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Sample records for antiserum

  1. Myasthenia gravis treated with purified antithymocyte antiserum.

    Science.gov (United States)

    Pirofsky, B; Reid, R H; Bardana, E J; Baker, R L

    1979-01-01

    The therapeutic effect of goat anti-human thymocyte antiserum globulin (ATG) was assessed in 10 patients with myasthenia gravis. All subjects had far-advanced, debilitating disease poorly responsive to anticholinesterase therapy. Prolonged, low-dose ATG therapy was used, with 1.0 to 2.6 gm ATG protein administered intramuscularly over a 28- to 73-day period. Therapeutic responses of varying degrees were noted in 8 of 10 patients. Completion of a course of ATG treatment and discontinuation of the drug did not lead to acute relapse. Follow-up examinations for over 5 years have been maintained. A mean remission period of approximately 2 years was observed. This therapy deserves further evaluation; subjects with progressive myasthenia gravis despite prior thymectomy may represent ideal candidates. PMID:311448

  2. Raising of Antiserum and development od IRMA serum ferritin

    International Nuclear Information System (INIS)

    Antiserum to human liver ferritin was developed by immunizing sheep with purified human liver ferritin. This antiserum has been purified using ammonium sulphate. A part of it was linked chemically to magnetisale particles, while the other part was adsorbed physically onto polystyrene beads in order to develop two IRMAs. The anti-ferritin antibody obtained was purified and diluted 200,000 folds before being coated to polystyrene beads, or coupled to magnetisable particles. Assay validation, sensitivity and accuracy tests for the two IRMAs were performed. The polystyrene beads IRMA system showed better performance than the magnetisable particles system. It was found that, the minimum detectable dose in the bead system was 0.6 ng/ml, whereas it was 6.0 ng/ml in the magnetisable one. In the beads system, the mean recovery of ferritin was found to be 98.5% while the linearity tests showed a correlation coefficient of 0.996. The comparison between our coated beads IRMA with NETRIA's IRMA serum ferritin showed a correlation coefficient of 0.982. (Author)

  3. Antigenic profile of human recombinant PrP: generation and chracterization of a versatile polyclonal antiserum

    NARCIS (Netherlands)

    Sachsamanoglou, M.; Paspaltzis, I.; Petrakis, S.; Verghese-Nikolakaki, S.; Panagiotidis, C.H.; Voitlander, T.; Budka, H.; Langeveld, J.P.M.; Sklaviadis, T.

    2004-01-01

    We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antise

  4. Production and Characterization of ZFP36L1 Antiserum against Recombinant Protein from Escherichia coli

    OpenAIRE

    Cao, Heping; LIN, RUI; Ghosh, Sanjukta; Anderson, Richard A.; Urban, Joseph F.

    2008-01-01

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over-expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were us...

  5. Neutralization of glucagon by antiserum as a tool in glucagon physiology. Lack of depression of basal blood glucose after antiserum treatment in rats

    DEFF Research Database (Denmark)

    Holst, J J; Galbo, H; Richter, Erik

    1978-01-01

    The method of producing experimental glucagon deficiency by administration of glucagon antiserum was evaluated in rats. A pool of antisera was prepared, the affinity of which exceeded that of the glucagon receptors of liver cell membranes, whereas the binding capacity of the volume used amounted ...... lowered beyond detection limit. The data indicate that the absolute concentration of glucagon in plasma is of minor importance for the maintenance of basal blood glucose in the rat....... exogenous glucagon was abolished. Antiserum treatment, however, had no effect on blood glucose in rats fasted for 3 and 10 h, in chemically sympathectomized and adrenomedullectomized rats, and in 48-h-fasted, acutely adrenalectomized rats. The antiserum was found to contain 460 nmol/liter of antibody......-bound glucagon, originating in the rabbit in which the antiserum was raised. However, antibody preparations from which the bound glucagon had been effectively removed were equally ineffective in lowering the basal blood glucose in rats, although in three-fourths of the rats the concentration of free glucagon was...

  6. Use of antiserum to neurotensin reveals a physiological role for the peptide in rat prolactin release.

    Science.gov (United States)

    Vijayan, E; Carraway, R; Leeman, S E; McCann, S M

    1988-12-01

    Previous studies have indicated that the brain peptide neurotensin can stimulate prolactin release by direct action on the pituitary gland, whereas its action within the hypothalamus is inhibitory. The inhibitory action is mediated by the release of dopamine into the hypophyseal portal veins, which deliver the neurotransmitter to the anterior pituitary gland to inhibit prolactin release. Our experiments were done to evaluate the physiologic significance of these neurotensin actions by injecting the globulin fraction of highly specific neurotensin antiserum either intravenously or intraventricularly. Injection into the third ventricle of either 1 or 3 microliter of neurotensin antiserum significantly increased plasma prolactin concentrations in (i) ovariectomized and (ii) ovariectomized estrogen- and progesterone-primed rats within 1 hr of injection. The response was more pronounced in the ovariectomized than in the ovariectomized estrogen- and progesterone-treated animals and was dose related. Intraventricular injection of these doses of neurotensin antiserum also evoked elevations in plasma prolactin in intact males, which were significant but smaller in magnitude than those seen in female rats. To evaluate the effect of the antiserum on the pituitary directly, the antiserum was injected intravenously at a dose of 40 microliter, which was sufficient to block the blood pressure-lowering effect of neurotensin. After the intravenous injection of antiserum, a highly significant suppression of plasma prolactin occurred, detectable when first measured at 1 hr after injection in both ovariectomized and ovariectomized estrogen- and progesterone-treated animals; however, the intravenous injection of antiserum had no significant effect on the prolactin release in males. These data indicate the physiological significance of the hypothalamic inhibitory actions of neurotensin on prolactin release, which are probably mediated by its stimulation of dopamine release that in turn

  7. Prolongation of Cardiac Allograft Survival in Rats by Treatment with Anti-Interleukin 2 Antiserum

    Directory of Open Access Journals (Sweden)

    Osaki,Toshihide

    1988-04-01

    Full Text Available Interleukin-2 (IL2 is the obligatory signal for both T cell mitogenesis and in vitro generation of alloreactive cytotoxic T lymphocytes (CTL. An investigation was made to determine whether an antibody directed against IL2 would suppress the rejection reaction of rat cardiac allografts. Rabbit anti-interleukin 2 (anti-IL2 antiserum was obtained by immunizing at 2 week intervals over a period of 8 weeks with 10(6 U of recombinant human IL2 along with complete Freund's adjuvant. The bioassay for inhibition of IL2 activity by anti-IL2 antiserum was carried out in conjunction with the IL2-dependent cytotoxic T cell (CTLL cell assay. Cardiac allografts of F344 rats were heterotopically transplanted into ACI rats. Seven daily doses of 1 ml of anti-IL2 antiserum were administered intravenously following transplantation. IL2-driven [3H]thymidine incorporation in CTLL cells was significantly inhibited by rabbit anti-IL2 antiserum. Graft survival in the anti-IL2 serum-treated group was significantly prolonged in a dose-dependent fashion compared to control groups. In conclusion, these results indicate that rabbit anti-IL2 antiserum may prove to be of significant value as an immunosuppressive agent in clinical organ transplantation.

  8. Specific antiserum to Leu-enkephalin and its use in a radioimmunoassay

    International Nuclear Information System (INIS)

    This paper describes the preparation of an immunogenic protein-conjugate of Leu-enkephalin and the specificity of an antiserum generated to this antigen in rabbits. Such an antiserum, when used in a radioimmunoassay as described herein, should provide a useful tool for studying the role of both Leu- and Met-enkephalin in the 'Pain Pathway'. According to a recent hypothesis, enkephalin levels in the central nervous system should determine not only the pain threshold but phenomena such as tolerance, physical dependence and the withdrawl syndrome as well. From this respect, the importance of a radioimmunoassay is obvious

  9. Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

    Institute of Scientific and Technical Information of China (English)

    Faiz MMT Marikar; Dingyuan Ma; Jianqiang Ye; Bo Tang; Weijuan Zheng; Jing Zhang; Min Lu; Zichun Hua

    2008-01-01

    The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichla coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refoided and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD不得 protein was allowed the production of high titre polycional antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.Cellular & Molecular Immunology. 2008;5(6):471-474.

  10. Cloning, Expression of Crocus sativus Phytoene Desaturase Gene and Preparation of Antiserum against It

    Institute of Scientific and Technical Information of China (English)

    Bai Jie; Miao Chen; Xu Ying; Tang Lin; Wang Zhi-tao; Chen Fang

    2004-01-01

    A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET -21a(+) and overexpressed in E. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma.

  11. Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum

    DEFF Research Database (Denmark)

    Bak, Hanne; Thomas, O.R.T.

    2007-01-01

    We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immumoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rProtein...... evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made....

  12. Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model.

    OpenAIRE

    Menzies, B E; Kernodle, D S

    1996-01-01

    A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with...

  13. Production of double antibody for radioimmunoassay (sheep anti-rabbit IgG antiserum)

    International Nuclear Information System (INIS)

    A second antibody (sheep anti-rabbit IgG antiserum) to be used in RIAs in which the first antibody is raised in rabbits was produced. For this production, initially the IgG was isolated from rabbit serum and purified by sodium sulphate precipitation followed by ion exchange chromatography on DEAE-cellulose. Four sheep were immunized with 500 u g of purified rabbit IgG, emulsified in Freund Complete Adjuvant and administered by multisite subcutaneous injections. These injections were repeated at 20-days intervals and blood samples (40 ml) were taken from the jugular vein 10 days after the boosts for the evaluation of the antisera title. After each four boosts a great bleeding was done by the same route. Approximately 500 ml of serum were obtained in each bleeding per animal. The antisera were evaluated by the human thyrotropin RIA developed at IPEN laboratories employing reagents provided by NIDDKD, USA. These evaluations referred to the determination of the antisera title and of the ideal concentration of carrier IgG, to the study of the kinetic of precipitation and to the confirmation of the inexistent cross-reactivity with human IgG, in comparison with a reference antiserum of know precipitation characteristics supplied by the Radioassay System Laboratories. Approximately 3,6 l of antiserum (sheep anti-rabbit IgG serum) were produced from the four sheep, which presented title and precipitation characteristics very similar to those exhibited by the imported commercial product, even presenting higher titles. The results obtained in this work indicated that it was created enough experience for the production of this biological reagent for RIA, that could be done integrally in the country in greater scale, and at a very reduced cost. (author). 81 refs, 36 figs, 33 tabs

  14. Prolongation of Cardiac Allograft Survival in Rats by Treatment with Anti-Interleukin 2 Antiserum

    OpenAIRE

    Osaki, Toshihide; Sakagami, Kenichi; Orita,Kunzo

    1988-01-01

    Interleukin-2 (IL2) is the obligatory signal for both T cell mitogenesis and in vitro generation of alloreactive cytotoxic T lymphocytes (CTL). An investigation was made to determine whether an antibody directed against IL2 would suppress the rejection reaction of rat cardiac allografts. Rabbit anti-interleukin 2 (anti-IL2) antiserum was obtained by immunizing at 2 week intervals over a period of 8 weeks with 10(6) U of recombinant human IL2 along with complete Freund's adjuvant. The bioassay...

  15. Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake venom

    Directory of Open Access Journals (Sweden)

    Ho Paulo L

    2009-03-01

    Full Text Available Abstract Background Micrurus corallinus (coral snake is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization. Results A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx (24% and phospholipases A2 (PLA2s (15%. However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA2 and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens. Conclusion Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA

  16. Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid

    International Nuclear Information System (INIS)

    Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid have been performed with a view to producing optimal assay conditions. Cholic acid-BSA was found to be the best immunogen to produce antibodies to conjugated cholic acid and the response was of an IgG type. Incorporating a spacer (hexanoic acid) between hapten and carrier protein resulted in a decrease in antiserum titre. Optimal conditions for the assay were found using [125I]histamine-glycocholic acid as ligand with a dilution of antiserum to produce 60% binding of ligand and a pH of 7.4. Using these assay conditions no serum effects were found; extraction of serum prior to assay was therefore unnecessary. The assay was sensitive enough to detect post-prandial increases in serum bile acid concentrations following a liquid test meal; no increase was observed throughout the same time period in a fasting control. (Auth.)

  17. Preparation of ferritin immunogen and raising of antiserum for immunoradiometric assay

    International Nuclear Information System (INIS)

    The increase demand for serum ferritin determination in Sudan, together with the short life of the tracer and the limited foreign currency resources lead to thinking of local production of such an assay. A purified human liver foreign was emulsified in Freunds adjuvant and used as immunogen in sheep to develop anti-ferritin antiserum. The harvested blood has been purified using 27% ammonium sulphate. A part of it was linked covalently o magnetizable particles using carbodimmidazole (CDI) as activator, while the other part was absorbed physically onto polystyrene beads with a dilution of 200000 folds in order to develop two immunoradiometric assays (IRMAs). Assay validation, sensitivity and accuracy tests for the two IRMAs were performed. The polystyrene beads IRMA system showed better performance than the magnetizable particles system. It was found that, the minimum detectable dose in the bead system was 0.6 ng/ml, whereas it was 6.6 ng/ml in the magnetizable one. In the beads system, the mean recovery of ferritin was found to be 98.5% while the linearity tests showed a correlation coefficient of 0.996. The comparison between our coated beads IRMA with North East Thames region for Immunoassay (NETRIA's) IRMA serum ferritin showed a correlation coefficient of 0.982. (Author)

  18. Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum

    Energy Technology Data Exchange (ETDEWEB)

    Marlow, R.; Freeman, S.J.

    1989-01-01

    Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-vesceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 ug/ml while AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 ul/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of /sup 125/I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to their previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on /sup 125/I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behavior is observed under similar experimental conditions.

  19. A comparative experiments for tube agglutination test of pullorum antiserum with gamma ray Co60 irradiated salmonella pullorum

    International Nuclear Information System (INIS)

    An agglutinability between naturally infected positive chicken serum of pullorum disease and hyperimmunized rabbit antiserum was compared. And the following results were obtained and summarized. On the agglutinability, Salmonella pullorum antigen which irradiated gamma-ray was better than another both formalized and heated antigen. Time of judgemented as positive titer in the tube agglutination test to the naturally infected positive chicken serum was it most suitable for 12 hours at 37°C. Agglutination titer of positive immune chicken serum against gamma-ray irradiate Salmonella pullorum were as 320 approximately 640x. (author).

  20. Antigen-induced and non-antigen-induced histamine release from rat mast cells sensitized with mouse antiserum.

    Directory of Open Access Journals (Sweden)

    Kurose,Masao

    1981-10-01

    Full Text Available Marked IgE-mediated histamine release from rat mast cells sensitized in vitro with mouse antiserum occurs in the presence of added Ca++ and phosphatidylserine (PS, although a considerable degree of antigen-induced histamine release which may utilize intracellular or cell-bound calcium is also observed. The decay in the responsiveness to Ca++ of the sensitized cells stimulated by antigen in Ca++-free medium in the presence of PS is relatively slow, and maximum release is produced by Ca++ added 1 min after antigen. Histamine release also occurs when Ca++ is added after PS in the absence of antigen to the sensitized cells suspended in Ca++-free medium. Unlike the antigen-induced release, the intensity of this non-antigen-induced release varies depending on both mast-cell and antiserum pools. A heat-labile factor(s, which is different from antigen-specific IgE antibody and is also contained in normal mouse serum, is involved in this reaction. In the antigen-nondependent (PS + Ca++-induced release, no decay in the responsiveness to Ca++ is observed after PS addition. Both the antigen-induced and non-antigen-induced release are completed fairly rapidly and are dependent of temperature, pH and energy.

  1. A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia.

    Science.gov (United States)

    Ratanabanangkoon, Kavi; Tan, Kae Yi; Eursakun, Sukanya; Tan, Choo Hock; Simsiriwong, Pavinee; Pamornsakda, Teeraporn; Wiriyarat, Witthawat; Klinpayom, Chaiya; Tan, Nget Hong

    2016-04-01

    Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a 'pan-specific' AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a 'pan-specific' AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund's adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a

  2. A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia

    Science.gov (United States)

    Ratanabanangkoon, Kavi; Tan, Kae Yi; Eursakun, Sukanya; Tan, Choo Hock; Simsiriwong, Pavinee; Pamornsakda, Teeraporn; Wiriyarat, Witthawat; Klinpayom, Chaiya; Tan, Nget Hong

    2016-01-01

    Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings

  3. Inhibition of atrial natriuretic peptide-induced cyclic GMP accumulation in the bovine endothelial cells with anti-atrial natriuretic peptide receptor antiserum

    International Nuclear Information System (INIS)

    Using an antiserum raised against the purified atrial natriuretic peptide (ANP) receptor that has a disulfide-linked homodimeric structure and represents one subtype of the multiple ANP receptors, we showed that the receptor is coupled to the guanylate cyclase activation; formerly, this type of ANP receptor is not considered to be coupled to the cyclase. The specificity of the antiserum was determined by immunoblot analysis and immunoprecipitation. The anti-receptor antiserum did not compete with 125I-ANP for binding to the receptor but it lowered the affinity of the receptor. When added to bovine endothelial cell cultures, the antiserum blocked the cyclic GMP response of the cells triggered by ANP. These results indicate that the subtype of the ANP receptor recognized by the antiserum is responsible for the activation of particulate guanylate cyclase as well as the double function type receptor that has been assumed to contain both the receptor domain and the catalytic domain for cGMP synthesis on the same molecule. The presence of dissociative complexes of ANP receptor and particulate guanylate cyclase was also demonstrated by radiation inactivation analysis

  4. Especificidade de anti-soro policlonal à Leifsonia xyli subsp. xyli Policlonal antiserum specifity against Leifsonia xyli subsp. xyli

    Directory of Open Access Journals (Sweden)

    Josil B. Carneiro Jr

    2004-12-01

    Full Text Available Detectar a presença da bactéria Leifsonia xyli subsp. xyli em material de propagação da cana-de-açúcar (Saccharum sp. é importante para direcionar o controle do raquitismo-da-soqueira. Neste trabalho, objetivou-se produzir anticorpo policlonal específico contra Leifsonia xyli subsp. xyli (Lxx, visando utilizá-lo em método sorológico para detecção do patógeno. Para isso, o antígeno foi preparado a partir de células intactas, após lavagem por centrifugação de cultura-pura em tampão fosfato salino 0,01 M (PBS e diálise em glutaraldeido 2% em PBS. O plano de imunização em coelho consistiu de duas injeções intramusculares da mistura 1:1 do antígeno com adjuvante Freund (completo e incompleto, a intervalos de 21 dias e duas injeções subcutâneas do antígeno puro, a intervalos de dez dias. O anti-soro foi testado pelo método de Dot Blot com revelação por peroxidase para se determinar: (i título do anticorpo e (ii reação contra Lxx, Xanthomonas axonopodis pv. vesicatoria e bactérias endofíticas de cana-de-açúcar (Azospirillum brasilense, A. lipoferum, Herbaspirillum rubrisubalbicans, H. seropedicae e Gluconacetobacter diazotrophicus. A maior diluição analisada do anti-soro 1:20.000 mostrou reação fortemente positiva e específica contra Lxx e ausência de reação contra as demais bactérias. A purificação da fração IgG (Imunoglobulina G não resultou em melhoria na reatividade e especificidade do anti-soro. Estimou-se o nível de detecção do método a partir de suspensão bacteriana em 2x10(6 células/ml.To detect the presence of the bacterium Leifsonia xyli subsp. xyli (Lxx in propagation material is important for the control of the ratoon stunting disease. The objective of this work was to improve a serological method for detection of the pathogen and to evaluate the policlonal antiserum specificity produced against Lxx. The antigen was prepared by resuspending pure culture in PBS and dialyzing it in

  5. Detection of an 85,000-dalton phosphoprotein in ts110 murine sarcoma virus-infected cells with antiserum against a v-mos peptide.

    OpenAIRE

    Papkoff, J; Hunter, T.

    1983-01-01

    We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P8...

  6. Development of specific antiserum against bovine follicle stimulating hormone (bFSH) and production of second antibody for bFSH radioimmunoassay

    International Nuclear Information System (INIS)

    Antisera against bovine follicle stimulating hormone (bFSH) and rabbit gamma globulins (RGG) were raised in male rabbits and male goats, respectively. The anti bFSH crossreacted with bovine luteinzing hormone (bLH), bovine thyroid stimulating hormone (bTSH) and normal calf serum (NCS). The crossreaction with bLH and bTSH was almost absent when anti bFSH serum was treated with NCS and the antigen (1 mg/ml) was diluted twice or more. Antibody titre curve determined by radioimmunoassay (RIA) demonstrated that NCS treated bFSH antiserum could bind approximately 52% and 28% with 1:2,000 and 1:10,000 final dilutions respectively. When the antiserum against RGG (ARGG) was tested against its antigen, ARGG serum showed very strong precipitin band. The ARGG serum of 1:16 final dilution preipitated the bound fraction maximum when used as a second antibody in RIA. (author)

  7. Production of antiserum to a non-structural potyviral protein and its use to detect narcissus yellow stripe and other potyviruses.

