--for the detection of Giardia lamblia and Cryptosporidium parvum antigens in faeces with conventional techniques. METHODS--Sensitivity and specificity were evaluated using preparations of cysts of G lamblia and purified oocysts of C parvum. Evaluation of 59 random faecal samples passing through the Department of Clinical Parasitology, Hospital for Tropical Diseases, London, was carried out for both organisms. RESULTS--The fluorescence staining techniques proved more sensitive than other tests routinely used for diagnosis. PMID:8331181

  17. Method and apparatus for detecting and quantifying bacterial spores on a surface

    NASA Technical Reports Server (NTRS)

    Ponce, Adrian (Inventor)

    2009-01-01

    A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

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

    PubMed Central

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

    2013-01-01

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

  19. Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells.

    PubMed

    Tanaka, Maiko; Hiragun, Takaaki; Tsutsui, Tomoko; Yanase, Yuhki; Suzuki, Hidenori; Hide, Michihiro

    2008-06-15

    Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors. PMID:18339533

  20. Use of peroxidase-labelled antigen for the detection of antibodies to Borrelia burgdorferi in human and animal sera.

    PubMed

    Eiffert, H; Lotter, H; Thomssen, R

    1991-01-01

    We have developed a modified ELISA for the detection of anti-Borrelia burgdorferi (Bb) antibodies based on a peroxidase enzyme labelled antigen (ELAT). Microtiter plates were coated with antigen of Bb. The immunoglobulins of the serum samples were bound to the antigen and specific antibodies were detected by an enzyme labelled antigen. The test principle facilitates the recognition of specific antibodies in different collectives of human and animal sera. We performed epidemiological studies with the ELAT on 231 sera from mothers in maternity wards (9.5% positive), 219 patient sera sent to the Bb routine diagnostics (15% positive) and 230 sera from forestry workers (21.3% positive). We further investigated sera from red deer from South Lower Saxony which remained 55% Bb-antibody positive; deer were 37% and fallow deer were 29% positive. PMID:2028231

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

    PubMed

    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 < or = 3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US dollar 143,000 less than if CCNA alone had been performed on all 5,887 specimens. PMID:16517916

  2. Citrinin detection using phage-displayed anti-idiotypic single-domain antibody for antigen mimicry.

    PubMed

    Xu, Yang; Xiong, Liang; Li, Yanping; Xiong, Yonghua; Tu, Zhui; Fu, Jinheng; Tang, Xiao

    2015-06-15

    Anti-idiotypic antibodies (AIds) can mimic antigen molecules and can thus offer an alternative to conventional antigens in immunoassays. In this study, citrinin (CIT) was chosen as a target analyte, and an anti-idiotypic single-domain antibody (VHH) was selected from a naïve alpaca VHHs library to serve as a surrogate for CIT hapten. The phage-displayed VHH was used as a signal-amplification carrier to develop an indirect competitive phage enzyme-linked immunosorbent assay (P-ELISA) for the sensitive detection of CIT. The half-inhibition concentration (IC50) of P-ELISA was 10.9 μg/kg, which was 9-fold better than that of conventional ELISA (IC50=102.1 μg/kg). Results on P-ELISA analysis of naturally contaminated samples were also consistent with those obtained by conventional ELISA. In conclusion, the proposed P-ELISA demonstrates the potential use of phage-displayed anti-idiotypic VHH as surrogate for small molecules and signal-amplification carrier to improve assay performance for more sensitive analyte detection in food safety monitoring. PMID:25660863

  3. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Glennon, Erin; Ching, Wei-Mei

    2014-01-01

    Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA) using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1) which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33) of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156) of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies. PMID:26904739

  4. Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity ▿

    PubMed Central

    Talha, Sheikh M.; Salminen, Teppo; Chugh, Deepti A.; Swaminathan, Sathyamangalam; Soukka, Tero; Pettersson, Kim; Khanna, Navin

    2010-01-01

    A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity. PMID:20089793

  5. Evaluation of Culture, Antigen Detection and Polymerase Chain Reaction for Detection of Vaginal Colonization of Group B Streptococcus (GBS) in Pregnant Women

    PubMed Central

    Konikkara, Kavitha Paul; Baliga, Shrikala; Shenoy, Suchithra; Bharati, B.