    Science.gov (United States)

    Mowat, W P; Dawson, S; Duncan, G H

    1989-08-01

    A protein, of apparent molecular weight 72,000, was purified from experimentally infected narcissus plants with yellow stripe symptoms utilising SDS-polyacrylamide gel electrophoresis. This protein was excised from the gels and used to prepare antiserum, which reacted specifically with cytoplasmic cylindrical inclusions in ultra-thin sections of virus-infected cells and, in immunoblots, with the 72 kDa protein in preparations containing cytoplasmic inclusions. The antiserum reacted in ELISA with leaf extracts from yellow stripe diseased plants of four narcissus cultivars but not with extracts from comparable symptomless plants. In tests with extracts of plants infected with seven definitive potyviruses, reactions were obtained with bean yellow mosaic and iris mild mosaic viruses. Virus-specific reactions in dot-blot ELISA were dependent on the presence of Tween 20 in the extraction buffer. In contrast, an antiserum to the putative cytoplasmic inclusion protein of alstroemeria mosaic virus reacted only with SDS-treated leaf extracts of infected plants. In limited tests, the method of purifying cytoplasmic inclusion protein was successfully applied to four definitive potyviruses, suggesting that it may be generally applicable to potyviruses and of use for preparing antisera when purification of virus particles is difficult. PMID:2778031

  8. New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

    Science.gov (United States)

    Watanabe, Eiki; Hoshino, Ryoko; Kanzaki, Yukiko; Tokumoto, Hiroshi; Kubo, Hiroaki; Nakazawa, Hiroyuki

    2002-06-19

    The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed

  9. Production of second antibody for insulin and related peptides radioimmunoassay (RIA) (sheep anti-serum and guinea pig anti-IgG)

    International Nuclear Information System (INIS)

    A good RIA separation technique is essential to develop precise assays and the double antibody separation method is one of the most widely employed, satisfying the majority of the criteria required by RIA. However, its high cost is its main disadvantage, which leads to employ less expensive techniques, that are not so efficient. Therefore, our institution is producing a second antibody to be used in insulin assays, in which the first antibody is raised in guinea pig. Three sheep were immunized with 500 μg of guinea pig IgG purified at our laboratory emulsified in Freund Complete Adjuvante and administered by multisite subcutaneous injection at 20 day intervals. Blood samples were taken from the jugular vein 10 days after boosts. After each four boosts a great bleeding was done by the same route. After these bleeding, the animals were subjected to a rest before being reimmunized. The antisera title were determined by the immuno diffusion method in comparison with a reference antiserum of know quality produced in goat by the Pel-Freez, USA. Approximately 3.5 L of antiserum were produced from the three sheep which presented title very similar to those exhibited by the commercial product, even presenting higher values. (author). 8 refs., 3 figs

  10. The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

    Science.gov (United States)

    Wallace, R; Ashton, F E; Ryan, A; Diena, B B

    1978-02-01

    An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures. PMID:417781

  11. Over-expression of 72 ku protein of wheat yellow mosaic virus in E.coli and preparation of its antiserum

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By reverse transcription-polymerase chain reaction (RT-PCR),cDNA fragment of wheat yellow mosaic virus (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic cells.BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level.The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation.In Western-blotting analysis,the antibodies reacted with the 72 ku protein expressed in E.coli.

  12. Radioimmunoassay of human epidermal growth factor (h-EGF) and characterization of domestically produced antiserum against h-EGF by genetic engineering

    International Nuclear Information System (INIS)

    An antiserum to h-EGF was obtained by immunizing rabbits with h-EGF produced by genetic engineering. Its titer amounted to 1 : 147000 with an affinity constant of 2.04 x 1011 L/mol. The specificity of the assay is very high, no cross-reaction being observed with gastrin, VIP, CCK. and others. When used in RIA, the sensitivity was 5.1 pg/tube, and the intra-assay and inter-assay coefficient of variation was 4.6 and 8.7%, respectively. Both serum and plasma h-EGF were measured in 68 healthy subjects, the plasma h-EGF values were 192.4 +- 106.1 μg/L and serum h-EGF values were 1032.2 +- 485.7 μg/L

  13. Anti-asialo GM1 antiserum treatment of lethally irradiated recipients before bone marrow transplantation: Evidence that recipient natural killer depletion enhances survival, engraftment, and hematopoietic recovery

    International Nuclear Information System (INIS)

    Natural killer (NK) cells are reported to have an important role in the resistance of lethally irradiated recipients to bone marrow transplantation (BMT). Therefore, we investigated the effects of recipient NK depletion on survival, chimerism, and hematopoietic reconstitution after lethal irradiation and the transplantation of limiting amounts of T-cell-deficient bone marrow (BM). When administered before BMT, anti-asialo GM1 (ASGM1) antiserum treatment, effective in depleting in vivo NK activity, was associated with a marked increase in survival in 3 of 3 allogeneic combinations (BALB/c into C3H/HeN, C57B1/6, or C3B6F1). This enhanced survival was independent of the susceptibility of each recipient strain to accept BALB/c BM. Moreover, recipient anti-ASGM1 treatment was also effective in increasing survival in recipients of syngeneic BM, suggesting that NK cells can adversely affect engraftment independent of genetically controlled polymorphic cell surface determinants. Analysis of chimerism in surviving animals 2 months post-BMT showed that recipient NK depletion significantly increased the level of donor engraftment when high doses of BM were transplanted. These studies also demonstrated that anti-ASGM1 pretreatment mainly resulted in an increase in extramedullary hematopoiesis in the second and third week after irradiation. Anti-ASGM1 treatment also dramatically accelerated the rate of appearance of donor-derived cells with a higher level of donor-cell engraftment apparent at a time when the differences in survival between NK-depleted and control BMT recipients became significant. Peripheral cell counts were also affected by NK depletion, with significantly enhanced platelet and red blood cell recovery and a moderate increase in granulocyte recovery

  14. 人乳头瘤病毒16型E2蛋白表达、纯化及抗血清制备%Expression and purification of HPV16 E2 protein and preparation of its antiserum

    Institute of Scientific and Technical Information of China (English)

    孙宇辉; 张沿君; 刘明明; 唐丽萍; 张光虹; 魏兰兰; 谷鸿喜; 商庆龙

    2013-01-01

    Objective To express and purity the human papillomavirus type 16 (HPV16) E2 protein in prokaryotic bacteria and prepare the antiserum of HPV16 E2.Methods After amplified by PCR,HPV16 E2 was inserted into pET21b vector.The recombinant pET21b-HPV16E2 vector was transfected into E.coli BL21 (DE3).Expression product was identified after induction.Through purification,denaturation and renaturation,soluble protein was obtained.With the HPV16 E2 protein,we immunized BALB/c mice and examined mouse IFN-γ,CD4+ T cells,CD8+ T cells,CD4/CD8 ratio and antiserum titer.Results Restriction digestion and DNA sequencing showed pET21b-HPV16E2 was constructed successfully.Relative molecular mass (Mr) of HPV16 E2 was 42 000 in SDS-PAGE and the specificity of the protein was confirmed with Western blotting.The antiserum could specifically bind with HPV16 E2 protein.In the immunized BALB/c mice,antiserum titre,CD4+ T cell count and CD4/CD8 ratio increased,while mouse IFN-γ did not change obviously.Conclusion Soluble HPV16 E2 protein was obtained successfully.The antiserum of high titer against HPV16 E2 was prepared in mice.%目的 表达人乳头瘤病毒16型(HPV16) E2蛋白,并制备小鼠抗HPV16 E2血清.方法 采用PCR技术扩增HPV16E2基因,构建入pET21b载体,重组表达载体pET21 b-HPV16E2经鉴定后转化大肠埃希菌BL21(DE3),诱导表达并鉴定表达产物.经纯化、变性和复性方法,制备可溶性HPV16 E2蛋白.免疫BALB/c小鼠制备抗血清,检测小鼠IFN-γ、CD4+T细胞、CD8+T细胞、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pET21b-HPV16 E2构建成功.表达蛋白相对分子质量(Mr)为42 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4+T细胞数量和CD4/CD8比值升高,小鼠IFN-γ无升高.结论 成功制备可溶性HPV16 E2蛋白和小鼠抗HPV16 E2高效价的抗血清.

  15. Prokaryotic Expression and Antiserum Preparation of the Coat Protein of Cymbidium Mosaic Virus%建兰花叶病毒CP基因的原核表达及抗血清制备

    Institute of Scientific and Technical Information of China (English)

    罗金水

    2009-01-01

    通过间接酶联免疫检测和电镜观察对从福建省漳州市采集的卡特兰病样进行检测,证明样品感染了建兰花叶病毒.设计一对特异性引物,扩增并克隆病毒分离物的外壳蛋白基因,随后将目的基因插入pET-29a(+)中构建相应的原核表达载体.目的蛋白经诱导表达及纯化后免疫家兔并获得了特异性抗血清.Westem blot检测结果表明.抗血清与诱导表达的CyMV外壳蛋白发生特异性反应.间接酶联免疫法检测结果表明,抗血清可检测病汁液的最低稀释度达1:51 200,最佳工作浓度为1:1000,病汁液灵敏度为0.39 mg/mL,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应.%Cymbidium mosaic virus (CyMV) is one of the most important and worldwide viruses attacking orchids. This virus causes the symptoms of mosaic,chlorosis,necrosis and malformation in the orchids,and has a high economic impact to the orchid industry. Cattleya plants contracted with a disease were collected as samples from Zhangzhou,Fujian,and were identified to be infected with Cymbidium mosaic virus by using ID -ELISA and electronic microscopy assay. One pair of specific primers was designed for amplification of the coat protein(CP) gene from the samples infected with CyMV. The open reading frame encoding CP of CyMV isolate obtained from Zhangzhou,Fujian is 672 bp,encoding a 23.6 ku protein with 223 aa. The expected CP gene was then inserted into the pET-29a(+)vector for prokaryotic expression. And the aimed protein was purified and used to immune the rabbit for antiserum preparation. According the result of ID-ELISA analysis,specific rabbit anti-CyMV serum was prepared with a high titre of 1:51 200,a working concentration of 1:1 000 and sap sensitivity of 0.39 mg/mL. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of CyMV. There were no cross reactions between the antiserums and 11 species of homologous or heterologous

  16. 抗恩诺沙星抗血清的制备及其ELISA检测%Preparation of enrofloxacin antiserum and its determination by enzyme - linked immunosorbent assay (ELISA)

    Institute of Scientific and Technical Information of China (English)

    刘兵; 曾华金; 杨冉; 雷利芳; 屈凌波

    2011-01-01

    Objective;To develop a rapid and sensitive enzyme - linked immunosorbent assay(ELISA) for determination of enrofloxacin( ENR). Method; Immunogen enrofloxacin - bovine serum albumin( Enrofloxacin - BSA) and coating antigen enrofloxcin -ovum albumin (Enrofloxacin - OVA) were prepared by a succinic anhydride method. By subcutaneous injection, the polyclonal antiserum was produced from big ear rabbits immunized with conjugates ENR -BSA. Result; After the assay procedure was optimized,the standard curve of ENR was established and the practical measuring range of the ELISA extended from 5 ng ? mL-1 to 2. 5 |xg ? mL '' ( R2 =0. 9935 ) . In a comparison of the assay results obtained by ELISA and HPLC, there is a good correlation between these two methods (R2 = 0. 9892, n = 9 ) . The proposed method has been satisfactorily applied for the determination of ENR in rat plasma and Pharmaceuticals with recoveries in the range of 98. 4% to 105. 8% for milk sample,91. 7% to 101. 2% for rat plasma and 97.0% to 110.3% for rat urine. Conclusion; The experimental data indicated that in some extent the ELISA method was more suitable for high throughput and real - time ENR analysis in biological samples and animal food with lower detection limit,low background and no requirement of sample pre -treatment.%目的:建立快速、灵敏的恩诺沙星残留酶联免疫检测方法(ELISA).方法:采用混合酸酐法,将恩诺沙星与牛血清白蛋白和卵清蛋白分别合成免疫抗原(Enrofloxacin-BSA)和包被抗原(Enrofloxacin-OVA).通过背部多点免疫法免疫日本大白耳兔,制备抗恩诺沙星的特异性抗血清.结果:利用所获得的特异性抗血清,建立的恩诺沙星ELISA检测方法,其线性范围为5 ng·mL.~2.5μg·mL(R=0.9935),且与HPLC方法具有良好的相关性(R=0.9892,n=9).牛奶、大鼠血浆和尿液中的加样回收率分别为98.4%~105.8%,91.7%~101.2%,97.0%~110.3%.运用所建立的方法,对大鼠体内血浆及药品

  17. Production of second antibody for insulin and related peptides radioimmunoassay (RIA) (sheep anti-serum and guinea pig anti-IgG); Producao de segundo anticorpo para radioimunoensaio (RIE) de insulina e peptideos relacionados (antissoro de carneiro anti-IgG de cobaia)

    Energy Technology Data Exchange (ETDEWEB)

    Castanheira, Maria do Carmo; Silva, Sandra Rosa da; Borghi, Vania Caira [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Wajchenberg, Bernardo Leo [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina

    1995-12-31

    A good RIA separation technique is essential to develop precise assays and the double antibody separation method is one of the most widely employed, satisfying the majority of the criteria required by RIA. However, its high cost is its main disadvantage, which leads to employ less expensive techniques, that are not so efficient. Therefore, our institution is producing a second antibody to be used in insulin assays, in which the first antibody is raised in guinea pig. Three sheep were immunized with 500 {mu}g of guinea pig IgG purified at our laboratory emulsified in Freund Complete Adjuvante and administered by multisite subcutaneous injection at 20 day intervals. Blood samples were taken from the jugular vein 10 days after boosts. After each four boosts a great bleeding was done by the same route. After these bleeding, the animals were subjected to a rest before being reimmunized. The antisera title were determined by the immuno diffusion method in comparison with a reference antiserum of know quality produced in goat by the Pel-Freez, USA. Approximately 3.5 L of antiserum were produced from the three sheep which presented title very similar to those exhibited by the commercial product, even presenting higher values. (author). 8 refs., 3 figs.

  18. 猪带绦虫TSO45W-4B-TSOL18融合基因在大肠埃希菌ArcticExpress(DE3)中的表达、纯化和兔抗血清的制备%Expression and purification of a fusion gene TSO45W-4B-TSOL18 of Taenia solium in Escherichia coli ArcticExpress(DE3) and preparation of rabbit antiserum

    Institute of Scientific and Technical Information of China (English)

    周必英; 周泠; 刘美辰; 刘晖; 贺莉芳

    2013-01-01

    Objective To construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18 of Taenia solium,to obtain purified recombinant protein TSO45W-4B-TSOL18 and to prepare rabbit antiserum against the recombinant fusion protein.Methods TSO45W-4B and TSOL18 encoding genes were connected with hydrophobic (Gly4Ser)3 linker.TSO45W-4B-TSOL18 fusion gene was synthesized and cloned into an expression vector pGEX-1λT to construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18.The positive recombinants were transformed into Escherichia(E.) coli ArcticExpress (DE3),and the expression of recombinant protein was induced with isopropylβ-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).The expressed recombinant fusion protein was purified through affinity chromatography.The purified recombinant protein was mixed with complete Freunds adjuvant at a dosage of 0.5 mg.Rabbit was intramuscularly and subcutaneously immunized in the hind leg and the back,respectively.After 2 weeks the rabbit was boosted with purified recombinant protein which was mixed with incomplete Freunds adjuvant at the same dosage.Rabbit can be boosted every 10 days until an adequate response was achieved.At the 6th day after the last immunization,blood was collected from the rabbit heart.Serum was separated to purify and prepare the antiserum.ELISA was applied to determine the titer of the antiserum and Western blot assay was used to determine the specificity of the antiserum.Results The size of the synthesized fusion gene TSO45W-4B-TSOL18 was 789 bp.The results of restriction enzyme digestion showed that the recombinant plasmid pGEX-TSO45W-4B-TSOL18 was successfully constructed.DNA sequencing showed that the size of the fusion gene TSO45W-4B-TSOL18 was 789 bp,which was consistent with expected result.As demonstrated by SDS-PAGE,relative molecular mass of the expressed recombinant fusion protein was approximately 55 × 103,and its purity was 85% after

  19. Use of antiserum to neurotensin reveals a physiological role for the peptide in rat prolactin release.

    OpenAIRE

    Vijayan, E; Carraway, R; Leeman, S E; McCann, S M

    1988-01-01

    Previous studies have indicated that the brain peptide neurotensin can stimulate prolactin release by direct action on the pituitary gland, whereas its action within the hypothalamus is inhibitory. The inhibitory action is mediated by the release of dopamine into the hypophyseal portal veins, which deliver the neurotransmitter to the anterior pituitary gland to inhibit prolactin release. Our experiments were done to evaluate the physiologic significance of these neurotensin actions by injecti...

  20. Experimental orchitis induced in rats by passive transfer of an antiserum to seminiferous tubule basement membrane.

    Science.gov (United States)

    Lustig, L; Denduchis, B; González, N N; Puig, R P

    1978-09-01

    A multifocal damage of the testis was obtained when rats were injected intravenously or under the tunica albuginea of the testis with a rabbit antiseminiferous tubule basement membrane serum. The damage was characterized by foci of perivascular and peritubular infiltrates of mononuclear round cells, infolding, thickening, and rupture of the seminiferous tubular wall and different degrees of injury of the germinal epithelium such as, cell disorganization, cell sloughing, and atrophy. Delamination and thickening of seminiferous tubule basement membrane and vacuolization of the Sertoli cell cytoplasm was often observed by electron microscopy. A linear deposit of rabbit gamma-globulin was detected by immunohistochemical techniques along the basement membranes of the seminiferous tubules and vessels. Testicular damage was not detected in rats injected with normal rabbit serum, used as control. In the kidneys of rats injected intravenously with the immune serum, a deposit of rabbit gamma-globulin was detected along glomerular basement membrane. Focal areas of mononuclear cell infiltrates, hypercellularity of glomeruli and thickening of glomerular capillary walls and Bowman's capsule were also observed. PMID:367304

  1. Efficacy of antiserum produced in goats and pigs to passively protect piglets against virulent transmissible gastroenteritis virus.

    OpenAIRE

    Woods, R D; Wesley, R D

    1992-01-01

    The protective effect of sera produced in swine and goats exposed to virulent transmissible gastroenteritis virus (TGEV) or modified-live TGEV was tested in hysterectomy-derived, colostrum-deprived three-day-old pigs. Pigs were given serum with their daily ration of milk, and their immunity to virulent TGEV was determined. The pigs were observed for ten days for clinical signs of TGEV infection. One of nine pigs receiving goat serum was protected whereas all three pigs receiving three doses o...

  2. Especificidade de anti-soro policlonal à Leifsonia xyli subsp. xyli Policlonal antiserum specifity against Leifsonia xyli subsp. xyli

    OpenAIRE

    Josil B. Carneiro Jr; Silvaldo F. da Silveira; de Souza Filho, Gonçalo A.; Olivares, Fabio L.; Éder A. Giglioti

    2004-01-01

    Detectar a presença da bactéria Leifsonia xyli subsp. xyli em material de propagação da cana-de-açúcar (Saccharum sp.) é importante para direcionar o controle do raquitismo-da-soqueira. Neste trabalho, objetivou-se produzir anticorpo policlonal específico contra Leifsonia xyli subsp. xyli (Lxx), visando utilizá-lo em método sorológico para detecção do patógeno. Para isso, o antígeno foi preparado a partir de células intactas, após lavagem por centrifugação de cultura-pura em tampão fosfato sa...

  3. Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein.

    Science.gov (United States)

    Vijayanandraj, S; Yogita, M; Das, Amrita; Ghosh, Amalendu; Mandal, Bikash

    2013-09-01

    Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renaturation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom. PMID:24426280

  4. Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor.

    OpenAIRE

    Bangalore, L; Tanner, A J; Laudano, A P; Stern, D F

    1992-01-01

    Rabbits were immunized with a synthetic phosphopeptide corresponding to a major autophosphorylation site of p185neu/erbB2 to determine the feasibility of producing tyrosine-phosphopeptide-specific antibodies. A series of adsorption and affinity chromatography steps were used to select antibodies with the desired reactivity. Immunoblot experiments showed that the resulting serum is highly specific for tyrosine-phosphorylated forms of p185 and the related epidermal growth factor receptor. The s...

  5. Utilization of a single antiserum for the direct radioimmunoassay of prostaglandins E and F in semen and prostaglandin F in amniotic fluid

    International Nuclear Information System (INIS)

    Antibodies to both prostaglandin F (PGF) and prostaglandin E (PGE) were raised in rabbits after they were immunized with prostaglandin F/sub 2a/ conjugated to bovine serum albumin (PGF/sub 2a/--BSA). The antisera were group specific although the antibodies to the F group of prostaglandins showed greater specificity than those to the E group. The antisera were sufficiently specific however to allow the direct radioimmunoassay of PGF and PGE in human semen and PGF in amniotic fluid during induced abortion. Specificity of the direct radioimmunoassay was checked by chromatographic separation of the prostaglandins prior to analysis. Estimation of the prostaglandins in the semen of 30 men attending the infertility clinic showed that 19 of the men had normal semen levels of PGE and PGF of 68 +- 7 (SE) and 6.0 +- 0.6 μg/ml respectively, as compared with data on normal fertile males, whilst the other 11 men had lower levels of 16 +- 2 (SE) and 0.8 +- 0.1 μg/ml respectively. Application of the method to amniotic fluid showed that the PGF concentration in amniotic fluid during the induction of abortion with extra-ovular saline increased from less than 0.6 ng/ml to 6.4 ng/ml when the induction-abortion intervals ranged from 6 to 48 hours. (U.S.)

  6. A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

    Science.gov (United States)

    Ramos, Henrique Roman; Junqueira-de-Azevedo, Inácio de Loiola M.; Novo, Juliana Branco; Castro, Karen; Duarte, Clara Guerra; Machado-de-Ávila, Ricardo A.; Chavez-Olortegui, Carlos; Ho, Paulo Lee

    2016-01-01

    Background Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. Methods and Findings In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. Conclusion Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity. PMID:26938217

  7. The Lassa fever virus L gene: nucleotide sequence, comparison, and precipitation of a predicted 250 kDa protein with monospecific antiserum

    OpenAIRE

    Lukashevich, Igor S.; Djavani, Mahmoud; Shapiro, Keli; Sanchez, Anthony; Ravkov, Eugene; Nichol, Stuart T.; Salvato, Maria S.

    1997-01-01

    The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other ...

  8. The Lassa fever virus L gene: nucleotide sequence, comparison, and precipitation of a predicted 250 kDa protein with monospecific antiserum

    Science.gov (United States)

    Lukashevich, Igor S.; Djavani, Mahmoud; Shapiro, Keli; Sanchez, Anthony; Ravkov, Eugene; Nichol, Stuart T.; Salvato, Maria S.

    2008-01-01

    The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS–lymphocytic choriomeningitis and Tacaribe–Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells. PMID:9049403

  9. Rabbit antiserum to brain-associated thymus antigens of mouse and rat. I. Demonstration of antibodies cross-reacting to T cells of both species

    Energy Technology Data Exchange (ETDEWEB)

    Peter, H.H.; Clagett, J.; Feldman, J.D.; Weigle, W.O.

    1973-04-01

    Immunization of rabbits with either mouse (CBA/ST) or rat (Le) brain homogenates results in the production of antisera which upon appropriate absorptions are T cell-specific for both mouse and rat. These antisera may be used effectively for direct cytotoxicity and detection of rat and mouse T cells by direct immunofluorescence. Furthermore, in the rat, it was demonstrated that antimouse brain serum, in the presence of complement, abrogates cell-mediated immunity in vitro, but does not affect antibody-forming cells.

  10. Preoaration and application of cucumber green mottle mosaic virus antiserum%黄瓜绿斑驳花叶病毒抗血清制备及应用

    Institute of Scientific and Technical Information of China (English)

    秦碧霞; 蔡健和; 黄金玲; 胡冬梅; 陆秀红; 刘志明

    2010-01-01

    用葫芦[Lagenaria siceraria(Molina)Stand.]作为黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的繁殖寄主,通过差速离心和PEG二次沉淀法进行病毒提纯,用提纯病毒液免疫新西兰兔制备CGMMV抗血清.制备的CGMMV抗血清经间接ELISA法测定效价为1:5120;利用制备的抗血清检测田间样品,证明其可以用于CGMMV的血清学及免疫捕获RT-PCR检测.研究结果对今后开展葫芦科作物CGMMV检测,及时发现疫情以便采取防控措施,避免病毒扩散为害,确保葫芦科作物的安全生产具有重要意义.

  11. Preparation of polyclonal antiserum against recombinant NSP2 protein of PRRSV HH08 strain and study on biological functions of the polyclonal antiserum%猪繁殖与呼吸综合征病毒HH08株NSP2蛋白多克隆抗体的制备及其生物学功能的研究

    Institute of Scientific and Technical Information of China (English)

    马玲; 李广兴; 洪琴; 任玉东; 任晓峰

    2013-01-01

    利用RT-PCR和SOE PCR技术扩增得到猪繁殖与呼吸综合征病毒(PRRSV) HH08株NSP2全长基因,经抗原性和亲水性分析,将NSP2部分序列成功亚克隆于pET-30a(+)和PVAX1栽体中.将阳性重组质粒pET30a-NSP2转化E.coli Rosetta(DE3)感受态细胞,经诱导表达获得107 ku的重组NSP2蛋白.Western-blot检测表明,重组蛋白能够与PRRSV参考阳性血清反应.以纯化的重组NSP2蛋白免疫新西兰白兔制备多克隆抗体,其ELISA效价达到1∶1015以上;Western-blot试验表明,其具有良好的反应性和特异性.间接免疫荧光试验显示,用抗NSP2多克隆抗体可以检测到PVAX-NSP2转染BHK 21细胞所表达的NSP2蛋白,且与经典型和高致病型PRRSV毒株均有很好的特异性反应.病毒感染抑制试验表明,多抗血清对PRRSV经典和高致病性毒株的抑制率可达到68%和53%.上述研究结果为PRRSV检测及NSP2蛋白功能的深入研究奠定了基础.%In this study,the complete PRRSV HH08 NSP2 gene was cloned and the partial gene sub-cloned into prokaryotic expression vector pET-30a( + ) and eukaryotic expression vector PVAX1 after hy-drophilicity plot and antigenic index analysis. E. coli Rosetta (DE3) was transformed with the recombinant plasmid pET30a-NSP2. The recombinant NSP2 protein that molecular weight is 107 ku was expressed. It could be recognized by specific PRRSV antisera in western blot. Then the purified recombinant NSP2 protein as antigen was used to immunize rabbit for preparation of anti-NSP2 polyclonal antibody. An indirect ELISA assays showed that the titer of anti-NSP2 polyclonal antibody was 1 : 1015 , and it had highly reactivity and specialty in Western-blot. Also.IFA test demonstrated that this polyclonal antibody could react with the BHK-21 cells which can express PRRSV NSP2 protein and the Marc-145 cells infected with PRRSV. Both attenuated and highly pathogenic PRRSV strains could be inhibited by the anti-NSP2 polyclonal antibody and the inhibition rates were 68% and 53% respectively. The rabbit anti-NSP2 protein polyclonal antibody obtained in this study laid a foundation for further functional research for NSP2 protein and detection of PRRSV.

  12. 鸡L-FABP抗血清制备及组织表达特性分析%Preparation of Antiserums against Chicken Liver-type Fatty Acid Binding Protein (L-FABP) and Tissue Expression Analyses of L-FABP

    Institute of Scientific and Technical Information of China (English)

    石慧; 王启贵; 王宇祥; 王宁; 李辉

    2008-01-01

    为制备鸡肝脏型脂肪酸结合蛋白(L-FABP)的多克隆抗血清,并分析L-FABP的组织表达特性,利用RT-PCR扩增L-FABP基因,构建鸡L-FABP基因的GST融合蛋白表达质粒pGEX-4T/L-FABP.将重组表达质粒转化大肠杆菌BL21,IPTG诱导产生GST/L-FABP融合蛋白,用亲和层析纯化目的蛋白,将纯化的GST/L-FABP融合蛋白免疫家兔制备抗血清,并利用此抗血清分析鸡L-FABP基因的组织表达特性.诱导得到了1个40 ku(14 ku L-FABP+ 26 ku GST)的融合蛋白,获得效价较高、特异性强的鸡L-FABP的抗血清.鸡L-FABP的组织表达特性研究结果表明,该基因在肝脏和小肠组织中有较高表达,但在心脏、脂肪、肌肉、肌胃、脾、肺和肾中没有检测到表达信号.

  13. 猪带绦虫HSP70-4的真核表达及其抗血清的制备%Eukaryotic expression and anti-serum preparation of HSP70-4 from Taenia solium

    Institute of Scientific and Technical Information of China (English)

    殷静; 王帅; 刘光学; 何伟; 才学鹏

    2016-01-01

    为了研究猪带绦虫(Taenia solium)热休克蛋白70-4 (TsHSP70-4)基因序列的特征及其真核表达蛋白的抗原性,参考GeneDB中猪带绦虫基因组注释信息,设计特异性引物,RT-PCR扩增TsHSP70-4ORF序列.根据毕赤酵母密码子偏好性,对TsHSP70-4基因密码子进行优化,连接载体pPIC9K,在毕赤酵母中进行诱导表达,通过Western-blot及质谱测序进行蛋白鉴定.将纯化后的TsHSP70-4表达蛋白免疫新西兰大白兔,制备抗血清.结果显示,成功克隆出大小为1 953 bp的TsHSP70-4ORF序列.在毕赤酵母中进行了高效表达,获得大小约为95 ku的重组蛋白.Western-blot检测和质谱鉴定表明,获得的重组蛋白即为TsHSP70-4.免疫新西兰大白兔,制备出效价高达1∶409 600的抗血清.该研究为猪囊尾蚴病新疫苗的研制提供了依据.

  14. 重组犬钩虫分泌蛋白抗血清的免疫反应性%Studies on Immunological Reaction of the Antiserum of Recombinant Secreted Protein from Ancylostoma Caninum

    Institute of Scientific and Technical Information of China (English)

    闻礼永; Hotez PJ

    2001-01-01

    目的分析重组犬钩虫分泌蛋白抗血清与钩虫不同种、期抗原的免疫反应性.方法用微型垂直电泳槽进行SDS-PAGE,以低分子量标准蛋白作参照.ELIB试验:以重组犬钩虫分泌蛋白-1(Ac-rAsp-1)或重组犬钩虫分泌蛋白-2(Ac-rAsp-2)免疫鼠血清作第一抗体,羊抗鼠IgG-HRP作第二抗体,用Western blotting发光底物试剂反应,全自动摄影,按照底片中显示带的位置测出相应分子量.结果与结论 Ac-rAsp-1组分为45kDa,其免疫血清能识别犬钩虫第Ⅲ期幼虫(Ac-L3)抗原和Ac-rAsp-1,不与十二指肠钩虫成虫(Ad-A)、十二指肠钩虫第Ⅲ期幼虫(Ad-L3)、美洲钩虫成虫(Na-A)、犬钩虫成虫(Ac-A)、巴西日圆线虫成虫(Nb-A)抗原和Ac-rAsp-2起反应;Ac-rAsp-2组分为24 kDa,其免疫血清能识别Ad-A、Ad-L3、Na-A、Ac-A、Ac-L3抗原和Ac-rAsp-2,不与Nb-A抗原和Ac-rAsp-1起反应.

  15. Preparation of Ciprofloxacin Antiserum and Its Determination by Enzyme-linked Immunosorbent Assay (ELISA)%抗环丙沙星抗血清制备及其ELISA检测

    Institute of Scientific and Technical Information of China (English)

    曾华金; 杨冉; 屈凌波; 李斐菲; 李建军

    2009-01-01

    采用碳二亚胺法和混和酸酐法,将环丙沙星与BSA和OVA分别合成免疫抗原(ciprofloxacin-BSA)和包被抗原(ciprofloxacin-OVA).利用合成的免疫抗原免疫家兔,获得了抗环丙沙星的特异性抗血清.在此基础上建立环丙沙星酶联免疫检测方法(ELISA),其线性范围为0.05~10μg/ml(r=0.998).在样品检测中与HPLC方法具有良好的相关性.

  16. Recombinant K1-3 expression of angiostatin and preparation of antiserum to expression protein%血管生成抑制素的功能结构域K1-3的融合表达与抗体制备

    Institute of Scientific and Technical Information of China (English)

    李福洋; 刘新平; 苏成芝; 张英起; 樊代明; 杨静华; 药立波

    1999-01-01

    目的: 重组表达angiostatin的功能结构域K1-3,并制备抗体,为其作用机理研究打下基础. 方法: 设计引物,通过PCR以人纤溶酶原cDNA为模板扩增出K1-3基因片段,克隆至pGEX-4T-1融合表达载体,IPTG诱导,挑出表达克隆,切下基因片段,克隆至pUC19进行序列测定,保留序列完全正确的克隆并进行诱导表达;从SDS-PAGE上切下表达的蛋白条带作为抗原免疫家兔,制备多克隆抗体, 并以Western blotting检测抗血清的结合特异性. 结果: DNA电泳结果显示PCR扩增出约780 bp的片段,序列测定表明为K1-3; K1-3可以与GST融合表达,产物存在于包含体中;抗血清与K1-3及煮沸变性的天然angiostatin特异地结合. 结论: 经PCR扩增并克隆到序列正确的K1-3片段,而且能够以融合形式表达,用其免疫家兔获得特异性抗体.

  17. 三种菜豆凝集素的分离纯化及其与黑皮扁豆凝集素抗血清的免疫交叉反应%Purification of Three Species of Phaseolus L. and Their Immune Cross Reaction with Antiserum Raised Against Dolichos Purpureus Lectin

    Institute of Scientific and Technical Information of China (English)

    李锋; 田立娟; 张年辉; 杜林方

    2006-01-01

    利用Tris-HCl缓冲液浸提、硫酸铵分级沉淀、离子交换和分子筛层析,分别从白花菜豆(Phaseolus albiflora L.var)、紫花菜豆(Phaseolus purpurea L.var)、红花菜豆(Phaseolus coccineus L.var)种子中纯化得到3种菜豆凝集素(PAL,白花菜豆凝集素;PPL,紫花菜豆凝集素;PCL,红花菜豆凝集素).纯化的3种菜豆凝集素经SDS-PAGE电泳检测都只有一个分子量约为32 kD的亚基,它们都能与黑皮扁豆凝集素抗血清发生免疫交叉反应.

  18. Ação do soro de cabra anti-soro de coelho imunizado ou não com células linfóides do doador sobre o alotransplante cardíaco em ratos: immunosupression of goat antiserum against rabbit serum immunized or not with donor lymphoid cells Cardiac allograft in rats

    Directory of Open Access Journals (Sweden)

    Haylton Jorge Suaid

    2002-01-01

    Full Text Available INTRODUÇÃO: A rejeição imunológica é uma das principais causas da perda de órgãos transplantados. A tentativa do controle da reação imunológica é clinicamente feita através da imunossupressão inespecífica e experimentalmente também por bloqueio específico. O alotransplante cardíaco em ratos pela técnica de ONO,K é um bom método para avaliação clínica da rejeição e de estudos voltados para o controle da rejeição. Objetivo : estudar o efeito de um anti-antisoro linfocitário, anti-linfócitos do doador sobre a rejeição do alotransplante cardíaco de ratos Wistar para ratos Holtzman. MÉTODOS: o soro anti-linfocitário (SAL foi obtido através da imunização de coelhos com linfócitos obtidos de gânglios linfáticos da cadeia mesentérica de ratos Wistar, em solução de Tyrode, contendo 3x10(9 células/ ml. A inoculação de 3 coelhos foi feita com 1 ml da suspensão celular e 1 ml de adjuvante completo de Freund. Duas semanas após a primeira inoculação fez-se 4 doses semanais de reforço. Os coelhos foram sangrados na 5ª semana, quando então foram separados os soros. A titulação dos soros foi realizada pelo teste de citotoxicidade, sendo verificado que ambos apresentaram título de 1:1024. A dosagem de proteínas mostrou albumina com 3,1 e 2,7 g% e globulinas com 3,5 e 2,9 g%, sendo o normal 3,7 e 2,2 g% respectivamente. Os dois SAL foram misturados. Duas cabras foram inoculados, com 3 ml da mistura desses SAL, associados a 2 ml de adjuvante de Freund. As doses de reforço com 5 ml do SAL foram iniciadas 2 semanas após. A cabra A recebeu 8 doses (1,4 g de globulinas. A cabra B recebeu 4 doses de reforço (0,7 g de globulinas. Uma semana após a última inoculação retirou-se 125 ml de sangue de cada cabra, fazendo a separação dos anti-soro anti-SAL (ASAL. Uma terceira cabra C foi imunizada com soro normal de coelho. A determinação de precipitinas foi feita pelo método de OUCHTERLONY. O ASAL A teve título de 1:64 e B e C título de 1:128. Os ASAL A e B foram capazes de bloquear "in vitro" a atividade citotóxica do SAL até a diluição de 1:2 do SAL. O soro de cabra anti-soro normal de coelho (SCANC não foi capaz de bloquear a citotoxicidade do SAL. Os animais submetidos a transplante cardíaco foram divididos em 2 grupos controles um normal com 10 ratos (C1 e outro (C2 com 5 ratos que recebeu 1,0 ml endovenoso de SCANC. O grupo de ratos testes A foi composto por 19 ratos distribuídos em 3 subgrupos. Subgrupo A1 com 5 ratos recebeu 0,5 ml do ASAL A, via endovenosa, logo após a cirurgia,o subgrupo A2 com 7 ratos recebeu 1.0 ml do ASAL A nas mesmas condições e o subgrupo A3 também com 7 ratos recebeu 1,0 ml no dia da cirurgia e 1,0 ml nos outros 2 dias consecutivos. O grupo de ratos testes B que recebeu o ASAL B foi igual ao grupo A. A avaliação dos corações transplantados foi diária através da palpação abdominal. O tempo máximo de seguimento foi de 243 dias. Os corações considerados rejeitados foram retirados e feito estudos histológicos. RESULTADOS: o período de rejeição dos grupos foi : controles C1 e C2 foram 11,9 e 14,6 dias, respectivamente; no subgrupo A1 apenas um rato teve sobrevida cardíaca significante (153 dias, nos demais ela variou de 9 a 15 dias; no subgrupo A2 a sobrevida do coração foi significante e variou de 23 a 230 dias; no subgrupo A3 apenas 5 corações tiveram sobrevida significante que variou de 29 a 190 dias. A sobrevida dos corações transplantados do grupo B foi significante para um animal de cada subgrupo (120,132 e 129 dias. Os corações com sobrevida longa foram retirados batendo. Os demais corações foram rejeitados dentro do período de variação dos grupos controles. CONCLUSÕES: O soro de cabra anti-soro anti-linfócitos do doador, com maior período de imunização, foi capaz de bloquear a resposta imune de rejeição dos corações transplantados nas doses de 1,0 e 3,0 ml. Os ratos que não promoveram a rejeição aguda dos corações transplantados não apresentaram anticorpos citotóxicos circulantes. O fator causador do bloqueio parace n��o estar vinculado aos bloqueios de citotoxicidade "in vitro" e do teor de precepitinas do SAL.OBJECTIVE: To study the immunosupression efficacy an specific anti-antilymphocytic serum prepared in goats in a model of cardiac allografts in rats. METHODS: Three rabbits were immunized with lymphoid cells obtained from mesenteric lymphatic nodes of Wistar rats. Each one received subcutaneously 3x10(9 cells mixed with Freund's adjuvant. After 2 weeks, they were injected with the same amount of cells at weekly intervals for 4 additional times. In the 5th week they were bled and their serum were mixed. This serum, which had a cytotoxic titer of 1:1024, was used to immunize 2 goats that gave rise to the anti-antilymphocytic serum (AAS-1 and AAS-2. As control we immunized 1 additional goat with normal rabbit serum (ANS. The gel diffusion technique (AAS x rabbit serum showed precipitation bands against till the following dilution: AAS-1 - 1/64, AAS-2 - 1/128 and ANS 1/124. Both AAS were able to block the in vitro lymphocytotoxity of goat antilymphocytic serum till dilution of 1:2 while ANS did not. The hearts from Wistar rats (donors were transplanted in Holtzman rats. The transplanted rats were divide in groups: C1 - 11 animals (control that received no serum; C2 - 5 animals (control that received 1ml of goat normal serum; A- 19 animals - A1 with 5 rats injected intravenously in the day of surgery with 0.5ml of AAS-1, A2 with 7 rats injected with 1ml of AAS-1 only in the of surgery, and A3 with 7 rats that received 1ml of AAS-1 in days 0, 1 and 2 postoperatively; and group B with 19 rats (B1, B2 and B3 treated as group A except with the AAS-2 serum. RESULTS: Mean heart survival in groups C1 and C2 was respectively 11.9 and 14.6 days Survival range in the subgroups A1 and A2 were respectively 9 to 230 days and 23 to 230 days. In subgroup A3 heart survival was prolonged till 29 to 190 days in 5 animals. In group B only 3 animals had prolonged (120, 130 and 129 days heart survival in comparison with the control groups. CONCLUSION: Anti-antilymphocytic serum against donor antigen is able to suppress rejection of cardiac allograft in rats.

  19. Preparation of Ruscogenin Affinity Chromatography Column and Its Application in the Purification of Antiserum%以鲁斯可皂苷元为配基的亲和层析柱的制备及其在纯化抗体中的应用

    Institute of Scientific and Technical Information of China (English)

    梁明; 刘楠; 刘吉华; 余伯阳

    2006-01-01

    目的:以鲁斯可皂苷元(ruscogenin,RUS)为配基制备亲和层析柱,并应用于免疫亲和纯化抗血清.方法:将鲁斯可皂苷元衍生为带有两个-COOH臂的丁二酸单酯衍生物(succinylated ruscogenin,RUS-2HS),与带有10碳原子链的琼脂糖凝胶EAH Sepharose 4B偶联,制成亲和层析柱;以制备的亲和层析柱纯化人工合成抗原RUS-2HS-BSA(牛血清白蛋白)免疫家兔产生的抗血清,得到特异性较高的抗体,并检测纯化抗体的纯度及特异性.结果:成功将RUS-2HS作为配体偶联至亲和介质上,偶联量为4.24 mg·mL-1,偶联率为53.25%;竞争性抑制试验显示纯化抗体交叉反应率下降,特异性提高;SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示单一蛋白质条带,其相对分子质量约为51.2 kD.结论:建立了以小分子天然活性产物为配基制备亲和柱的方法,应用于抗血清的纯化,获得的特异性抗体为探索该类难检测的活性成分的药理作用机制提供新的技术手段.

  20. Elaboración y evaluación de un antisuero para la detección inmunohistoquímica del virus de la rabia en tejido cerebral fijado en aldehídos Production and evaluation of an antiserum for immunohistochemical detection of rabies virus in aldehyde fixed brain tissues

    OpenAIRE

    Orlando Torres-Fernández; Ladys Sarmiento; Gerardo Santamaría; Lina María Ortega; Nina Paola Lamprea

    2010-01-01

    Introducción. El procedimiento estándar para el diagnóstico de la rabia requiere de muestras frescas del cerebro infectado, que se estudian mediante las técnicas de inmunofluorescencia directa y la inoculación en ratones. No obstante, a veces se necesita estudiar cerebros infectados con rabia mediante inmunohistoquímica de material fijado en aldehídos, pero los anticuerpos comerciales que se requieren son es...

  1. Inhibition of toxic actions of phospholipase A2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds

    OpenAIRE

    Gomes, Antony; Bhattacharya, Shamik; Mukherjee, Sanghamitra; Inn-ho-Tsai,; Gomes, Aparna

    2012-01-01

    Background & objectives: Phospholipase A2 (PLA2) is one of the major constituents of krait venom associated with several pathophysiological actions like myotoxicity, cardiotoxicity, neurotoxicity, etc. As there was no specific antiserum available against Bungarus fasciatus venom, this study was done with synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum to neutralize the PLA2 induced toxicities in experimental models. Methods: B. fasciatus ...

  2. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

    OpenAIRE

    Gordon, R D; Fieles, W E; Schotland, D L; Hogue-Angeletti, R; Barchi, R. L.

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane protein...

  3. Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties.

    OpenAIRE

    Jackman, D M; Bartlett, F M; Patel, T R

    1983-01-01

    A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic det...

  4. Antibody specific for Escherichia coli J5 cross-reacts to various degrees with an Escherichia coli clinical isolate grown for different lengths of time.

    OpenAIRE

    McCallus, D E; Norcross, N. L.