    2014-01-01

    Background and Objective: Group B Streptococcal infection is an important cause of neonatal morbidity and mortality. Early detection of perinatal vagino-rectal (VR) carriage of Group B Streptococcus (GBS) is important in the management of newborn infections. The objective of the study was to evaluate Culture, antigen detection and Polymerase chain reaction for detection of GBS in Pregnant women. Settings and Design: Observational descriptive study was done in a tertiary care hospital in Southern India. Materials and Methods: VR swabs were collected from 50 women at 35 to 37 weeks of gestation. Culture in a selective Lim enrichment broth with subsequent culture on 5% sheep blood agar, Conventional PCR assay and antigen detection method were performed. The performance of antigen detection and PCR methods were compared with culture. Statistical analysis: Statistical analysis was performed by Chi-Square test. Results: GBS cultures were positive for 16% of the specimen (8 out of 50). Considering culture as a gold standard, Sensitivity, Specificity, Positive predictive value and Negative predictive value of antigen detection was 100%, 92.86%, 72.73%, 100% and similarly for that of PCR was 100%, 45.23%, 25.80%, 100%, respectively. Conclusion: Antigen detection method was the rapid, sensitive and specific test for the detection of GBS colonizers during pregnancy. PMID:24701479

  6. Highly sensitive nanoparticle-based immunoassays with elemental detection: Application to Prostate-Specific Antigen quantification.

    PubMed

    Garcia-Cortes, Marta; Encinar, Jorge Ruiz; Costa-Fernandez, Jose M; Sanz-Medel, Alfredo

    2016-11-15

    One of the major challenges in developing novel assay methods for the detection of biomolecules is achieving high sensitivity, because of the ultralow concentrations typically in clinical samples. Here, a Mn-doped ZnS quantum dots-based immunoassay platform is presented for highly sensitive detection of cancer biomarkers. Ultrahigh sensitivity is achieved through gold deposition on the surface of the nanoparticle tags acting as catalytic seeds, thus effectively amplifying the size of the metallic nanoparticles after the immunoassay and before the tag detection. Elemental mass spectrometry measurement of the gold content allowed detection of Prostate-Specific Antigen (PSA) at the low attog mL(-1) level. Moreover, the developed method showed not only an extremely high sensitivity for PSA detection but also a broad dynamic range, higher than 8 orders of magnitude, particularly useful for clinical studies involving quantitative detection of diverse biomarkers at their very different relevant concentration levels. Its applicability to discriminate small differences in PSA concentrations at low levels (few pgmL(-1)) in real serum samples was successfully evaluated. PMID:27162143

  7. Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen.

    PubMed

    Nourani, Sara; Ghourchian, Hedayatollah; Boutorabi, Seyed Mehdi

    2013-10-01

    An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP-HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3. PMID:23831477

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

    PubMed

    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

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

    PubMed

    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

  10. Detection of bovine group a rotavirus using rapid antigen detection kits, rt-PCR and next-generation DNA sequencing.

    PubMed

    Minami-Fukuda, Fujiko; Nagai, Makoto; Takai, Hikaru; Murakami, Toshiaki; Ozawa, Tadashi; Tsuchiaka, Shinobu; Okazaki, Sachiko; Katayama, Yukie; Oba, Mami; Nishiura, Naomi; Sassa, Yukiko; Omatsu, Tsutomu; Furuya, Tetsuya; Koyama, Satoshi; Shirai, Junsuke; Tsunemitsu, Hiroshi; Fujii, Yoshiki; Katayama, Kazuhiko; Mizutani, Tetsuya

    2013-12-30

    We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick 'Eiken' Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 10⁰-10³-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes. PMID:23912876

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

    PubMed

    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

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

    PubMed

    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

  13. Comparison of Stool Antigen Detection Kits to PCR for Diagnosis of Amebiasis ▿

    PubMed Central

    Stark, D.; van Hal, S.; Fotedar, R.; Butcher, A.; Marriott, D.; Ellis, J.; Harkness, J.