    1987-01-01

    Rabbit antiserum raised against the rough mutant of Escherichia coli O111:B4, designated J5, was examined for cross-reactivity to an E. coli clinical isolate (A2385). In whole-cell enzyme-linked immunosorbent assays, J5 antiserum reacted to a greater extent with A2385 grown for 5 h than with the same bacteria grown for 19 h, while the homologous antiserum reacted similarly with bacteria grown for different lengths of time. J5 antiserum reacted to the greatest extent with lipopolysaccharide (L...

  5. Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Tegtmeier, Conny

    2001-01-01

    The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6 ml...

  6. Prevention of edema disease in pigs by passive immunization

    DEFF Research Database (Denmark)

    Johansen, M.; Andresen, Lars Ole; Thomsen, L.K.; Busch, M.E.; Wachmann, H.; Jorsal, Sven Erik Lind; Gyles, C.L.

    2000-01-01

    The effect of treatment with verotoxin 2e (VT2e) specific antiserum was evaluated in 3 Danish pig herds with edema disease (ED). The antiserum was prepared by immunizing horses with a VT2e toroid. The study was performed as a randomized blind field trial with parallel treatment and control groups...

  7. Comparison of antibody production against aflatoxin B1 in goats and rabbits.

    OpenAIRE

    Gaur, P K; El-Nakib, O; Chu, F. S.

    1980-01-01

    Antibody production against aflatoxin B1 was compared in three rabbits and one goat. Titers obtained were 20 times higher in the rabbits than in the goat. The goat antiserum appeared to have a higher degree of cross-reactivity for other aflatoxins and related metabolites than did the rabbit antiserum.

  8. Immunochemical Properties of Glucosyltransferases from Streptococcus mutans

    Science.gov (United States)

    Fukui, Kazuhiro; Kokeguchi, Susumu; Kato, Keijiro; Miyake, Yoichiro; Nogami, Ryuzo; Moriyama, Takafumi

    1983-01-01

    Antiserum against purified mutansynthetase (EC 2.4.1.?) of Streptococcus mutans 6715 (serotype g), which is responsible for the synthesis of water-insoluble glucan (ISG) in the presence of both sucrose and water-soluble glucan, was prepared. The specificity of the antiserum was tested by using crude enzyme preparations (CEPs) of S. mutans strains of various serotypes. On immunodiffusion, the antiserum cross-reacted with CEPs from strains of serotypes a (HS-6 and AHT), d (OMZ176), and g (OMZ65 and KIR), but not with those from strains of serotypes b (BHT and FA-1) and c (GS-5 and Ingbritt). The antiserum inhibited the synthesis of ISG by crude or purified mutansynthetase of S. mutans 6715. The activities of ISG synthesis by CEPs from the strains antigenically related in the foregoing immunodiffusion were inhibited by the antiserum against strain 6715 mutansynthetase. The antiserum, however, also inhibited the enzyme activity of the strains of serotype b. The finding that the antiserum against purified dextransucrase of S. mutans HS-6 inhibited ISG synthesis by a CEP of strain HS-6 and also by CEPs of antigenically related strains suggested that dextransucrase activity is involved in ISG synthesis. Images PMID:6187685

  9. Thyroxine (T4)-antisera

    International Nuclear Information System (INIS)

    The required antiserum for radioimmunoassay of the thyroid hormone T4, can be stimulated by injection of thyroglobulin into experimental animals, with a large chance of success. The frequency of occurrence of antisera with a very high strength could perhaps be increased by suppressing the thyroid hormone production in the animal. Different approaches to improving the quality of T4 antiserum, obtained with low strength and low binding affinity, are discussed. When extremely low concentrations of T4 need to be measured, repeated immunisation of the animal offers a possibility of acquiring a similar antiserum. (C.F.)

  10. Prevention of edema disease in pigs by passive immunization

    DEFF Research Database (Denmark)

    Johansen, M.; Andresen, Lars Ole; Thomsen, L.K.;

    2000-01-01

    The effect of treatment with verotoxin 2e (VT2e) specific antiserum was evaluated in 3 Danish pig herds with edema disease (ED). The antiserum was prepared by immunizing horses with a VT2e toroid. The study was performed as a randomized blind field trial with parallel treatment and control groups...... control groups was statistically significant (P <0.0001). It was not possible to establish an effect of dose (2, 4, or 6 mt) of anti-VT2e serum, because only one pig died of ED in the treatment groups. It was concluded that passive immunization by intramuscular injection of a VT2e-specific antiserum can...

  11. Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

    OpenAIRE

    Klootwijk, Willem; Sleddens-Linkels, Esther; Boer, Remco; Jansen, C.A.; Autar, R.; Herder, Wouter; Boeve, E.R.; Visser, Ton; Greef, W.J.

    1997-01-01

    textabstractTRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors ...

  12. Detection of circulating antibodies against c-myc protein in cancer patient sera.

    OpenAIRE

    Ben-Mahrez, K.; Thierry, D.; Sorokine, I.; Danna-Muller, A.; Kohiyama, M

    1988-01-01

    We have partially purified an archaebacterial protein of 84 kD which shares common epitopes with the human c-myc protein as shown by its cross-reactivity with a commercialized anti-human c-myc antiserum. An antiserum raised against the 84 kD protein recognizes a 60 kD protein from HL-60 nuclei. This protein is also recognized by the anti-human c-myc antiserum. Using this archaebacterial protein as antigen for Western blot analysis, we found that the human c-myc oncogene product could be immun...

  13. Distinct localization of FMRFamide- and bovine pancreatic polypeptide-like material in the brain, retrocerebral complex and suboesophageal ganglion of the cockroach Periplaneta americana L

    DEFF Research Database (Denmark)

    Verhaert, P; Grimmelikhuijzen, C J; De Loof, A

    1985-01-01

    One bovine pancreatic polypeptide (BPP) antiserum and two FMRFamide antisera were applied in the peroxidase-antiperoxidase (PAP) immunohistochemical technique on a complete series of sections of brains, suboesophageal ganglia (SOG), corpora cardiaca (CC) and corpora allata of Periplaneta americana...

  14. DEMONSTRATION OF MULTIPLE ANTIGENIC DETERMINANTS ON 'MYCOPLASMA PNEUMONIAE' ATTACHMENT PROTEIN BY MONOCLONAL ANTIBODIES

    Science.gov (United States)

    Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum ...

  15. Foodborne outbreak of shigellosis caused by an unusual Shigella strain.

    OpenAIRE

    Huq, I; Alam, A K; Morris, G K; Wathen, G; Merson, M

    1980-01-01

    A family outbreak of foodborne shigellosis caused by an unusual strain of Shigella is described. The strain was a mannitol-positive variant of Shigella dysenteriae and agglutinated in antiserum prepared against provisional serotype 3341-55.

  16. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    OpenAIRE

    Akagi,Tadaatsu; Sonobe, Hiroshi; Miyoshi, Isao; Yoshimoto,Shizuo

    1982-01-01

    Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL) line (BALL-1). The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1) and T-ALL (TALL-1) cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or n...

  17. Isolation and characterization of type IV group B Streptococcus capsular polysaccharide.

    OpenAIRE

    Wessels, M R; Benedí, W J; Jennings, H J; Michon, F; DiFabio, J L; Kasper, D L

    1989-01-01

    An antigenically distinct serotype, type IV, has recently been added to the recognized serotypes of group B streptococci (GBS). We isolated and purified the capsular polysaccharide antigen from a prototype type IV GBS strain. The type IV capsular polysaccharide formed a precipitin line with rabbit antiserum to type IV GBS organisms but not with antiserum to organisms of GBS serotype Ia, Ib, II, or III. Enzyme-linked immunosorbent assay inhibition experiments showed no cross-reaction between t...

  18. gamma-Glutamyltranspeptidase from Proteus mirabilis: localization and activation by phospholipids.

    OpenAIRE

    Nakayama, R; Kumagai, H.; Tochikura, T

    1984-01-01

    Antiserum was prepared against the purified gamma-glutamyltranspeptidase (EC 2.3.2.2) of Proteus mirabilis. The antiserum inactivated the gamma-glutamyltranspeptidase activities of both purified enzyme and intact cells. Native cells were agglutinated with the antibody. Immunocytochemical studies with indirect immunofluorescence and electron microscopy analysis suggested that gamma-glutamyltranspeptidase is localized on the surface of the cell. Its distribution in the cell wall or periplasmic ...

  19. Renal localization of heparan sulfate proteoglycan by immunohistochemistry.

    OpenAIRE

    Klein, D. J.; Oegema, T.R.; Eisenstein, R.; Furcht, L.; Michael, A. F.; Brown, D M

    1983-01-01

    Glomerular localization of heparan sulfate proteoglycan (HS-proteoglycan) has been studied immunohistochemically with a highly purified antiserum to bovine aorta HS-proteoglycan core protein. The specificity of the antiserum was enhanced by consecutive fibronectin and chondroitin sulfate-dermatan sulfate proteoglycan (CS-DS proteoglycan) affinity chromatography. The affinity-purified HS-proteoglycan antibody lacked cross-reactivity by enzyme-linked immunosorbent assays (ELISA) with CS-DS prot...

  20. The effect of antidiuretic stimuli on the morphology of the lateral intercellular spaces in the medullary collecting duct of the rat.

    OpenAIRE

    Rowen, D; Law, R O

    1981-01-01

    An investigation has been carried out into the formation of dilated lateral intercellular spaces in the medullary collecting duct of the rat kidney following water deprivation or infusion of vasopressin. Dilation is conspicuous under these conditions, by comparison with normally hydrated controls, but its appearance is prevented by prior treatment of rats with antiserum raised against urinary (renal) hyaluronidase. Antiserum against testicular hyaluronidase is without effect. The presence or ...

  1. Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin.

    OpenAIRE

    Didier, E S; Rogers, L B; Brush, A D; Wong, S.; Traina-Dorge, V; Bertucci, D

    1996-01-01

    A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. ...

  2. Immunohistochemical analysis of amylase isoenzymes in thyroid cancer.

    OpenAIRE

    Higashiyama, M; Doi, S; Tomita, N; Monden, T.; Murotani, M.; Kawasaki, Y.; Kobayashi, T.; Shimano, T; Ogawa, M; Takai, S

    1991-01-01

    The expression of amylase in various histological types of thyroid cancer was studied by an immunohistochemical technique, using a polyclonal antiamylase antiserum and two monoclonal antibodies specific for salivary and pancreatic-type amylases, respectively. Amylase was expressed in 21 of 24 (88%) thyroid cancers by polyclonal antiserum analysis. Analysis by monoclonal antibodies, however, showed that only 13 (54%) cases and three (13%) cases contained salivary-type and pancreatic-type amyla...

  3. Isolation and identification of a Giardia lamblia-specific stool antigen (GSA 65) useful in coprodiagnosis of giardiasis.

    OpenAIRE

    Rosoff, J D; Stibbs, H H

    1986-01-01

    A Giardia lamblia-specific antigen (GSA 65) was isolated from stools of G. lamblia-positive patients by crossed- and line-immunoelectrophoresis and counterimmunoelectrophoresis (CIE) in agarose by using rabbit antiserum prepared against G. lamblia cysts. CIE with rabbit anti-GSA 65 monospecific antiserum revealed that GSA 65 was present in aqueous stool eluates of giardiasis patients and in cysts and trophozoites of the parasite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of im...

  4. Cloning and characterization of Trypanosoma cruzi antigens bearing repetitive epitopes

    International Nuclear Information System (INIS)

    The genes of Trypanosoma cruzi, the causative agent of Chagas Disease, were cloned in the expression vector lambda gt11, and screened with a trypamastigote antiserum. Twelve clones expressing fusion proteins reacting to the antiserum were selected and further analysed in the western blot. One clone (7/2) expressing an antigen present in the cytoplasm of the parasite was found to react specifically with chagasic sera and may have potential as an immunodiagnostic reagent. (author). 11 refs, 4 figs

  5. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

    OpenAIRE

    Shi, J.; Dixon, R A; Gonzales, R A; Kjellbom, P; Bhattacharyya, M K

    1995-01-01

    An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive ...

  6. Expression of surface hydrophobic proteins by Candida albicans in vivo.

    OpenAIRE

    Glee, P M; Sundstrom, P; Hazen, K C

    1995-01-01

    Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The po...

  7. Immunochemical characterization of brain and pineal tryptophan hydroxylase.

    OpenAIRE

    Chung, Y. I.; Park, D. H.; Kim, M.; Baker, H; Joh, T H

    2001-01-01

    Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, a...

  8. Group B streptococcal Ibc protein antigen: distribution of two determinants in wild-type strains of common serotypes.

    OpenAIRE

    Johnson, D R; Ferrieri, P

    1984-01-01

    Studies were carried out on the distribution of the Ibc protein antigenic marker in wild-type strains of group B streptococci of diverse serotypes isolated from epidemiological studies. Rabbits were immunized with group B streptococcal strain H36B, a prototype Ib strain, to produce antibody to the Ibc protein antigens. One antiserum (no. 970) contained antibody only against the trypsin-sensitive (TS) portion of the Ibc antigen. A second antiserum (no. 973), however, contained antibody to both...

  9. Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

    OpenAIRE

    1984-01-01

    Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protect...

  10. Exclusive presence of lactose-sensitive fimbriae on a typical strain (WVU45) of Actinomyces naeslundii.

    OpenAIRE

    Cisar, J O; David, V. A.; Curl, S H; Vatter, A. E.

    1984-01-01

    Lactose-sensitive fimbriae were identified as the only fimbriae present on Actinomyces naeslundii WVU45 (ATCC 12104). A single antigen reactive with antiserum against WVU45 cells was detected by cross immunoelectrophoresis of isolated fimbriae, and a monospecific antiserum against this antigen reacted with all fimbriae observed on the bacterial surface by immunoelectron microscopy. Moreover, the loss of one cell surface antigen by a spontaneous mutant of A. naeslundii WVU45 (WVU45M), isolated...

  11. Megaloblastic hematopoiesis in vitro. Interaction of anti-folate receptor antibodies with hematopoietic progenitor cells leads to a proliferative response independent of megaloblastic changes.

    OpenAIRE

    Antony, A C; Briddell, R A; Brandt, J E; Straneva, J E; Verma, R S; M. E. Miller; Kalasinski, L A; R. HOFFMAN

    1991-01-01

    We tested the hypothesis that anti-placental folate receptor (PFR) antiserum-mediated effects on hematopoietic progenitor cells in vitro of increased cell proliferation and megaloblastic morphology were independent responses. We determined that (a) purified IgG from anti-PFR antiserum reacted with purified apo- and holo-PFR and specifically immunoprecipitated a single (44-kD) iodinated moiety on cell surfaces of low density mononuclear cells (LDMNC); (b) when retained in culture during in vit...

  12. Epithelial membrane antigen in cells from the uterine cervix: immunocytochemical staining of cervical smears.

    OpenAIRE

    Valkova, B; Ormerod, M G; Moncrieff, D.; Coleman, D V

    1984-01-01

    Smears made from cervical scrapes have been stained immunocytochemically for epithelial membrane antigen using a polyclonal antiserum and two monoclonal antibodies. With the polyclonal antiserum malignant cells and those showing dysplasia consistently expressed the antigen. Normal cells were generally negative, with the exception of some metaplastic cells. The monoclonal antibodies, although they stained the abnormal cells less consistently, gave the same pattern of staining. All three antibo...

  13. Neutralizing antibody response of rabbits and goats to caprine arthritis-encephalitis virus.

    OpenAIRE

    Klevjer-Anderson, P; McGuire, T C

    1982-01-01

    Rabbits were immunized with purified caprine arthritis-encephalitis virus and examined for neutralizing activity. Analysis of virus-antiserum interaction at 37 degrees C demonstrated little loss of viral infectivity after incubation with heat-inactivated rabbit antiserum for 60 min. However, sensitization of virus (as assessed by the addition of complement) occurred almost immediately and was 95% complete after 10 min. The complement-dependent neutralizing activity was associated with the imm...

  14. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, R.D.; Fieles, W.E.; Schotland, D.L.; Hogue-Angeletti, R.; Barchi, R.L.

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

  15. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    International Nuclear Information System (INIS)

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure

  16. Optimization of the radioimmunoassays for measuring fentanyl and alfentanil in human serum

    International Nuclear Information System (INIS)

    Measurement of serum fentanyl and alfentanil concentrations by radioimmunoassay (RIA) may result in significant errors and high variability when the technique described in the available fentanyl and alfentanil RIA kits is used. The authors found a 29-94% overestimation of measured fentanyl and alfentanil serum levels when 3H-fentanyl or 3H-alfentanil was added lastly to the mixture of antiserum and sample. This finding is related to a reduction in binding sites for the labeled compounds after preincubation of sample and antiserum. If this sequence is used, it becomes necessary to extend the incubation period up to 6 h for fentanyl and up to 10 h for alfentanil in order to achieve equilibration between unlabeled and labeled drug with respect to antiserum binding. However, when antiserum is added lastly to the mixture of sample and labeled drug, measurement accuracy and precision for fentanyl and alfentanil serum concentrations are enhanced markedly. In addition, it is important to perform the calibration curves and sample measurements using the same medium (i.e., serum alone or a serum/buffer dilution). In summary, to optimize the RIA for fentanyl and alfentanil, the authors recommend the following: 1) adding the antiserum lastly to the mixture of sample and labeled drug; 2) performing calibration curves using patient's blank serum when possible; 3) carefully examining and standardizing each step of the RIA procedure to reduce variability, and, finally; 4) comparing results with those of other established RIA laboratories

  17. Optimization of the radioimmunoassays for measuring fentanyl and alfentanil in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Schuettler, J.; White, P.F.

    1984-09-01

    Measurement of serum fentanyl and alfentanil concentrations by radioimmunoassay (RIA) may result in significant errors and high variability when the technique described in the available fentanyl and alfentanil RIA kits is used. The authors found a 29-94% overestimation of measured fentanyl and alfentanil serum levels when 3H-fentanyl or 3H-alfentanil was added lastly to the mixture of antiserum and sample. This finding is related to a reduction in binding sites for the labeled compounds after preincubation of sample and antiserum. If this sequence is used, it becomes necessary to extend the incubation period up to 6 h for fentanyl and up to 10 h for alfentanil in order to achieve equilibration between unlabeled and labeled drug with respect to antiserum binding. However, when antiserum is added lastly to the mixture of sample and labeled drug, measurement accuracy and precision for fentanyl and alfentanil serum concentrations are enhanced markedly. In addition, it is important to perform the calibration curves and sample measurements using the same medium (i.e., serum alone or a serum/buffer dilution). In summary, to optimize the RIA for fentanyl and alfentanil, the authors recommend the following: 1) adding the antiserum lastly to the mixture of sample and labeled drug; 2) performing calibration curves using patient's blank serum when possible; 3) carefully examining and standardizing each step of the RIA procedure to reduce variability, and, finally; 4) comparing results with those of other established RIA laboratories.

  18. Germ tube-specific antigens of Candida albicans cell walls

    International Nuclear Information System (INIS)

    Studies were performed to characterize the surface differences between blastospores and germ tubes of the pathogenic, dimorphic yeast, Candida albicans, and to identify components of yeast cells responsible for these differences. Investigation of surfaces differences of the two growth forms was facilitated by the production of rabbit antiserum prepared against Formalin-treated yeast possessing germ tubes. To prepare antiserum specific for germ tubes, this serum was adsorbed with stationary phase blastospores. Whereas the unadsorbed antiserum reacted with both blastospore and germ tube forms by immunofluorescence and Enzyme-Linked Immunosorbent Assay, the adsorbed antiserum did not react with blastospores but detected germ tube-specific antigens in hyphal forms. The differences between blastospores and germ tubes of Candida albicans, were further studied by comparing enzymatic digests of cell walls of both growth forms in radiolabeled organisms. Organisms were labeled either on the surface with 125I, or metabolically with [35S] methionine or [3H] mannose. Three-surface-located components (as shown by antibody adsorption and elution experiments) were precipitated from Zymolase digests. All three components were mannoproteins as shown by their ability to bind Concanavalin A, and to be labeled in protein labeling procedures, and two of these (200,000 and 155,000 molecular weight) were germ tube specific, as shown by their ability to be precipitated by germ tube-specific antiserum. Monoclonal antibodies were prepared to C. albicans, using blastospores bearing germ tubes as immunogen

  19. Production of anti-estradiol serum in sheep

    International Nuclear Information System (INIS)

    Anti estradiol serum has been raised in sheep by immunization. Two adult sheep were actively immunized against 17 beta-Estradiol-17 Hemisuccinate-BSA conjugate. The emulsified antigen in 0.9% NaCl-Freund's adjuvant 1:1 (1.5 mg/ml estradiol) was injected subcutaneously at pelvic area. Every week is blood samples were obtained from the immunized animals from the Jugular's Veins. Blood samples were allowed to clot and serum was separated by centrifugation. Titer of antiserum was measured by RIA method as described by Abraham. The results indicated that the highest antiserum titers were noted 2 weeks after booster injection (47% and 56%) for antiserum 1/1000. (author). 4 refs, 1 fig, 1 tab

  20. Preparation and Application of Polyclonal Antibody against a Recombinant Laccase

    Institute of Scientific and Technical Information of China (English)

    Yinghai Xu; Yuzhi Hong; Yazhong Xiao; Wei Fang

    2007-01-01

    A laccase gene from Trametes sp. 420 was recombinantly expressed in Pichia pastoris, producing the enzyme rLacD.Six mutant enzymes were produced by site-directed mutation at six potential glycosylation sites in the enzyme rLacD respectively. To probe the mutants with lower activities sensitively and specifically, the antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits, about 4-month-old and 2 kilogram weight, using pure rLacD as an immunogen. Antibodies were collected after the fifth immunization injection. The antiserum had titres of 1:32 in double immunodiffusion test and of 1:128,000 in enzyme-linked immunosorbent assay (ELISA). The results obtained by Western blot analysis showed that the antiserum could react with rLacD and its mutants with highly specific and sensitive affinities.

  1. Detection of feline coronavirus using microcantilever sensors

    Science.gov (United States)

    Velanki, Sreepriya; Ji, Hai-Feng

    2006-11-01

    This work demonstrated the feasibility of detecting severe acute respiratory syndrome associated coronavirus (SARS-CoV) using microcantilever technology by showing that the feline coronavirus (FIP) type I virus can be detected by a microcantilever modified by feline coronavirus (FIP) type I anti-viral antiserum. A microcantilever modified by FIP type I anti-viral antiserum was developed for the detection of FIP type I virus. When the FIP type I virus positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the FIP type I virus by the antiserum on the surface of the microcantilever. A negative control sample that does not contain FIP type I virus did not cause any bending of the microcantilever. The detection limit of the sensor was 0.1 µg ml-1 when the assay time was <1 h.

  2. Identification and role of plasma membrane aquaporin in maize root

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antiserum against expressed aquaporin fusion protein, GST-RD28, the distribution of aquaporin in the plasma membrane of maize root protoplasts has been examined under confocal laser scanning microscopy by indirect fluorescence staining. Results indicate that there are abundant aquaporins in maize roots, which are distributed in plasma membrane unevenly. Western blotting analysis of total protein solubilized from maize root plasma membrane shows that antiserum against GST-RD28 can cross-react with one protein around 55 ku. Another 28 ku protein can also be detected when the concentration of SDS and DTT in SDS-PAGE sample buffer is increased. The 55 and 28 ku proteins may be dimeric and monomeric of aquaporin respectively. Functional experiments show that aquaporin blocker HgCl2 and aquaporin antiserum can suppress the swelling of maize root protoplasts in hypotonic solution, indicating that aquaporin in plasma membrane of protoplast facilitates rapid transmembrane water flow.

  3. Heterogeneous distribution of a basement membrane heparan sulfate proteoglycan in rat tissues

    DEFF Research Database (Denmark)

    Couchman, J R

    1987-01-01

    buoyant density HSPG from the murine Engelbreth-Holm swarm tumor. It was, however, confirmed that only a single population of antibodies was present in the serum. Despite the presence of similar epitopes on these two proteoglycans of different hydrodynamic properties, it was apparent that the PYS-2 HSPG...... recognized by this antiserum. Those basement membranes that lacked the HSPG strongly stained with antisera against laminin and type IV collagen. The striking distribution pattern is possibly indicative of multiple species of basement membrane HSPGs of which one type is recognized by this antiserum. Further...... represents a basement membrane proteoglycan of distinct properties reflected in its restricted distribution in vivo....

  4. Identification of murine complement receptor type 2.

    OpenAIRE

    Fingeroth, J D; Benedict, M A; Levy, D.N.; Strominger, J L

    1989-01-01

    A rabbit antiserum reactive with the human complement component C3d/Epstein-Barr virus receptor (complement receptor type 2, CR2) immunoprecipitates a Mr 155,000 murine B-cell surface antigen. The apparent molecular weight and cellular distribution of this murine antigen are similar to those of human CR2. Cells expressing the murine protein bind sheep erythrocytes coated with antibody and murine C1-C3d but do not bind Epstein-Barr virus at all. The monospecific antiserum to human CR2 together...

  5. Characterization of anti-glucagon sera elicited against a C-terminal fragment of pancreatic glucagon and their use in glucagon radioimmunoassay

    International Nuclear Information System (INIS)

    Experimental results indicate that antiserum OAL-123 raised in the rabbit against a C-terminal fragment of pancreatic glucagon possesses immunological properties similar to those of antiserum 30 K and that it is useful for specific measurement of pancreatic glucagon. A radioassay was developed using OAL-123 which showed the highest sensitivity in the assay system used. It utilised human pancreatic monocomponent glucagon as standard and monoradioionated glucagon as tracer. Cross reactivities of extracts from dog jejuunm and stomach mucosa and of glucagen-related peptides and immunoreactivities in dog tissues and human blood were examined. (Auth./C.F.)

  6. Pneumocystis pneumonia: importance of gallium scan for early diagnosis and description of a new immunoperoxidase technique to demonstrate Pneumocystis carinii

    Energy Technology Data Exchange (ETDEWEB)

    Levin, M.; McLeod, R.; Young, Q.; Abrahams, C.; Chambliss, M.; Walzer, P.; Kabins, S.A.

    1983-07-01

    Pneumocystis pneumonia presented in a homosexual with fever, a normal chest radiograph, and pulmonary gallium uptake. Bronchial washings yielded Mycobaterium tuberculosis, but despite antituberculosis therapy he remained febrile, and gallium uptake in the lung increased. Subsequently, silver stain of transbronchial lung biopsy obtained 2 months earlier at the time that tuberculosis was diagnosed showed many Pneumocystis cysts in alveolar spaces. In contrast to Pneumocystis cysts in infected lung tissue from other humans, our patient's Pneumocystis cysts reacted more avidly with antiserum to rat Pneumocystis than with antiserum to human pneumocystis, raising the possibility that organisms that infect humans may have varied surface antigenic properties.

  7. Neurotensin-like immunoreactivity in the nervous system of hydra

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Carraway, R E; Rökaeus, A;

    1981-01-01

    well with an antiserum directed against sequence 1-8 and an antiserum directed against sequence 6-13 of neurotensin. This fraction elutes also at the position of neurotensin and might closely resemble the mammalian peptide. A fraction eluting with the void volume crossreacts preferentially with...... various region-specific antisera towards mammalian neurotensin, show the presence of multiple neurotensin-related peptides. The amounts of these peptides vary between 1 and 350 pmol per gram wet weight. Gel filtration on Sephadex G-25 reveals a fraction of neurotensin-like peptides that crossreact equally...

  8. Pneumocystis pneumonia: importance of gallium scan for early diagnosis and description of a new immunoperoxidase technique to demonstrate Pneumocystis carinii

    International Nuclear Information System (INIS)

    Pneumocystis pneumonia presented in a homosexual with fever, a normal chest radiograph, and pulmonary gallium uptake. Bronchial washings yielded Mycobaterium tuberculosis, but despite antituberculosis therapy he remained febrile, and gallium uptake in the lung increased. Subsequently, silver stain of transbronchial lung biopsy obtained 2 months earlier at the time that tuberculosis was diagnosed showed many Pneumocystis cysts in alveolar spaces. In contrast to Pneumocystis cysts in infected lung tissue from other humans, our patient's Pneumocystis cysts reacted more avidly with antiserum to rat Pneumocystis than with antiserum to human pneumocystis, raising the possibility that organisms that infect humans may have varied surface antigenic properties

  9. The changes in antigenic components of Vibrio cholerae strains isolated in Vietnam

    OpenAIRE

    Ha, Thi Quyen; Dinh, Duy Khang

    2015-01-01

    Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kD...

  10. Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    T. L. Kipnis

    1992-01-01

    Full Text Available A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG1 and one IgG2a recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.

  11. Affinity purification of antibodies

    Science.gov (United States)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  12. USE OF IMMUNOFLUORESCENCE AND PHASE-CONTRAST MICROSCOPY FOR DETECTION AND IDENTIFICATION OF 'GIARDIA' CYSTS IN WATER SAMPLES

    Science.gov (United States)

    A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia ...

  13. IMMUNOFLUORESCENCE DETECTION OF 'CRYPTOSPORIDIUM' OOCYSTS IN FECAL SMEARS

    Science.gov (United States)

    An indirect fluorescent antibody (IFA) procedure was developed for the detection of Cryptosporidium sp. oocysts in human, nonhuman primate, and bovine fecal smears. The procedure, which takes about 90 min to perform, involves the use of a rabbit antiserum against Cryptosporidium ...

  14. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø;

    1997-01-01

    inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  15. Labelling of human gastric inhibitory polypeptide with 125I using conjunction method

    International Nuclear Information System (INIS)

    Human gastric inhibitory polypeptide (GIP) was labelled with the conjunction method. The results showed that the conjunction method succeeds in protecting the immunoactivity of human GIP. The 125I-GIP was able to combine with its GIP antibody specifically. The binding rate was 50% at an antiserum dilution of 1.3 x 104

  16. Antigens in human glioblastomas and meningiomas: Search for tumour and onco-foetal antigens. Estimation of S-100 and GFA protein

    DEFF Research Database (Denmark)

    Dittmann, L; Axelsen, N H; Norgaard-Pedersen, B;

    1977-01-01

    (glia specific); monospecific anti-GFA (glial fibrillary acidic protein), (astroglia specific); polyspecific anti-foetal brain (12-16th week of gestation); a polyspecific anti-glioblastoma antiserum, absorbed with insolubilized serum, haemolysate and normal brain extract; polyspecific anti...

  17. Tomato necrotic ring virus (TNRV), a recently described tospovirus species infecting tomato and pepper in Thailand

    NARCIS (Netherlands)

    Mehraban, A.; Cheewachaiwit, S.; Relevante, C.; Kormelink, R.J.M.; Peters, D.

    2011-01-01

    Two tospovirus isolates collected from tomato and bell pepper in Thailand were studied. The isolates induced severe necrotic mottling and/or necrotic spots and rings on the leaves and fruits of the respective plants as confirmed by back-inoculation. A polyclonal antiserum raised against its nucleoca

  18. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø; Christiansen, Gunnar; Klemm, Per

    inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  19. Distribution and characterization of two non-opiod peptides with morphine modulating activity in brain

    International Nuclear Information System (INIS)

    Two peptides which are immunoreactive to Phe-Met-Arg-Phe-NH2 (FMRF-NH2) antiserum were purified from bovine brain, chemically characterized and synthesized. These two peptides, octapeptide octadecapeptide, can reduce rat tail flick latencies; octapeptide can also attenuate morphine induced analgesia. The distribution of these two peptides in brain was studied by radioimmunoassay. Octapeptide antiserum shows high affinity for synthetic octapeptide, PYY and FMRF-NH2. The antiserum is almost totally inactive to γ1-MSH. Octadecapeptide antiserum shows high affinity for octadecapeptide, some cross-reaction for the octapeptide (50%), γ1-MSH (10%) and FMRF-NH2 (0.01%). NPY, PYY, Met5-Enk-Arg-Phe and CCk are inactive. Octa and Octadecapeptide immunoreactivities are unevenly distributed in bovine brain. The highest concentrations (pmol g-1) are found in dorsal spinal cord (9.8 and 16.4 respectively), periaqueductal gray (8.6 and 6.8) and pons medulla (7.0 and 8.9); non-detectable amounts in cortex and cerebellum. The enrichment of these peptides in dorsal cord and PAG, areas important in opoid-mediated pain perception, suggest that they may play a role in mediating antinociception. Furthermore, using these two antisera, it is now possible to differentiate mammalian FMRF-NH2-like and NPY

  20. Distribution and characterization of two non-opiod peptides with morphine modulating activity in brain

    Energy Technology Data Exchange (ETDEWEB)

    Majane, E.A.; Yang, H.Y.T.

    1986-03-05

    Two peptides which are immunoreactive to Phe-Met-Arg-Phe-NH/sub 2/ (FMRF-NH/sub 2/) antiserum were purified from bovine brain, chemically characterized and synthesized. These two peptides, octapeptide octadecapeptide, can reduce rat tail flick latencies; octapeptide can also attenuate morphine induced analgesia. The distribution of these two peptides in brain was studied by radioimmunoassay. Octapeptide antiserum shows high affinity for synthetic octapeptide, PYY and FMRF-NH/sub 2/. The antiserum is almost totally inactive to ..gamma../sub 1/-MSH. Octadecapeptide antiserum shows high affinity for octadecapeptide, some cross-reaction for the octapeptide (50%), ..gamma../sub 1/-MSH (10%) and FMRF-NH/sub 2/ (0.01%). NPY, PYY, Met/sup 5/-Enk-Arg-Phe and CCk are inactive. Octa and Octadecapeptide immunoreactivities are unevenly distributed in bovine brain. The highest concentrations (pmol g/sup -1/) are found in dorsal spinal cord (9.8 and 16.4 respectively), periaqueductal gray (8.6 and 6.8) and pons medulla (7.0 and 8.9); non-detectable amounts in cortex and cerebellum. The enrichment of these peptides in dorsal cord and PAG, areas important in opoid-mediated pain perception, suggest that they may play a role in mediating antinociception. Furthermore, using these two antisera, it is now possible to differentiate mammalian FMRF-NH/sub 2/-like and NPY.

  1. Human C-peptide. Pt. 1

    International Nuclear Information System (INIS)

    Synthetic human C-peptide bearing a tyrosine group at its amino end is labelled with 125iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results of the two methods are compared. Antiserum to synthetic human C-peptide (without tyrosine), which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pips concerning antibody production. The so-called 'hook effect' phenomenon is observed when setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated using the anion exchange resin amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum. (orig.)

  2. Stability evaluation of CNBr-Sepharose 4 B for using as solid matrix in immunoradiometric assay antibodies coupling; Avaliacao da estabilidade da CNBr-sepharose 4B para emprego como matriz solida no acoplamento de anticorpos especificos de ensaios imunorradiometricos

    Energy Technology Data Exchange (ETDEWEB)

    Haber, Esther Piltcher; Silva, Sandra Rosa da; Borghi, Vania Caira [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Wajchenberg, Bernardo Leo [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina

    1995-12-31

    The present work verifies the stability of a CNBr-Sepharose 4 B product (Pharmacia) stored at our laboratory one year after its expire date in view of its application as solid phase antibodies in the development of an immunoradiometric assay for measurement of serum human proinsulin. From rabbit IgG antiserum previously purified and concentrated by ultrafiltration (Publication IPEN 294, 1990) the antibodies were isolated by affinity chromatography. Sheep antiserum anti-rabbit IgG were coupled to cyanogen bromide activated Sepharose 4 B and the rabbit IgG which were bound to the immunosorbent could be obtained by elution with stepwise pH gradient from pH 7.0 to pH 2.5. The complying efficiency of the sheep antiserum to this solid phase material was 97%. The elution profile obtained shows identify of the sample related to the antiserum anti-rabbit IgG by affinity chromatography. These results suggest that this CNBr-Sepharose 4 B lot can be used satisfactorily to attach antibodies for use in the two-site immunoradiometric assay. (author). 7 refs., 1 fig.

  3. Stability evaluation of CNBr-Sepharose 4 B for using as solid matrix in immunoradiometric assay antibodies coupling

    International Nuclear Information System (INIS)

    The present work verifies the stability of a CNBr-Sepharose 4 B product (Pharmacia) stored at our laboratory one year after its expire date in view of its application as solid phase antibodies in the development of an immunoradiometric assay for measurement of serum human proinsulin. From rabbit IgG antiserum previously purified and concentrated by ultrafiltration (Publication IPEN 294, 1990) the antibodies were isolated by affinity chromatography. Sheep antiserum anti-rabbit IgG were coupled to cyanogen bromide activated Sepharose 4 B and the rabbit IgG which were bound to the immunosorbent could be obtained by elution with stepwise pH gradient from pH 7.0 to pH 2.5. The complying efficiency of the sheep antiserum to this solid phase material was 97%. The elution profile obtained shows identify of the sample related to the antiserum anti-rabbit IgG by affinity chromatography. These results suggest that this CNBr-Sepharose 4 B lot can be used satisfactorily to attach antibodies for use in the two-site immunoradiometric assay. (author). 7 refs., 1 fig

  4. Subcellular localization of casein kinase I

    DEFF Research Database (Denmark)

    Grankowski, N; Issinger, O G

    1990-01-01

    An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

  5. Use of protein A-positive Staphylococci as adsorbent in a radioimmunoassay for cyclic AMP and cyclic GMP

    International Nuclear Information System (INIS)

    A reproducible (SD4) was 10 μl. The blank value was below 2% and separation could be run at room temperature or at 40C as convenient. The acetylation or succinylation procedure of the sample totally eliminated interference of test sample immunoglobulins with anti-serum binding to the adsorbent. (author)

  6. Anti-brain antisera bind primarily to non-T cells in mouse bone marrow

    Energy Technology Data Exchange (ETDEWEB)

    Press, O.W.; Rosse, C.; Clagett, J.

    1977-12-01

    Anti-brain heteroantisera are widely employed for the delineation of T lymphocytes in lymphomyeloid organs despite compelling evidence demonstrating the cross reactivity of such reagents with unrelated hemopoietic cells. The experiments described in the paper utilized absorptions of a fluoresceinated goat anti-rat brain antiserum with thymocytes or nude bone marrow cells to determine the validity of using such an antiserum to identify T cells in the thymus, lymph nodes, spleen, and bone marrow of CBA/J mice. Thymocyte absorption of this fluoresceinated antiserum removed virtually all of the staining of thymus, lymph node, and spleen cells by this reagent. In contrast, the percentage of labeled marrow cells was unchanged by thymocyte absorption of the anti-rat brain antiserum, although the staining could be eliminated by absorption with nude bone marrow. These results suggest that anti-brain antisera can be employed as dependable T-cell probes with suspensions of the thymus or peripheral lymphoid organs. However, the vast majority of marrow cells staining with the anti-brain antisera are not T cells. Therefore, anti-brain antisera should not be utilized to discern T lymphocytes in this tissue unless additional absorptions (with nude marrow) or special controls are performed.

  7. Tissue-type plasminogen activator in somatostatin cells of rat pancreas and hypothalamus

    DEFF Research Database (Denmark)

    Kristensen, P; Larsson, L I; Danø, K;

    1987-01-01

    -PA, and immunoblotting analysis demonstrated one band with a similar electrophoretic mobility. No urokinase-type PA immunoreactivity was found in the rat endocrine pancreas. A granular t-PA immunoreactivity resembling that found in adjacent sections with somatostatin antiserum was found in the median eminence...

  8. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Solberg, H; Løber, D; Eriksen, J;

    1992-01-01

    -hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand...

  9. A radioimmunoassay for the Hydra head activator

    International Nuclear Information System (INIS)

    A highly-sensitive and specific radioimmunoassay for the head activator has been developed which utilises tritiated head activator. The assay is sensitive in the range of 40-200 fmol. Immunochemical studies showed that the antiserum recognised the intact molecule better than any of the fragments or derivatives produced by enzymatic treatment or chemical synthesis. (Auth.)

  10. Denmark: botulism in an infant or infant botulism?

    DEFF Research Database (Denmark)

    Paerregaard, A; Angen, O; Lisby, M;

    2008-01-01

    was noted. Botulism was suspected and confirmed by testing of patient serum in a bioassay. The condition of the patient improved following administration of botulism antiserum. The clinical picture was suggestive of intestinal (infant) botulism. However, botulism acquired from consumption of food...

  11. Denmark: Botulism in an infant or infant botulism?

    DEFF Research Database (Denmark)

    Pærregaard, A; Angen, Øystein; Lisby, M;

    2008-01-01

    was noted. Botulism was suspected and confirmed by testing of patient serum in a bioassay. The condition of the patient improved following administration of botulism antiserum. The clinical picture was suggestive of intestinal (infant) botulism. However, botulism acquired from consumption of food...

  12. Avian Follicular and Interdigitating Dendritic Cells: Isolation and Morphologic, Phenotypic, and Functional Analyses

    Science.gov (United States)

    An antiserum against Eimeria tenella sporozoites was used to localize and isolate Ag-binding cells in intestinal cecal tonsils of parasite-infected chickens. Based on their tissue localization, ultrastructural features, and expression of surface markers, two subpopulations of cells were isolated, C...

  13. Expression of the recA gene of Pseudomonas aeruginosa PAO is inducible by DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.V.; Kokjohn, T.A.

    1988-05-01

    Western (immunoblot) analysis using Escherichia coli anti-RecA antiserum revealed that expression of the RecA protein of Pseudomonas aeruginosa PAO is induced upon exposure of the bacterium to UV irradiation or norfloxacin, a quinolone related to nalidixic acid.

  14. Is amyloid A (AA) amyloidosis always secondary?

    OpenAIRE

    Maury, C P; Törnroth, T; Wegelius, O

    1985-01-01

    The case is reported of a patient with systemic AA amyloidosis associated with non-specific mesenteric lymphadenitis and chronic sideropenia. Renal, small bowel, and rectal biopsies showed amyloid deposits containing AA protein, as defined by potassium permanganate sensitivity and by reactivity with AA antiserum. Reversal of the nephrotic syndrome occurred during steroid-azathioprine therapy.

  15. The measurement of triclosan in water using a magnetic particle enzyme immunoassay

    Science.gov (United States)

    A sensitive magnetic particle-based immunoassay to determine triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)hexanoic acid-keyhole limpet hemocya...

  16. Materials, methods and quality control, ch. 3

    International Nuclear Information System (INIS)

    A description of the chemical reagents, the 125I-labelled angiotensin I, the antiserum and the standards is given. A modified measuring method with the New England Nuclear kit for angiotensin I radioimmunoassay is presented as well as the quality control data

  17. Non-chromatographic radioimmunoassay for serum dehydroepiandrosterone using a mixture of antisera

    International Nuclear Information System (INIS)

    A simplified method for evaluating serum dehydroepiandrosterone (DHEA) without chromatography was developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ5 antiserum and anti-11-deoxy-17-ketosteroid antiserum, in which cross-reactivity of each antiserum is reduced to a negligible amount. Serum (20 μl) was extracted with 1 ml of n-hexane. One milliliter of 80 percent methanol was added to the n-hexane extract, which was stirred and centrifuged. The n-hexane layer was discarded, and the methanol layer was evaporated to dryness. The residue was incubated with an antiserum mixture containing DHEA-7α-3H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α-3H. The accuracy, precision, sensitivity, and specificity were satisfactory. Good agreement was found between the serum DHEA levels obtained by the present radioimmunoassay and those obtained by radioimmunoassay with paper chromatography, making this method suitable for routine use. (U.S.)

  18. Nerve ring of the hypostome in hydra. I. Its structure, development, and maintenance

    DEFF Research Database (Denmark)

    Koizumi, O; Itazawa, M; Mizumoto, H;

    1992-01-01

    The anatomy and developmental dynamics of the nerve ring in the hypostome of Hydra oligactis were examined immunocytochemically with an antiserum against a neuropeptide and with neuron-specific monoclonal antibodies. The nerve ring is unique in the mesh-like nerve net of hydra. It is a distinct...