    2008-01-01

    The present study was conducted to compare two stool antigen detection kits with PCR for the diagnosis of Entamoeba histolytica infections by using fecal specimens submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, and the Institute of Medical and Veterinary Science, Adelaide, Australia. A total of 279 stool samples containing the E complex (E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii) were included in this study. The stool specimens were tested by using two commercially produced enzyme immunoassays (the Entamoeba CELISA PATH and TechLab E. histolytica II kits) to detect antigens of E. histolytica. DNA was extracted from all of the samples with a Qiagen DNA stool mini kit (Qiagen, Hilden, Germany), and a PCR targeting the small-subunit ribosomal DNA was performed on all of the samples. When PCR was used as a reference standard, the CELISA PATH kit showed 28% sensitivity and 100% specificity. The TechLab ELISA (enzyme-linked immunosorbent assay) kit did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR. With the TechLab kit, cross-reactivity was observed for three specimens, one of which was positive for both E. dispar and E. moshkovskii while the other two samples contained E. moshkovskii. Quantitative assessment of the PCR and ELISA results obtained showed that the ELISA kits were 1,000 to 10,000 times less sensitive, and our results show that the CELISA PATH kit and the TechLab ELISA are not useful for the detection of E. histolytica in stool samples from patients in geographical regions where this parasite is not endemic. PMID:18367563

  14. Rapid detection of bacterial growth in blood samples by a continuous-monitoring electrical impedance apparatus.

    PubMed Central

    Specter, S; Throm, R; Strauss, R; Friedman, H

    1977-01-01

    A growth detection method utilizing an automated apparatus capable of rapidly detecting bacterial growth by measuring changes of electrical impedance in bacteriological medium was utilized with "mock" blood cultures containing various gram-negative and gram-positive bacteria. Measurement of changes of electrical impedance was found to ba as accurate and comparable for time of growth detection as the radiometric method for detection of the same bacteria using mock blood cultures. In a limited clinical trial the use of the electrical impedance apparatus detected in 1 positive specimen from 40 clinical blood specimens as rapidly as by radiometric measurement. Both methods were considerably faster for detecting bacterial growth as compared with conventional culture methods. The selected species of gram-positive and -negative organisms tested were all detected by the electrical impedance method, including aerobes and anerobes. However, addition of 5% CO2 to the incubation atmosphere enhanced detection of gram-positive organisms. PMID:336642

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo.

    PubMed

    Rauch, Isabella; Tenthorey, Jeannette L; Nichols, Randilea D; Al Moussawi, Khatoun; Kang, James J; Kang, Chulho; Kazmierczak, Barbara I; Vance, Russell E

    2016-05-01

    NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]-containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1(-/-) and Naip2(-/-) mice, as well as Naip1-6(Δ/Δ) mice lacking all functional Naip genes. By challenging Naip1(-/-) or Naip2(-/-) mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5(-/-) mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6(Δ/Δ) mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo. PMID:27045008

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

    PubMed

    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

  18. The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria.

    PubMed

    Tam, Frankie C H; Ling, Thomas K W; Wong, Kam Tak; Leung, Danny T M; Chan, Raphael C Y; Lim, Pak Leong

    2008-03-01

    TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8-31 mug ml(-1)) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2-4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9(+) Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9 x 10(8) organisms ml(-1). Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials. PMID:18287294

  19. Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus

    PubMed Central

    2013-01-01

    Background Outbreaks in poultry involving influenza virus from H7 subtype have resulted in human infections, thus causing a major concern for public health, as well as for the poultry industry. Currently, no efficient rapid test is available for large-scale detection of either antigen or antibody of H7 avian influenza viruses. Results In the present study, a dual function ELISA was developed for the effective detection of antigen and antibody against H7 AIVs. The test was established based on antigen-capture-ELISA and epitope blocking ELISA. The two Mabs 62 and 98 which were exploited in the assay were identified to recognize two conformational neutralizing epitopes on H7 HA1. Both of the epitopes exist in all of the human H7 strains, including the recent H7N9 strain from China and > 96.6% of avian H7 strains. The dual ELISA was able to detect all of the five H7 antigens tested without any cross reaction to other influenza subtypes. The antigen detection limit was less than 1 HA unit of H7. For antibody detection, the sensitivity and specificity of the dual ELISA was evaluated and compared to HI and microneutralization using immunized animal sera to different H7 strains and different subtypes of AIVs. Results indicated that antibodies to H7 were readily detected in immunized animal sera by the dual ELISA whereas specimens with antibodies to other AIVs yielded negative results. Conclusions This is the first dual-function ELISA reported for either antigen or antibody detection against H7 AIVs. The assay was highly sensitive and 100% specific in both functions rendering it effective for H7 diagnosis. PMID:24083616

  20. Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures

    PubMed Central

    Pasteran, Fernando; Tijet, Nathalie; Melano, Roberto G.