  19. Immunofixation electrophoresis: a technique for the study of protein polymorphism. Vox Sang 1969:17:445-52.

    Science.gov (United States)

    Alper, C A; Johnson, A M

    1993-01-01

    A technique is described which allows direct visualization of individual proteins in mixtures by specific antiserum after electrophoresis. By minimizing diffusion it permits rapid, direct, and clear detection of genetic polymorphism and 'conversion' of proteins in the complement and coagulation systems. PMID:8362522

  20. Production of polyclonal antibodies to a recombinant coat protein of Potato virus Y

    Czech Academy of Sciences Publication Activity Database

    Kmoníčková, Jitka; Plchová, Helena; Moravec, Tomáš; Hoffmeisterová, Hana; Dědič, P.; Čeřovská, Noemi

    2008-01-01

    Roč. 53, č. 5 (2008), s. 438-442. ISSN 0015-5632 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : ESCHERICHIA-COLI * ANTISERUM * ELISA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.172, year: 2008

  1. Antibodies directed to Neisseria gonorrhoeae impair nerve growth factor-dependent neurite outgrowth in Rat PC12 cells.

    Science.gov (United States)

    Reuss, B

    2014-03-01

    In children born from mothers with prenatal infections with the Gram-negative bacterium Neisseria gonorrhoeae, schizophrenia risk is increased in later life. Since cortical neuropil formation is frequently impaired during this disease, actions of a rabbit polyclonal antiserum directed to N. gonorrhoeae on neurite outgrowth in nerve growth factor-stimulated PC12 cells were investigated here. It turned out that 10 μg/ml of the antiserum leads indeed to a significant reduction in neurite outgrowth, whereas an antiserum directed to Neisseria meningitidis had no such effect. Furthermore, reduction in neurite outgrowth could be reversed by the neuroleptic drugs haloperidol, clozapine, risperidone, and olanzapine. On the molecular level, the observed effects seem to include the known neuritogenic transcription factors FoxO3a and Stat3, since reduced neurite outgrowth caused by the antiserum was accompanied by a reduced phosphorylation of both factors. In contrast, restitution of neurite outgrowth by neuroleptic drugs revealed no correlation to the phosphorylation state of these factors. The present report gives a first hint that bacterial infections could indeed lead to impaired neuropil formation in vitro; however, the in vivo relevance of this finding for schizophrenia pathogenesis remains to be clarified in the future. PMID:24203572

  2. Identification of an EF-Tu protein that is periplasm-associated and processed in Neisseria gonorrhoeae.

    Science.gov (United States)

    Porcella, S F; Belland, R J; Judd, R C

    1996-09-01

    A 44 kDa protein is a dominant component of periplasmic extracts of Neisseria gonorrhoeae. Peptide sequence generated from a cyanogen-bromide-cleaved fragment of this protein indicated sequence homology with elongation factor-Tu (EF-Tu). Polyclonal antiserum was made against the 44 kDa protein purified from periplasm extracts of N. gonorrhoeae. The preabsorbed antiserum was immunoblotted against whole-cell lysates on two-dimensional gels. A 44 kDa protein and a smaller 37 kDa protein were recognized by this antiserum. A N. gonorrhoeae gamma phage DNA library was screened and a clone expressing a 44 kDa protein was identified. The DNA insert in this clone contained several genes homologous to genes contained in the str operon of Escherichia coli. One ORF product with a calculated molecular mass of 43 kDa was highly homologous to the EF-TuA of E. coli. A synthetic peptide antiserum specific for a portion of the C terminus of EF-Tu confirmed that the 37 kDa protein in whole-cell lysates of N. gonorrhoeae was a processed form of EF-Tu. Deletion of the tufA gene homologue in N. gonorrhoeae was attempted but was unsuccessful. PMID:8828215

  3. A potential role for tetranectin in mineralization during osteogenesis

    DEFF Research Database (Denmark)

    Wewer, U M; Ibaraki, K; Schjørring, P; Durkin, M E; Young, M F; Albrechtsen, R

    and immunoprecipitation. Both control PC12 cells and PC12-tet cells injected into nude mice produced tumors containing bone material, as evidenced by von Kossa staining for calcium and immunostaining with bone sialoprotein and alkaline phosphatase antiserum. Nude mice tumors established from PC12-tet...

  4. Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.

    Directory of Open Access Journals (Sweden)

    Wei Cun See Too

    Full Text Available BACKGROUND: Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β. Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor and liver (15-fold lower in tumor samples. CONCLUSION/SIGNIFICANCE: Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.

  5. Effect of IgG subclasses on in vivo bioavailability and metabolic fate of immune-complexed insulin in Lewis rats

    International Nuclear Information System (INIS)

    The bioavailability, distribution, and metabolic fate of 125I-labeled insulin complexed to antibodies in guinea pig antiserum, purified guinea pig IgG1, IgG2, a mixture of IgG1 and IgG2, and homologous Lou/m rat antiserum were studied in inbred Lewis rats. 125I-insulin complexed to purified guinea pig IgG2 antibodies was rapidly cleared from the blood and sequestered in increasing amounts with time in the liver. Large amounts of the 125I-insulin complexed to guinea pig IgG1 antibodies remained in the blood for at least 30 min. The bioavailability of 125I-insulin bound to IgG1 and IgG2 antibodies was inhibited for at least 30 min because significantly less was available for rapid binding to insulin receptors on hepatocytes and renal tubular cells and its subsequent rapid degradation. The bioavailability of 125I-insulin was further decreased when bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies compared with the 125I-insulin complexed to either purified IgG1 or IgG2 antibodies alone. The 125I-insulin bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies was distributed in vivo in a manner reflecting the relative concentrations of the IgG1 and IgG2 antibodies present. The bioavailability, distribution, and metabolic fate of 125I-insulin in immune complexes prepared with homologous Lou/m rat insulin antiserum was qualitatively similar to that observed with immune complexes prepared with guinea pig insulin antiserum. It appears that the Lewis rat can be used as an in vivo model to study the bioavailability,distribution,and metabolic fate of insulin bound to xenogenic or homologous insulin antibodies

  6. The simple methods for radioimmunoassay of unconjugated and sulfate conjugated dehydroepiandrosterone

    International Nuclear Information System (INIS)

    It is well-known that dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the major C19-secretory products of the human adrenal cortex. The measurement of these steroids in plasma may serve as a possibly superior index in assessing biologic function of adrenal. However one of the disadvantages of competitive protein binding assays and radioimmunoassay (RIA), which have usually been used, exists in the need for preliminary extraction or troublesome purification of steroids. Now, RIA method which do not require any extraction or chromatographic purification have been developed. Antiserum I and II were derived from rabbits immunized by 11α-succinoyloxy-DHEA-BSA and DHEA 3-succinate-BSA, respectively. Antiserum I was highly specific to DHEA and was used for determination of DHEA, and its reactivity titer was 1:2,000. Antiserum II was used for DHEA-S assay at dilution of 1:60,000. Comparison experiment on plasma samples were carried out as follows. DHEA in plasma was determined by RIA using antiserum I for samples obtained by the method A and B. A: DHEA was extracted from plasma with ether and purified by chromatography (Sephadex LH-20). B: The sample was obtained by extraction as a similar described above (without chromatography). DHEA-S in plasma was determined using antiserum I (C) and antiserum II (D and E) for samples obtained by the method C, D and E. C: DHEA-S was hydrolyzed by acid and DHEA thus obtained was purified by the method A. D: A saturated ammonium sulfate was added to plasma and DHEA-S was extracted with ethyl acetate. E: Plasma was directly used for RIA. All results were found to be fully reliable. Coefficient of correlation in A-B, C-D and C-E were 0.965, 0.858 and 0.880, respectively. It was found that RIA methods for obtained by method B and E are very useful for clinical application. (auth.)

  7. Plasma levels of vitellogenin in Chrysemys picta during the annual gonadal cycle: Measurement by specific radioimmunoassay

    International Nuclear Information System (INIS)

    A RIA for turtle (Chrysemys picta) vitellogenin is described. After dimethylformamide precipitation of vitellogenin from the plasma of estrogen-treated female turtles, antibodies were developed in rabbits. The dimethylformamide precipitate was further purified by o-triethylaminoethyl cellulose column chromatography; the vitellogenin component eluted as a single peak. This material was used for iodination by a mild chloramine method. Antibodies to turtle vitellogenin did not cross-react with plasma from male turtles or vitellogenic females of other vertebrate groups, including lizards and snakes. Limited cross-reactivity exists among the chelonians, however. Using a 1:5000 dilution of antiserum, the limit of detection was 15 ng, and the midrange was 320 +- 45 ng. For an antiserum dilution of 1:1000, these figures were 30 and 600 +- 37 ng, respectively. Using this assay, the seasonal pattern of plasma vitellogenin in the turtle has been described, and preliminary studies on in vitro hepatic vitellogenesis have been performed

  8. High sensitivity solid phase exchange radioimmunoassay for morphine

    International Nuclear Information System (INIS)

    An immobilized antibody technique is described which has several advantages over other opiate measuring methods. It is exceptionally useful in determining morphine concentrations in biological fluids and tissues. Antiserum was prepared by injection of a morphine-6-succinyl bovine serum albumen suspension into rabbits. Strips of water swollen unsintered polyvinylidene fluoride film were submerged in antiserum solution for 16 hr. The impregnated strips were washed, air-dried and stored. The activity of the immobilized antibody did not change significantly over 3 months. This very firm binding is believed to comprise physical absorption aided by entanglement of the protein in the strands of the polymer. Results of tests of radioimmunoassay capability are given. (author)

  9. Immunological studies on BaH1 and BaP1, two hemorrhagic metalloproteinases from the venom of the snake Bothrops asper.

    Science.gov (United States)

    Rucavado, A; Borkow, G; Ovadia, M; Gutiérrez, J M

    1995-08-01

    No immunological cross-reactivity was observed between BaH1 and BaP1, two hemorrhagic metalloproteinases isolated from B. asper venom, by gel immunodiffusion, Western blotting and neutralization studies. Cross-reactivity was detected with antisera against these toxins in several crotaline and viperine snake venoms by ELISA, whereas no reactivity was observed with either antiserum against the venoms of Bothrops nummifer, Crotalus durissus terrificus, Vipera russelli and several elapid venoms. Antiserum against native BaH1 neutralized hemorrhagic activity of the venoms of B. asper, B. atrox, B. jararaca, Crotalus atrox, C. durissus durissus, Echis carinatus and Trimeresurus flavoviridis, being ineffective against the venoms of Agkistrodon bilineatus and Lachesis muta. PMID:8533144

  10. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y;

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from the...... isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from the...... presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved in...

  11. Production of biological reagents for radioimmunoassay second antibody

    International Nuclear Information System (INIS)

    The experimental production of second antibody to be used in hormonal assays, in which the first antibody is raised in rabbits, is described. Four sheep were immunized with the rabbit immunoglobulin prepared at IPEN-CNEN laboratory. Their antisera were evaluated by the human thyrotropin radioimmunoassay employing materials provided by the National Hormone and Pituitary Program (USA), in comparison with a reference antiserum of known quality, produced in goat by the Radioassay Systems Laboratories - RSL (USA). From the fourth booster injection the animals developed antiserum with titer similar to that exhibited by the commercial product, even presenting higher values. These antisera are now being examinated for the optimal conditions of precipitation before be packed for future use and distribution. (author)

  12. Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture

    DEFF Research Database (Denmark)

    Sandgren, Katarina; Lin, Zhong; Svenningsen, Åsa Fex;

    2003-01-01

    Several motility disorders originate in the enteric nervous system (ENS). Our knowledge of factors governing survival of the ENS is poor. Changes in the expression of vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in enteric neurons occur after neuronal injury and in intestinal...... adaptation. The aim of this study was to evaluate whether VIP and nitric oxide (NO) influence survival of cultured, dissociated myenteric neurons. Neuronal survival was evaluated after 0, 4, and 8 days in culture. Influence of VIP and NO on neuronal survival was examined after culturing in the presence of...... VIP, NO donor, VIP antiserum, or NOS inhibitor. A marked loss of neurons was noted during culturing. VIP and NO significantly promoted neuronal survival. Corroborating this was the finding of an enhanced neuronal cell loss when cultures were grown in the presence of VIP antiserum or NOS inhibitor....

  13. A sensitive radioimmunoassay for fentanyl

    International Nuclear Information System (INIS)

    Antiserum to fentanyl was obtained in rabbits repeatedly injected with carboxyfentanyl conjugated to bovine serum albumin. Using the antiserum, a highly sensitive radioimmunoassay has been developed, based on the dextran-coated charcoal method. It proved possible to assay the drug directly in plasma, in amounts as small as 30 picogram in 0.5 ml. The antibody was highly specific for fentanyl and no cross-reaction was observed with its major metabolites. This sensitive and specific radioimmunoassay method was employed to determine fentanyl in plasma from six volunteers after an intravenous bolus of 0.2 mg, and in plasma from dogs treated both intravenously and subcutaneously with 0.02 mg/kg. The plasma level of fentanyl could be followed for up to 6 h after a therapeutic dose in dogs and man. (orig.)

  14. Radioimmunoassay for nortriptyline (and other tricyclic antidepressants) in plasma

    International Nuclear Information System (INIS)

    The radioimmunoassay for nortriptyline described here can detect as little 1 μg/liter of plasma. Within-day precision and day-to-day precision (CV) were +-6 and +-11%, respectively, over the concentration range 100 to 200 μg/liter. The major metabolite hydroxy-nortriptyline does not cross react with the antiserum. Results so obtained correlate closely with results by a double-isotope derivative dilution technique. The major advantages of this technique over currently available methods are its sensitivity, convenience (many samples can be processed in one day), simplicity, and cost. Further, prior extraction of plasma samples is not required. Cross-reactivity studies have been carried out with all other available tricyclic antidepressants. The antiserum has the ability to bind these drugs, thus radioimmunoassay for all the tricyclic antidepressant drugs can be set up because concurrent use of more than one of these drugs is rare

  15. [Preparation and Identification of High Immunogenic A/PR/8/34 Maternal Strain HA Protein for Influenza Virus Classical Reassortment].

    Science.gov (United States)

    Tang, Jing; Xin, Li; Guo, Junfeng; Zhu, Wenfei; Zhang, Heyuan; Lang, Shaohui; Wang, Dayan; Shu, Yuelong

    2016-03-01

    Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China. PMID:27396155

  16. Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Jianke Ren; Long Wang; Guoxiang Liu; Wen Zhang; Zhejin Sheng; Zhugang Wang; Jian Fei

    2008-01-01

    Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibodypreparation.

  17. Multiple Serotypes of the Moderate Thermophile Thiobacillus caldus, a Limitation of Immunological Assays for Biomining Microorganisms.

    Science.gov (United States)

    Hallberg, K B; Lindstrom, E B

    1996-11-01

    Phylogenetic and phenotypic analysis indicates that a moderately thermophilic isolate, C-SH12, from Australia belongs to the species Thiobacillus caldus. Antiserum generated against whole cells of T. caldus KU recognized protein antigens common to cell lysates of the three T. caldus strains KU, BC13, and C-SH12 but did not recognize whole cells of isolate C-SH12. Differences in the lipopolysaccharide (LPS) of strain C-SH12 and those of the other two T. caldus strains were found, and the anti-KU antiserum did not recognize the LPS from strain C-SH12. These data indicate that this T. caldus isolate belongs to a serotype different from that of strains KU and BC13. PMID:16535449

  18. 3H and 125I radioimmunoassays of haloperidol compared with fluoroimmunoassay involving antibody coupled to magnetizable solid phase

    International Nuclear Information System (INIS)

    Radioimmunoassays for haloperidol are described, involving use of tritium-or 125I-labeled drug or tritium-labeled spiroperidol, and a rabbit antiserum to a drug/bovine serum albumin conjugate. The 125I-labeled drug was prepared by the Chloramine T iodination technique. A fluoroimmunoassay for haloperidol is also described in which the antiserum is coupled to magnetizable solid-phase medium, and fluorescein-labeled haloperidol is used. The assays have acceptable accuracy, precision, and reproducibility, and are specific for haloperidol and similar butyrophenones, with no significant interference from known metabolites and other drugs. Only the radioimmunoassays have sufficient sensitivity to cover the whole range of haloperidol concentrations in serum. The fluoroimmunoassay can be used to monitor high concentrations of haloperidol in 150 μL samples or the complete concentration range of 1-mL serum samples that are extracted and concentrated before assay

  19. 3H and 125I radioimmunoassays of haloperidol compared with fluoroimmunoassay involving antibody coupled to magnetizable solid phase

    International Nuclear Information System (INIS)

    Radioimmunoassays for haloperidol are described, involving use of tritium- or 125I-labeled drug or tritium-labeled spiroperidol, and a rabbit antiserum to a drug/bovine serum albumin conjugate. The 125I-labeled drug was prepared by the Chloramine T iodination technique. A fluoroimmunoassay for haloperidol is also described in which the antiserum is coupled to magnetizable solid-phase medium, and fluorescein-labeled haloperidol is used. The assays have acceptable accuracy, precision, and reproducibility, and are specific for haloperidol and similar butyrophenones, with no significant interference from known metabolites and other drugs. Only the radioimmunoassays have sufficient sensitivity to cover the whole range of haloperidol concentrations in serum. The fluoroimmunoassay can be used to monitor high concentrations of haloperidol in 150-microL samples or the complete concentration range of 1-mL serum samples that are extracted and concentrated before assay

  20. Counterimmunoelectrophoresis in the diagnosis of bacterial meningitis

    DEFF Research Database (Denmark)

    Colding, H; Lind, I

    1977-01-01

    The aim of the present study was to investigate whether counterimmunoelectrophoresis (CIE) would facilitate the rapid, etiological diagnosis of bacterial meningitis when used in parallel with other routine methods in a medical bacteriological laboratory. Of 3,674 consecutive specimens of cerebros......The aim of the present study was to investigate whether counterimmunoelectrophoresis (CIE) would facilitate the rapid, etiological diagnosis of bacterial meningitis when used in parallel with other routine methods in a medical bacteriological laboratory. Of 3,674 consecutive specimens of...... culture-negative specimens. CSF specimens from 21 patients with bacterial meningitis caused by other species were all negative in CIE, except four, three of which contained Escherichia coli antigen reacting with antiserum to N. meningitidis group B and one E. coli antigen reacting with antiserum to H...

  1. Radioimmunoassay (RIA) technique of steroid hormones in the laying hens, Gallus domesticus

    International Nuclear Information System (INIS)

    The principle of radioimmunoassay (RIA) has been applied to many organic compounds of biological interest. In this work, commercially available antisera developed for various steroid hormones were used in the analysis of steroid hormones in the laying hens. The RIA procedure for plasma steroid hormones was divided into three phases: sample preparation, incubation of the antibody-3H-steroid complex with prepared samples and a standard curve and separation of antibody bound 3H-steroid from free 3H-steroid. Results showed that it is possible to use commercially available antiserum source for the determination of steroid hormones in this species. This approach has the advantage of savings in both time and money, by eliminating time losses in screening potential animals producing steroid antiserum and the costs of maintaining these animals

  2. Identification, isolation, and molecular cloning of a hookworm protease: an approach towards a defined vaccine for ancylostomiasis

    Energy Technology Data Exchange (ETDEWEB)

    Hotez, P.J.

    1985-01-01

    The hookworm Ancylostoma caninum was shown to release in vitro a 37 kDa protease that catalyzed the hydrolysis of fibrinogen, plasminogen, and elastin. The enzyme was purified from parasite extracts by ion-exchange chromatography, followed by gel filtration and hydrophobic interaction chromatography. An amino-terminal sequence was determined. When assayed with radiolabeled fibrin as substrate, the enzyme displayed optimal activity at pH 9-11; it was inactivated by dialysis against ethylenediamine tetraacetic acid. Antiserum raised against the protease in rabbits cross-reacted on western blots with soluble antigen from the infective larval stage of the parasite. A cDNA library from hookworm mRNA was constructed in the expression vector bacteriophage lambdagtll. A positive clone was identified with the rabbit antiserum that was shown to contain an 800-bp insert. The insert was mapped, subcloned into M13, and sequenced, revealing an open reading frame of 789 nucleotides corresponding to 263 amino acids.

  3. Methadone radioimmunoassay: two simple methods

    International Nuclear Information System (INIS)

    Two simple and economical radioimmunoassays for methadone in blood or urine are described. Haemolysis, decomposition, common anticoagulants and sodium fluoride do not affect the results. One assay used commercially-available [1-3H](-)-methadone hydrobromide as the label, while the other uses a radioiodinated conjugate of 4-dimethylamino-2,2-diphenylpentanoic acid and L-tyrosine methyl ester. A commercially-available antiserum is used in both assays. Normethadone and α-methadol cross-react to a small extent with the antiserum while methadone metabolites, dextropropoxyphene, dipipanone and phenadoxone have negligible cross-reactivities. The 'cut-offs' of the two assays as described are 30 and 33 ng ml-1 for blood, and 24 and 21 ng ml-1 for urine. The assay using the radioiodinated conjugate can be made more sensitive if required by increasing the specific activity of the label. (author)

  4. Methadone radioimmunoassay: two simple methods

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, K.; Smith, R.N. (Metropolitan Police Forensic Science Laboratory, London (UK))

    1983-09-01

    Two simple and economical radioimmunoassays for methadone in blood or urine are described. Haemolysis, decomposition, common anticoagulants and sodium fluoride do not affect the results. One assay used commercially-available (1-/sup 3/H)(-)-methadone hydrobromide as the label, while the other uses a radioiodinated conjugate of 4-dimethylamino-2,2-diphenylpentanoic acid and L-tyrosine methyl ester. A commercially-available antiserum is used in both assays. Normethadone and ..cap alpha..-methadol cross-react to a small extent with the antiserum while methadone metabolites, dextropropoxyphene, dipipanone and phenadoxone have negligible cross-reactivities. The 'cut-offs' of the two assays as described are 30 and 33 ng ml/sup -1/ for blood, and 24 and 21 ng ml/sup -1/ for urine. The assay using the radioiodinated conjugate can be made more sensitive if required by increasing the specific activity of the label.

  5. Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F StrainVP1 Protein

    Institute of Scientific and Technical Information of China (English)

    Jian-qiang Li; Jun-jie Yang; Xiu-juan Fan; Zhen-peng Sun; Yan Sun; Huan Li; Zi-xin Meng; Wei Li

    2012-01-01

    Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand,foot,and mouth disease (HFMD) in children.Different strains from Gansu were cloned and the P1 protein was sequenced and analysed.Results indicate that there are three kinds of EV71 infections prevalent in Gansu.The VP1 protein from one of these strains,55F,was expressed.The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody,the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay.These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.

  6. A radioimmunoassay for arthropod moulting hormones, introducing a novel method of immunogen coupling

    International Nuclear Information System (INIS)

    Inokosteron-26-oic acid was coupled to thyroglobulin in aqueous pyridine by a watersoluble carbodiimide. After exhaustive dialysis and gel filtration on Sephadex G-25 in the presence of sodium dodecylsulfate, a coupling ratio of 164 haptens per molecule of thyroglobulin was determined. In all three animals injected with the conjugate, ecdysone-binding antibodies were detected. After one booster injection the antiserum could be diluted 1:5000 (1:4000, or 1:2000) in order to get a 50% binding of [3H]ecdysone. The dissociation constant was calculated as 5.8 x 10-10 mol/l. The antiserum has a greater affinity for ecdysone and 22-isoecdysone than for all other ecdysteroids and steroids tested. (orig.)

  7. Studies to optimize radioimmunoassay for progesterone and estradiol (E2) with reference to its application in the ovulatory stimulation with HMG and HCG

    International Nuclear Information System (INIS)

    In this dissertation a quick method for progesterone radioimmunoassay is presented for routine daily use. The high specificity of the antiserum enables the progesterone content to be determined directly from the plasma by this method, without the need for extraction and chromatography. A maximum of 8 hrs. are required for the determination of 10 (max. 15) double samples. 10 μl plasma/sample is needed, the tracer count 5000 cpm, antiserum dilution 1:2000 and the incubation time 1 hr at 210C. 0.5 ml carbon-dextran suspension (20 mg carbon/ml) is required for adsorption of the free hormones. Measurement time for each sample is 2 min. The results achieved by this method are comparable to those obtained by other quick methods. (orig./MG)

  8. The majority of lamina propria CD4(+) T-cells from scid mice with colitis undergo Fas-mediated apoptosis in vivo

    DEFF Research Database (Denmark)

    Bregenholt, S; Petersen, T R; Claesson, Mogens Helweg

    2001-01-01

    . Immunol. 28:3655 (1998)). Here we investigate the apoptosis-inducing mechanism in these lamina propria infiltrating CD4(+) T-cells. We observe that freshly isolated lamina propria CD4(+) T-cells can kill Fas transfected P815 mastocytoma cells in a TCR/CD3 redirected chromium-release assay, but do not......-FasL antiserum for 12 h blocked the apoptotic process in lamina propria CD4(+) T-cells by more than 65% compared to mice treated with control antiserum. Together, these results point towards the Fas-FasL and not the TNF-alpha-TNF-alpha receptor system as the primary apoptosis-inducing mechanism of lamina propria...

  9. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    International Nuclear Information System (INIS)

    To explain the development mechanism of mutation by radiation, DNA polymerase molecules repairing DNA should be identified. In this study, plasmid was constructed in order to express anti sense DNA of DNA polymerase in the cell and it was introduced into the cell by the calcium phosphate method. Polyclonal antibody of DNA polymerase δ and ε were produced so as to prove no existence of specific polymerase molecules in the cell. When center part of polymerase ε was immunized, antiserum with high antibody titer was obtained. Near terminal C of polymerase δ was immunized, then antiserum was obtained. We discovered very interesting fact that base sequence of polymerase ε published by Syvaoja was not correct. (S.Y.)

  10. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    Directory of Open Access Journals (Sweden)

    Akagi,Tadaatsu

    1982-10-01

    Full Text Available Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL line (BALL-1. The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1 and T-ALL (TALL-1 cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or normal B cell line (KO-HL-3, it reacted only with BALL-1 cells and did not react with other leukemia/lymphoma and normal B cell lines. The serum absorbed with tonsillar cells reacted only with BALL-1 and some B cell lines. Thus we were able to obtain antisera with specificity to B cell antigen, B-ALL antigen, and B cell line antigen.

  11. The development of determining human prostatic acid phosphatase by radioimmunoassay

    International Nuclear Information System (INIS)

    We purified human prostatic acid phosphatase (hPAP) from prostatic tissues by affinity chromatography, DEAE cellulose and gel filtration and also examined physicochemical properties of highly purified PAP. We developed a double-antibody radioimmunoassay for hPAP in serum, with use of antiserum raised in rabbit against highly purified PAP. The antiserum did not cross react with acid phosphatase from platelets and red blood cells. Experimental detail are outlined to assess the reproducibility and reliability of the method under various conditions. The upper limit of the serum PAP levels in the present assay was set at 3.0 ng/ml by 162 determinations of samples. The serum PAP levels of 2 untreated patients with prostatic carcinoma were higher than 3.0 ng/ml and 39 patients with benign prostatic hyperplasia were an average value of 1.9 ng/ml. (author)

  12. Development and applications of a radioimmunoassay to human plasma kallikrein

    International Nuclear Information System (INIS)

    A radioimmunoassay to human plasma kallikrein (Hu PK) was developed using specific antibodies against the purified activates enzyme. The antiserum showed identity in the precipitation, double immuno-diffusion, immobility, immuno-electrophoresis, using as normal plasma as purified kallikrein. The protein 125 I-kallikrein was prepared by chloramine-t method and presented homogeneity, stability, immunoactivity and high specific activity, so that a sensitive assay was assured. 193 refs., 19 figs., 4 tabs

  13. Localization of the neuropeptide NGIWYamide in the holothurian nervous system and its effects on muscular contraction

    OpenAIRE

    M. Inoue; Birenheide, R.; Koizumi, O.; Kobayakawa, Y.; Muneoka, Y.; Motokawa, T

    1999-01-01

    NGIWYamide is a peptide recently isolated from the sea cucumber Apostichopus japonicus. It stiffens the connective tissue of the holothurian body wall. Localization of NGIWYamide was investigated by immunohistochemical staining with antiserum raised against NGIWYamide. In holothurian nervous systems NGIWYamide-like immunoreactivity (NGIWYa-LI) was observed in the hyponeural and ectoneural regions of the radial nerve cord, as well as in the circumoral nerve ring, podial nerves, tentacular nerv...

  14. Isolation of coronaviruses antigenically indistinguishable from bovine coronavirus from wild ruminants with diarrhea.

    OpenAIRE

    Tsunemitsu, H; el-Kanawati, Z R; Smith, D R; Reed, H H; Saif, L J

    1995-01-01

    Diarrheal feces from three sambar deer and one waterbuck in a wild animal habitat and one white-tailed deer on a wildlife farm in Ohio contained coronavirus particles which were agglutinated by antiserum to bovine coronavirus (BCV) in immune electron microscopy. Three coronavirus strains were isolated in human rectal tumor cells from the feces of the sambar and white-tailed deer and the waterbuck, respectively. Hemagglutination, receptor-destroying enzyme activity, indirect immunofluorescence...

  15. Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples.

    OpenAIRE

    Sauch, J F

    1985-01-01

    A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other micr...

  16. C4d Presence in Kidney Allograft Biopsy: Sensitivity and Specifity of Immunoperoxidase vs Immunofluorescence

    OpenAIRE

    Viana, H; Carvalho, F.; Santos, A.; Galvão, MJ; Nolasco, F.

    2009-01-01

    OBJECTIVES: Evaluate the sensitivity/specificity of immunoperoxidase method in comparison with the standard immunofluorescence. MATERIAL AND METHODS: Retrospective review of 87 biopsies made for allograft dysfunction. Immunofluorescence (IF) was performed in frozen allograft biopsies using monoclonal antibody anti-C4d from Quidel®. The indirect immunoperoxidase (IP) technique was performed in paraffin-embebbed tissue with polyclonal antiserum from Serotec®. Biopsies were independen...

  17. Appraisal of Antiophidic Potential of Marine Sponges against Bothrops jararaca and Lachesis muta Venom

    OpenAIRE

    Guilherme Muricy; Eládio Flores Sanchez; Suzi Ribeiro; Eduardo Coriolano De Oliveira; Camila Nunes Faioli; Thaisa Francielle Souza Domingos; Andre Lopes Fuly

    2013-01-01

    Snakebites are a health problem in many countries due to the high incidence of such accidents. Antivenom treatment has regularly been used for more than a century, however, this does not neutralize tissue damage and may even increase the severity and morbidity of accidents. Thus, it has been relevant to search for new strategies to improve antiserum therapy, and a variety of molecules from natural sources with antiophidian properties have been reported. In this paper, we analyzed the ability ...

  18. Radioimmunoassay of dehydroepiandrosterone sulfate

    International Nuclear Information System (INIS)

    The development of a radioimmunological method for the measurement of dehydroepiandrosterone sulfate in serum is described. For the immunization of rabbits, a DHA-3-hemissuccinate-bovine serum albumin conjugate was synthetized and a highly specific anti-serum was produced. The method developed requires only simple dilution prior to assay and the normal values for the different age groups were determined in 146 normal individuals. (Author)

  19. Characterization of antigens specific to the surface of germ tubes of Candida albicans by immunofluorescence.

    OpenAIRE

    Sundstrom, P M; Kenny, G. E.

    1984-01-01

    To characterize germ tube-specific antigens of Candida albicans, rabbit antiserum prepared to Formalin-treated yeast possessing germ tubes was adsorbed with stationary-phase blastospores. By immunofluorescence and enzyme-linked immunosorbent assay, this antibody did not react with blastospores but detected germ tube-specific antigens in hyphal forms. Germ tube-specific antigens appeared 30 min after placing blastospores in appropriate conditions for germ tube formation. Hyphae, formed by allo...

  20. Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis.

    OpenAIRE

    van der Meer, J.; Dorssers, L; Zabel, P

    1983-01-01

    We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii...

  1. Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli.

    OpenAIRE

    Berry, A M; Lock, R A; Thomas, S M; Rajan, D P; Hansman, D.; Paton, J C

    1994-01-01

    A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert,...

  2. C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast

    OpenAIRE

    1986-01-01

    Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. O...

  3. Development and validation of a radioimmunoassay for thyrotropin in cattle

    OpenAIRE

    Guyot, Hugues; Sulon, Joseph; Beckers, Jean-François; Closset, Jean; Lebreton, Pascal; Alves de Oliveira, Laurent; Rollin, Frédéric

    2007-01-01

    In mammals, thyrotropin, or thyroid-stimulating hormone (TSH), assay is used for the diagnosis of primary hypothyroidism. Hypothyroidism is the most common type of thyroid disorder in cattle. The aim of this study was to develop and validate, under physiologic and pathologic conditions, a radioimmunoassay (RIA) for bovine TSH (bTSH). Double RIA was performed with purified bTSH and specific bovine antiserum. Laboratory validation included research of minimal detection limit, accuracy, and repr...

  4. Value of passive immune hemolysis for detection of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli.

    OpenAIRE

    Tsukamoto, T; Kinoshita, Y; Taga, S; Takeda, Y; Miwatani, T

    1980-01-01

    The method of passive immune hemolysis of Evans and Evans (Infect. Immun. 16:604-609, 1977) for detection of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli was modified. A total of 373 strains of E. coli were tested by this method using materials obtained by treating the cells with polymyxin B and rabbit antiserum against cholera enterotoxin, purified by affinity gel column coupled with purified cholera enterotoxin, in N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid bu...

  5. Cingulin, a specific protein component of tight junctions, is expressed in normal and neoplastic human epithelial tissues.

    OpenAIRE

    Citi, S.; Amorosi, A; Franconi, F.; Giotti, A.; Zampi, G.

    1991-01-01

    Cingulin is a 140-kd protein localized on the cytoplasmic face of avian tight junctions. The expression of cingulin in human normal and neoplastic colonic tissue has been investigated with an antiserum against chicken cingulin. Human cingulin shares its apparent molecular mass and localization with avian cingulin. In normal colonic epithelium, villous adenomas, and differentiated adenocarcinomas, cingulin staining is observed in the junctional region of the polarized cells lining the surface,...

  6. Glial origin of rapidly adhering amniotic fluid cells.

    OpenAIRE

    Aula, P; von Koskull, H; Teramo, K; Karjalainen, O; Virtanen, I.; Lehto, V P; Dahl, D

    1980-01-01

    Rapidly adhering cells (RA cells) from the amniotic fluid of a pregnancy with fetal anencephaly were investigated by immunofluorescence assay with an antiserum against glial cells. After 24 hours' cultivation a high proportion of the cells showed positive glial-specific fluorescence, whereas no staining was seen in cells from samples of normal amniotic fluid. At the 24th week the mother was delivered of a stillborn infant with anencephaly. Immunofluorescence staining of RA cells with glial-sp...

  7. Serological differentiation of foot-and-mouth disease virus on electron microscope grids coated with protein A and antibody.

    OpenAIRE

    Polatnick, J; Wool, S

    1981-01-01

    A serological technique using electron microscope grids coated with protein A and antiserum was able to detect foot-and- mouth disease virus particles in oesophageal-pharyngeal fluids from infected cattle without the need for prior concentration of the sample. The technique was adapted to differentiate serologically among foot-and-mouth disease virus types A, O and C with antigen-adsorbed sera. When grids were coated with heterotypic antigenadsorbed antisera, the homotypic antigen could be ob...

  8. Wood Degradation by White Rot Fungi: Cytochemical Studies Using Lignin Peroxidase-Immunoglobulin-Gold Complexes

    OpenAIRE

    Garcia, Susana; Latge, Jean Paul; Prevost, Marie Christine; Leisola, Matti

    1987-01-01

    Using an anti-lignin peroxidase antiserum-protein A-gold complex, we found lignin peroxidase mainly intracellularly in several white rot fungi colonizing sawdust under laboratory conditions. This enzyme was also present in fungi found in naturally decayed wood. However, in all cases, lignin peroxidase was located mainly inside the fungal cells. Labeled lignin peroxidase did not bind to the lignocellulosic samples tested, with the exception of poplar milled-wood lignin. These results are discu...

  9. Indiscriminate recombination in simian virus 40-infected monkey cells.

    OpenAIRE

    Winocour, E; Keshet, I

    1980-01-01

    DNA transfection of African green monkey BSC-1 cells with simian virus 40 (SV40) DNA and bacterial virus phi X174 replicative form DNA ("cotransfection") yielded stocks containing SV40/phi X174 recombinant virus, which was detected by an infectious-center in situ plaque hybridization procedure and which was sensitive to anti-SV40 antiserum. The recombinant virus replicated during serial passage. Restriction endonuclease cleavage of the SV40/phi X174 DNA indicated that several different types ...

  10. MEDICINAL PLANTS ACTIVE AGAINST SNAKE ENVENOMATION

    OpenAIRE

    Kanojia Anita; Chaudhari Kishor Shivaji; Gothecha Vinod Kumar

    2012-01-01

    Snakebite is an important cause of morbidity and mortality and is one of the major health problems in India. About 30000 to 40,000 persons die each year from venomous snake bite. Russell’s viper or daboia (Viper russelli) appears to be the commonest cause of fatal snakebite in Southern India, Pakistan, Bangladesh, Sri Lanka, Burma and Thailand. Intravenous administration of anti-snake venom neutralizes the systemic actions, however, antiserum does not provide enough protection against venom i...

  11. Updates on tetanus toxin: a fundamental approach

    Directory of Open Access Journals (Sweden)

    Md. Ahaduzzaman

    2015-03-01

    Full Text Available Clostridium tetani is an anaerobic bacterium that produces second most poisonous protein toxins than any other bacteria. Tetanus in animals is sporadic in nature but difficult to combat even by using antibiotics and antiserum. It is crucial to understand the fundamental mechanisms and signals that control toxin production for advance research and medicinal uses. This review was intended for better understanding the basic patho-physiology of tetanus and neurotoxins (TeNT among the audience of related field.

  12. Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1.

    OpenAIRE

    Valvano, M A; Crosa, J H

    1989-01-01

    We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material whi...

  13. Detection of two growth hormone receptor mRNAs and primary translation products in the mouse

    OpenAIRE

    Smith, W.C.; Linzer, D I; Talamantes, F

    1989-01-01

    Two mouse growth hormone-receptor primary translation products of Mr 95,900 and 31,800 were identified from in vitro-translated late pregnant mouse liver mRNA. RNA isolated from mouse liver was translated in a rabbit reticulocyte lysate system containing [35S]methionine, and the growth hormone receptor primary translation products were identified by immunoprecipitation with anti-mouse growth hormone receptor antiserum followed by sodium dodecyl sulfate/PAGE and fluorography. Detectable amount...

  14. Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications†,‡

    OpenAIRE

    Cao, Heping

    2004-01-01

    Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding act...

  15. A distinct tospovirus causing necrotic straek on alstroemeria sp. in Colombia

    OpenAIRE

    Mehraban, A.; Botermans, M.; Verhoeven, J.Th.J.; Meekes, E.; Saaijer, J.; Peters, D.; Goldbach, R.W.; Kormelink, R.J.M.

    2010-01-01

    A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5' untranslated region and a part of the intergenic region...

  16. Identification and characterization of potyviruses of Alstroemeria spp

    OpenAIRE

    Bouwen, L.; Vlugt, van der, R.A.A.

    1996-01-01

    Several viruses have been reported to infect Alstroemeria. The two most important ones are considered to be Alstroemeria mosaic (AIMV) and Alstroemeria streak virus (AISV). Both AIMV and AISV react in ELISA with a commonly used antiserum to AIMV. There are indications that potyviruses other than AIMV and AISV also occur in Alstroemeria. The present study was initiated to identify and characterise these potyviruses. A strategy was developed that is aimed at separation of these viruses and thei...

  17. A distinct tospovirus causing necrotic streak on Alstroemeria sp. in Colombia

    OpenAIRE

    Hassani-Mehraban, Afshin; Botermans, Marleen; Verhoeven, J.Th.J.; Meekes, Ellis; Saaijer, Janneke; Peters, Dick; Goldbach, Rob; Kormelink, Richard

    2010-01-01

    A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5′ untranslated region and a part of the intergenic region...

  18. Production and characterization of recombinantly derived peptides and antibodies for accurate determinations of somatolactin, growth hormone and insulin-like growth factor-I in European sea bass (Dicentrarchus labrax).

    Science.gov (United States)

    de Celis, S Vega-Rubín; Gómez-Requeni, P; Pérez-Sánchez, J

    2004-12-01

    A specific radioimmunoassay (RIA) for European sea bass (Dicentrarchus labrax) growth hormone (GH) was developed and validated. For this purpose, a stable source of GH was produced by means of recombinant DNA technology in a bacteria system. The identity of the purified protein (ion exchange chromatography) was demonstrated by Western blot and a specific GH antiserum was raised in rabbit. In Western blot and RIA system, this antiserum recognized specifically native and recombinant GH, and it did not cross-react with fish prolactin (PRL) and somatolactin (SL). In a similar way, a specific polyclonal antiserum against the now available recombinant European sea bass SL was raised and used in the RIA system to a sensitivity of 0.3 ng/ml (90% of binding of tracer). Further, European sea bass insulin-like growth factor-I (IGF-I) was cloned and sequenced, and its high degree of identity with IGF-I peptides of barramundi, tuna, and sparid fish allowed the use of a commercial IGF-I RIA based on barramundi IGF-I antiserum. These assay tools assisted for the first time accurate determinations of SL and GH-IGF-I axis activity in a fish species of the Moronidae family. Data values were compared to those found with gilthead sea bream (Sparus aurata), which is currently used as a Mediterranean fish model for growth endocrinology studies. As a characteristic feature, the average concentration year round of circulating GH in growing mature males of European sea bass was higher than in gilthead sea bream. By contrast, the average concentration of circulating SL was lower. Concerning to circulating concentration of IGF-I, the measured plasma values for a given growth rate were also lower in European sea bass. These findings are discussed on the basis of a different energy status that might allowed a reduced but more continuous growth in European sea bass. PMID:15560873

  19. Radioimmunoassay for the determination of low insulin levels

    International Nuclear Information System (INIS)

    The assay system was set up in such a way as to increase the sensitivity of the reaction, reducing the tracer concentration from the usual 1.000 cpm/ml to 500 cpm/ml levels of radioactivity (specific activity aproximately 200 mCi/mg) and/or increasing the antiserum dilution by one thirty of the usual values in the assay of the final volume of incubation of 2,5 ml. (author)

  20. Reduction of 3-mercaptopyruvate in rat liver is catalyzed by lactate dehydrogenase.

    OpenAIRE

    Ohta,Jun; Ubuka,Toshihiko

    1989-01-01

    It has been assumed that the in vivo reduction of 3-mercaptopyruvate, an intermediate of cysteine metabolism, to 3-mercaptolactate is catalyzed by lactate dehydrogenase (EC 1.1.1.27) though no definitive evidence has been presented. In order to examine this assumption, reduction of 3-mercaptopyruvate and its inhibition were studied using rat liver homogenate, lactate dehydrogenase purified from rat liver and anti-lactate dehydrogenase antiserum. Reduction of 3-mercaptopyruvate was actively ca...

  1. Comparative study of the second antibody for radioimmunoassay totally produced in the country to a similar imported one (sheep serum anti-rabbit IgG)

    International Nuclear Information System (INIS)

    This work compares a second antibody for radioimmunoassay (RIA) produced at IPEN-CNEN/SP with a commercial one of known quality, produced by Radioassay Systems Laboratories, U.S.A.. This antiserum, sheep serum anti-rabbit IgG produced in its totality in the country, presented title and precipitation characteristics similar to those exhibited by the commercial product, being as suitable for the RIA separation as its imported similar. (author)

  2. Close Vicinity of PrP Expressing Cells (FDC) with Noradrenergic Fibers in Healthy Sheep Spleen

    OpenAIRE

    Bencsik, A.; Lezmi, S.; Hunsmann, G; Baron, T.