    2015-01-01

    We compared carbapenemase detection among 266 Gram-negative bacilli (161 carbapenemase producers) using the Carba NP tests issued by the CLSI (CNPt-CLSI) and a novel protocol (CNPt-direct) designed for carbapenemase detection direct from bacterial cultures (instead of bacterial extracts required by the CLSI tests). The specificities were comparable (100%), but the CNPt-direct was more sensitive (98% versus 84%). The CNPt-direct was easier to perform due to the direct use of colonies and offered a more robust detection of carbapenemase producers. PMID:26424841

  1. 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

    PubMed Central

    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

  2. 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.

    PubMed

    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

  3. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

    PubMed Central

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  4. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

    PubMed

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  5. Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffin-embedded tissues

    USGS Publications Warehouse

    Ochiai, T.; Yasutake, W.T.; Gould, R.W.

    1985-01-01

    The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

  6. Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

    PubMed

    Sakuma, Shinji; Yu, James Y H; Quang, Timothy; Hiwatari, Ken-Ichiro; Kumagai, Hironori; Kao, Stephanie; Holt, Alex; Erskind, Jalysa; McClure, Richard; Siuta, Michael; Kitamura, Tokio; Tobita, Etsuo; Koike, Seiji; Wilson, Kevin; Richards-Kortum, Rebecca; Liu, Eric; Washington, Kay; Omary, Reed; Gore, John C; Pham, Wellington

    2015-03-01

    Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level. PMID:25052906

  7. Diagnosis of Giardia lamblia infections by detection of parasite-specific antigens.

    PubMed Central

    Janoff, E N; Craft, J C; Pickering, L K; Novotny, T; Blaser, M J; Knisley, C V; Reller, L B

    1989-01-01

    Antigen detection methods may facilitate diagnosis of Giardia lamblia in stool specimens. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and immunoblotting, G. lamblia cysts and trophozoites share several antigens, especially in the 65-kilodalton and 30- to 34-kilodalton regions. By using blind methods, we compared results obtained by counterimmunoelectrophoresis using cyst-immune rabbit serum and by enzyme-linked immunosorbent assay (ELISA) using trophozoite-immune rabbit serum with results obtained by microscopic examination of a preserved, concentrated, and permanently stained stool specimen. Results were similar when these three methods were used to examine 118 stool specimens from clinical microbiology laboratories (53 specimens with G. lamblia) and specimens from 239 day-care-center toddlers (39 specimens with G. lamblia). Compared with microscopy, we found, for counterimmunoelectrophoresis and ELISA, respectively: sensitivity, 88 versus 94%; specificity, 97 versus 95%; positive predictive value, 86 versus 76%; negative predictive value, 98 versus 97%; and concordance, 89%. The false-positive rate by ELISA was 24% (10 of 42) in day-care-center toddlers but only 3% (1 of 32) in healthy adults (P less than 0.04) as corroborated by microscopy. This discrepancy suggests that the ELISA may be more sensitive than microscopy, which is considered the reference standard, and that results may be dependent, in part, on the epidemiology of the infection in the study subjects. Images PMID:2715318

  8. Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells

    PubMed Central

    Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

    2015-01-01

    A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated. PMID:26274954

  9. Asymptomatic females: detection of antibody activity to gonococcal pili antigen by radioimmunoassay.

    PubMed Central

    Oates, S A; Falkler, W A; Joseph, J M; Warfel, L E

    1977-01-01

    A gonococcal pili antigen preparation was used to detect antibody activity sera obtained from 322 culture-positive asymptomatic females and 150 negative controls. Pili were obtained from a culture of type 2 Neisseria gonorrhoeae (strain 2686) and labeled with 125I for use in a double-antibody radioimmunoassay test system. Of the 322 sera obtained from culture-positive, asymptomatic females, 276 (85.7%) showed antibody activity greater than or equal to 1.8 mug/ml. Negative controls were obtained from three different groups of individuals, and 130 (86.7%) had undetectable antibody activity. Sera from asymptomatic, culture-positive females were absorbed with three different strains of N. gonorrhoeae, one of these strains being the organism used for pili antigen preparations. The absorbed sera were tested for antibody activity, and in each case the activity in the absorbed sera dropped to an undetectable level. When the same sera were absorbed with N. meningitidis, N. catarrhalis, N. perflava, Escherichia coli, Herellea vaginicola, Mima polymorpha, Staphylococcus aureus, and Candida albicans, little, if any, decline in the level of anti-pili antibody activity was observed. PMID:401829

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

    NASA Astrophysics Data System (ADS)

    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.