    2001-01-01

    In naturally and experimentally occurring scrapie in sheep, prions invade the immune system and replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleens of 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentially supporting prion replication, as well as their relationship with autonomic innervation. PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directed against AA 108–123 portion of the bovine...

  3. Antibody against Porcine Parvovirus VP2 Protein Differentiated This Virus from Other Viruses

    OpenAIRE

    Weijuan Zhu§, Yudong Ren§1, Guangxing Li, Dingding Su2, Qing Yang3 and Xiaofeng Ren*

    2012-01-01

    Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and responsible for eliciting neutralizing antibodies in immunized animals. In this study, the gene encoding VP2 of PPV was amplified by PCR. The VP2 gene was then cloned into the prokaryotic expression vector, pET-32a followed by expression in Escherichia coli Rosetta. The VP2 protein expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit polyclonal antiserum was gene...

  4. Development of porcine rotavirus vp6 protein based ELISA for differentiation of this virus and other viruses

    OpenAIRE

    ZHU, JIAYI; Yang, Qing; Cao, Liyan; Dou, Xiujing; Zhao, Jianguo; Zhu, Weijuan; Ding, Fan; Bu, Ri-e; Suo, Siqingaowa; Ren, Yudong; Li, Guangxing; Ren, Xiaofeng

    2013-01-01

    Background The context and purpose of the study included 1) bacterial expression of viral protein 6 (VP6) of porcine rotavirus (PRV) and generation of rabbit polyclonal antiserum to the VP6 protein; 3) establishment of a discrimination ELISA to distinguish PRV from a panel of other porcine viruses. Results The VP6 gene of PRV isolate DN30209 amplified by reverse transcription-PCR was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. Sequence comparison and phylo...

  5. Immunohistochemical localization of granzyme B antigen in cytotoxic cells in human tissues.

    OpenAIRE

    Hameed, A.; Truong, L D; Price, V; Kruhenbuhl, O.; Tschopp, J

    1991-01-01

    Human granzyme B antigen is expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme B was generated using a prokaryotic expression vector under the control of T7 transcription and translation signals. The 25-kd recombinant protein (granzyme B) was used to develop a rabbit polyclonal antiserum. Purified anti-granzyme B antibodies were used to detect the antigen expression in cytotoxic cells in human tissues. Using the avidin-biotin-...

  6. Effects of Static Magnetic Field on Growth of Leptospire, Leptospira interrogans serovar canicola: Immunoreactivity and Cell Division

    OpenAIRE

    Triampo, Wannapong; Doungchawee, Galayanee; Triampo, Darapond; Wong-Ekkabut, Jirasak; Tang, I-Ming

    2004-01-01

    The effects of the exposure of the bacterium, Leptospira interrogans serovar canicola to a constant magnetic field with magnetic flux density from a permanent ferrite magnet = 140 mT were studied. Changes in Leptospira cells after their exposure to the field were determined on the basis of changes in their growth behavior and agglutination immunoreactivity with a homologous antiserum using darkfield microscopy together with visual imaging. The data showed that the exposed Leptospira cells hav...

  7. Brain-derived neurotrophic factor and cocaine addiction

    OpenAIRE

    McGinty, Jacqueline F.; Whitfield, Timothy W.; Berglind, William J.

    2009-01-01

    The effects of brain-derived neurotrophic factor (BDNF) on cocaine-seeking are brain region-specific. Infusion of BDNF into subcortical structures, like the nucleus accumbens and ventral tegmental area, enhances cocaine-induced behavioral sensitization and cocaine seeking. Conversely, repeated administration of BDNF antiserum into the nucleus accumbens during chronic cocaine self-administration attenuates cocaine-induced reinstatement. In contrast, BDNF infusion into the dorsomedial prefronta...

  8. Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 beta-converting enzyme.

    OpenAIRE

    Petit, F.; Bertagnoli, S.; Gelfi, J; Fassy, F; Boucraut-Baralon, C; MILON, A.

    1996-01-01

    A genomic library of myxoma virus (MV) DNA, a leporipoxvirus that causes myxomatosis, was constructed and screened by in vitro transcription-translation. A clone was selected on the basis of its strong reactivity with MV antiserum. Analysis of the corresponding DNA sequence and the deduced amino acid sequence revealed an open reading frame coding for a 34-kDa protein with strong homologies to members of the serpin superfamily. The gene encoding this new protein, called serp2, was localized on...

  9. Expression of Two Members of the pMGA Gene Family of Mycoplasma gallisepticum Oscillates and Is Influenced by pMGA-Specific Antibodies

    OpenAIRE

    Markham, Philip F.; Glew, Michelle D.; Browning, Glenn F.; Whithear, Kevin G.; Ian D. Walker

    1998-01-01

    Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the express...

  10. Studies on the expression of the microtubule-associated protein, tau, during mouse brain development, with newly isolated complementary DNA probes

    OpenAIRE

    1984-01-01

    Tau protein is a collection of closely related polypeptides that associate with microtubules in vivo and stimulate their assembly in vitro. Using an affinity-purified antiserum against bovine brain tau protein, we found that the number and amount of tau polypeptides changes dramatically during mouse brain development. The different forms appear to result from changes in tau mRNA since in vitro translation products reflect the qualitative and quantitative changes found in vivo. To study the mR...

  11. A novel approach for preparation of the antisera reagent for potency determination of inactivated H7N9 influenza vaccines

    OpenAIRE

    Schmeisser, Falko; Jing, Xianghong; Joshi, Manju; Vasudevan, Anupama; Soto, Jackeline; Li, Xing; Choudhary, Anil; Baichoo, Noel; Resnick, Josephine; Ye, Zhiping; McCormick, William; Weir, Jerry P.

    2016-01-01

    Background The potency of inactivated influenza vaccines is determined using a single‐radial immunodiffusion (SRID) assay and requires standardized reagents consisting of a Reference Antigen and an influenza strain‐specific antiserum. Timely availability of reagents is a critical step in influenza vaccine production, and the need for backup approaches for reagent preparation is an important component of pandemic preparedness. Objectives When novel H7N9 viruses emerged in China in 2013, candid...

  12. Assessment of multiple types of DNA damage in human placentas from smoking and nonsmoking women in the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Pratt, M. M.; King, L. C.; Adams, L. D.; John, K.; Sirajuddin, P.; Olivero, O. A.; Manchester, D. K.; Šrám, Radim; DeMarini, D. M.; Poirier, M. C.

    2011-01-01

    Roč. 52, č. 1 (2011), s. 58-68. ISSN 0893-6692 R&D Projects: GA MŽP(CZ) SP/1B3/8/08 Institutional research plan: CEZ:AV0Z50390512 Keywords : automated cellular imaging system * immunohistochemistry * BPDE-DNA antiserum Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.709, year: 2011

  13. Effects of aspirin, prednisolone and indomethacin on nephrotoxic serum nephritis in the rat.

    OpenAIRE

    Kurokawa, H; Sakamoto, K.

    1982-01-01

    1 The effects of aspirin, prednisolone, and indomethacin on nephrotoxic serum nephritis in rats was studied. The nephritis was induced by a single intravenous injection of nephrotoxic serum (NTS, rabbit anti-serum against the water-soluble renal antigen of the rat). The injection of NTS induced the heterologous phase of proteinuria (within a day after NTS injection) and then the autologous phase (5 to 7 days after NTS injection). The effect of drugs given before the NTS (i.e. prophylactically...

  14. Dopamine- and Tyrosine Hydroxylase-Immunoreactive Neurons in the Brain of the American Cockroach, Periplaneta americana

    Science.gov (United States)

    Hamanaka, Yoshitaka; Minoura, Run; Nishino, Hiroshi; Miura, Toru; Mizunami, Makoto

    2016-01-01

    The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine’s actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach, Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase (TH, an enzyme required for dopamine synthesis), and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of “dopaminergic” neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory

  15. Promotion of remyelination by polyclonal immunoglobulin in Theiler's virus-induced demyelination and in multiple sclerosis.

    OpenAIRE

    van Engelen, B. G.; Miller, D.J.; Pavelko, K. D.; Hommes, O R; Rodriguez, M.

    1994-01-01

    Spontaneous remyelination occurs in experimental models of demyelination and in patients with multiple sclerosis, although to a limited extent. This enables the search for factors that promote remyelination. Using the Theiler's virus model of central nervous system demyelination, promotion of remyelination was observed after passive transfer of CNS-specific antiserum and transfer of CNS-specific purified immunoglobulin. Mouse polyclonal immunoglobulin from normal non-syngeneic mice, comparabl...

  16. Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

    Institute of Scientific and Technical Information of China (English)

    罗依惠; 严杰; 毛亚飞; 李淑萍

    2004-01-01

    Objective: To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups. The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence, and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972). OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results: All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa. A positive protein fragment with approximately 32 KDa confirmed by Western blot, was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira. Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

  17. Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

    Institute of Scientific and Technical Information of China (English)

    罗依惠; 严杰; 毛亚飞; 李淑萍

    2004-01-01

    Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

  18. Modification of the lipid moiety of the enterobacterial common antigen by the "Pseudomonas factor".

    OpenAIRE

    Kuhn, H M; Neter, E; Mayer, H

    1983-01-01

    Pseudomonas aeruginosa produces a factor (PF) which affects the enterobacterial common antigen (ECA); resulting in failure of the antigen to modify erythrocytes for hemagglutination by ECA antibodies. In the present study the nature of PF was determined. Pronase treatment abolished its activity, indicating the protein nature of PF. PF-treated ECA no longer coated erythrocytes but still reacted with ECA antibodies in immunoelectrophoresis tests with monospecific antiserum to ECA, although diff...

  19. Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection

    OpenAIRE

    Mehdy, Mona C.; Lamb, Christopher J.

    1987-01-01

    The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene encoding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A λgt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 105 recombinants with an antiserum to purified bean CHI. ...

  20. Detection of p53 overexpression in routinely paraffin-embedded tissue of human carcinomas using a novel target unmasking fluid.

    OpenAIRE

    van den Berg, F M; Baas, I O; Polak, M. M.; Offerhaus, G. J.

    1993-01-01

    With the aid of a newly developed target unmasking fluid (TUF), p53 overexpression was visualized by immunohistochemistry on recent and archival paraffin-embedded tissue samples of colon, stomach, and pancreas neoplasms. Using monoclonal anti-p53 antibody pAb1801 as well as polyclonal antiserum to p53 CM1, TUF-mediated immunohistochemistry was fully concordant for p53 overexpression in paraffin-embedded carcinoma samples compared with freshly frozen tissue from the same tumors. Thus, prognost...

  1. Ultrastructural and molecular confirmation of the development of Sarcocystis neurona tissue cysts in the central nervous system of southern sea otters (Enhydra lutris nereis)

    OpenAIRE

    Miller, M.A.; Barr, B C; Nordhausen, R.; James, E R; Magargal, S.L.; Murray, M.; Conrad, P A; Toy-Choutka, S.; Jessup, D.A.; Grigg, M. E.

    2009-01-01

    In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona-associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts from sea otter 1 (SO1) stained positively using anti-S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two imm...

  2. Antibody-mediated neutralization of virus is abrogated by mycoplasma.

    OpenAIRE

    Dickson, C; Elkington, J; Hales, A.; Weiss, R.

    1980-01-01

    The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasm...

  3. Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

    OpenAIRE

    Ossewaarde, J M; de Vries, A; Bestebroer, T; Angulo, A F

    1996-01-01

    Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by th...

  4. Identification of three coated vesicle components as alpha- and beta- tubulin linked to a phosphorylated 50,000-dalton polypeptide

    OpenAIRE

    1983-01-01

    Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precip...

  5. Characterization of Rabbit Polyclonal Sera against Recombinant Shiga Toxin and its Subunits for Detection of Stx-Producing E. coli

    OpenAIRE

    Mana Oloomi; Saeid Bouzari

    2011-01-01

    Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera...

  6. Cell-free translation of bovine viral diarrhea virus RNA.

    OpenAIRE

    Purchio, A F; Larson, R.; Torborg, L L; Collett, M S

    1984-01-01

    Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to...

  7. Regulation of murine macrophage Ia-antigen expression by products of activated spleen cells

    OpenAIRE

    1980-01-01

    This investigation examined the effects of mediators derived form activated spleen cells on macrophage Ia-antigen expression and function. Incubation of adherent thioglycollate-induced murine peritoneal macrophages(> 90% Ia-) with concanavalin A (Con A)- stimulated spleen cell supernate (Con A sup) resulted in a dose- dependent increase in the percentage of Ia-containing (Ia+) phagocytic cells, as detected by antiserum-and-complement-mediated cytotoxicity. The Ia-antigen expression of macroph...

  8. Detection of Rickettsia rickettsii antibodies in human sera by crossed immunoelectrophoresis.

    OpenAIRE

    Anacker, R. L.; Philip, R N; Wilfert, C M; Kleeman, K T; Turner, L; MacCormack, J. N.; Hechemy, K E

    1983-01-01

    To identify Rickettsia rickettsii antigens of immunological importance, we examined sera from patients with serologically confirmed cases of Rocky Mountain spotted fever by crossed immunoelectrophoresis for antibodies to antigens extracted from the R strain of R. rickettsii with the detergent Triton X-100. Sixteen antigens were identified in the detergent extract by crossed immunoelectrophoresis with a hyperimmune rabbit serum raised against whole rickettsiae. When the rabbit antiserum was pl...

  9. Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins.

    OpenAIRE

    Konkel, M E; Mead, D J; Cieplak, W

    1996-01-01

    A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequenc...

  10. Entwicklung eines Biosensors zum Nachweis von Antibiotika und Sulfonamiden in Milch- Herstellung der immunchemischen Komponenten

    OpenAIRE

    Strasser, Angelika

    2003-01-01

    This paper describes the development and application of enzyme immunoassays for the detection of antimicrobials in milk, aiming at the establishment of a biosensor system for the on-line analysis of drug residues in milk. For the development of group-specific antibodies against penicillins rabbits were immunized with an ampicillin-BSA-conjugate. The resulting antiserum was employed for the development of a direct competitive enzyme immunoassay (EIA), giving a detection limit of 1 ng/ml f...

  11. Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera : a study of paraspecificity

    OpenAIRE

    Archundia, I. G.; de Roodt, A. R.; Ramos-Cerrillo, B.; Chippaux, Jean-Philippe; Olguin-Perez, L.; Alagon, A.; Stock, R. P.

    2011-01-01

    We conducted an extensive study of neutralization of lethality of 11 species and one subspecies of snakes of the genus Vipera, and of five species of Macrovipera, by two experimental equine antisera. One antiserum was a trivalent preparation raised against the venoms of Vipera aspis aspis, Vipera berus berus and Vipera ammodytes ammodytes; the other was a pentavalent preparation that also included venoms of Vipera (now Montivipera) xanthina and Macrovipera lebetina obtusa. We measured specifi...

  12. Schistosoma mansoni circulating polysaccharide and protein antigens recognized by sheep antisera in patients with different clinical forms of schistosomiasis before and after treatment Antígenos polissacarídicos e proteicos circulantes de Schistosoma mansoni reconhecidos por antissoros de carneiro em pacientes com diferentes formas clínicas da esquistossomose antes e após o tratamento

    OpenAIRE

    Julia Maria Costa-Cruz; Sumie Hoshino-Shimizu; Antonio Walter Ferreira; Mario E. Camargo; Thales de Brito; Luiz Caetano da Silva

    1994-01-01

    Two sheep antisera, one of which raised against polysaccharide (Po) and other against protein (Pt) components of Schistosoma mansoni adult worms, were assessed by ELISA for their ability to detect circulating parasite antigens in patients with different clinical forms of chronic schistosomiasis mansoni. The former antiserum detected parasite antigens in liver granulomata and the latter in renal glomeruli from schistosomiasis patients and mice experimentally infected with S. mansoni. In genera...

  13. Lymphoid leukosis: detection of group specific viral antigen in chicken spleens by immunofluorescence and complement fixation.

    OpenAIRE

    Spencer, J.L.; Gilka, F

    1982-01-01

    Monospecific antiserum obtained from rabbits hyperimmunized against homogeneous p27 group specific protein purified from avian myeloblastosis virus was commercially procured and was then conjugated with fluorescein isothiocyanate. The conjugate was applied to spleens from naturally or experimentally infected chickens that had no evidence of lymphoid tumors. Fluorescence was usually localized in connective tissue of sheathed capillaries giving it a ring-like appearance. Sites of fluorescence c...

  14. Mechanism of interferon action: Simian virus 40-specific early polypeptides synthesized in untreated and interferon-treated monkey kidney cells

    OpenAIRE

    Kingsman, Susan M.; Smith, Mark D; Samuel, Charles E.

    1980-01-01

    The effect of interferon treatment on proteins synthesized in simian virus 40 (SV40)-infected cells in the presence of cytosine arabinoside was investigated. The following results were obtained: (i) In addition to previously described large tumor (T) antigen (94 kilodaltons) and small tumor (t) antigen (19 kilodaltons), a 62-kilodalton polypeptide was immunoprecipitated by SV40 anti-T antiserum from extracts of infected CV-1 and BSC-1 monkey kidney cells and transformed SV3T3 mouse cells. The...

  15. [Viper envenomation by Echis coloratus].

    Science.gov (United States)

    Gilon, D; Mann, G; Shalev, O

    1991-06-01

    Clinical and therapeutic experience with 24 cases of envenomation by Echis coloratus, the Mideast saw-scaled viper, is reported. These cases were seen between 1979-1989 at this hospital (Mt. Scopus). A clinical classification is proposed, based on severity of the bleeding diathesis and platelet count at presentation. It is suggested as a predictor of clinical outcome and as a guide to whether antiserum should be administered. PMID:1937209

  16. Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin.

    OpenAIRE

    Tweten, R K; Collier, R J

    1983-01-01

    Two restriction fragments from corynebacteriophage beta vir tox+ that encode peptides similar to diphtheria toxin fragment A and the chain termination fragment, CRM45, have been cloned into Escherichia coli in plasmid pBR322. Clones containing the recombinant plasmids produced gene products that were active in catalyzing the ADP ribosylation of elongation factor 2 and were reactive with diphtheria toxin antiserum. Toxin-related peptides were found primarily in the periplasmic compartment and ...

  17. Characterization of Rabbit Polyclonal Sera Against Recombinant Shiga Toxin and its Subunits for Detection of Stx-Producing E. Coli

    OpenAIRE

    Mana Oloomi; Saeid Bouzari

    2011-01-01

    Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera...

  18. cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.

    OpenAIRE

    Myerowitz, R; Proia, R L

    1984-01-01

    We have isolated a cDNA clone containing sequences complementary to mRNA encoding the alpha-chain of the lysosomal enzyme beta-hexosaminidase. RNA from a human lung fibroblast strain, IMR90, was enriched for beta-hexosaminidase messenger by polysome immunoselection with antiserum against beta-hexosaminidase A. This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure. After transformation of Escherichia coli, 385 ampicillin-resistant colonies...

  19. Characterization of antibody-mediated inhibition of Pseudomonas aeruginosa adhesion to epithelial cells.

    OpenAIRE

    Sexton, M; Reen, D J

    1992-01-01

    An enzyme-linked immunosorbent assay system was developed and used to study adhesion of Pseudomonas aeruginosa to human epithelial cells and the abilities of specific antibodies to inhibit this process. Human buccal epithelial cells coated onto microtiter plates were incubated with P. aeruginosa suspensions, and adherent bacteria were detected by using anti-P. aeruginosa serum and a horseradish peroxidase-conjugated secondary antiserum. Adhesion, quantitated as an increase in A405, varied lin...

  20. Molecular properties of succinate dehydrogenase isolated from Micrococcus luteus (lysodeikticus).

    OpenAIRE

    Crowe, B A; Owen, P.(Imperial College London, London, United Kingdom)

    1983-01-01

    Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectively precipitated from Triton X-100-solubilized membranes by using specific antiserum. The precipitated enzyme contained equimolar amounts of four polypeptides with apparent molecular weights of 72,000, 30,000, 17,000, and 15,000. The 72,000 polypeptide possessed a covalently bound flavin prosthetic group and appeared to be strongly antigenic as judged by immunoprinting experiments. Low-temperature absorption spectroscopy ...

  1. Hyaluronidase, phospholipase A2 and protease inhibitory activity of plants used in traditional treatment of snakebite-induced tissue necrosis in Mali, DR Congo and South Africa

    DEFF Research Database (Denmark)

    Schmidt, Marianne Molander; Nielsen, Line Hagner; Søgaard, Søren Vinter;

    2014-01-01

    ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation, every year, causes estimated 5-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone. Antiserum is not easily accessible in these regions or doctors are simply not available, thus more than 80% of all p...... patients seek traditional practitioners as first-choice. Therefore it is important to investigate whether the plants used in traditional medicine systems contain compounds against the necrosis-inducing enzymes of snake venom....

  2. Ultrastructural immunocytochemical localization of renin and angiotensin II in the juxtaglomerular cells of the ischemic kidney in experimental renal hypertension.

    OpenAIRE

    Cantin, M; J. Gutkowska; Lacasse, J.; Ballak, M.; Ledoux, S.; Inagami, T; Beuzeron, J.; Genest, J.

    1984-01-01

    Partial ligation of the rat aorta between the renal arteries induces acute hypertension with atrophy of the left (ischemic) kidney, intense stimulation of juxtaglomerular cell (JGC) secretory activity, and significant increases in renal cortical renin activity, in plasma renin activity, and in the plasma levels of angiotensin I (AI) and angiotensin II (AII). With the unlabeled antibody technique at the light-microscopic level and various dilutions of renin antiserum, immunoreactive renin can ...

  3. Antigenic cross-reactivity between Treponema pallidum and other pathogenic members of the family Spirochaetaceae.

    OpenAIRE

    Baker-Zander, S A; Lukehart, S A

    1984-01-01

    The antigenic cross-reactivity between Treponema pallidum and several pathogenic members of the family Spirochaetaceae was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Blots of T. pallidum antigens were incubated with antiserum from rabbits infected or immunized with T. pallidum, Treponema paraluiscuniculi, Treponema hyodysenteriae (strains B204 and T22), Borrelia hermsii serotype 7, or Leptopsira interrogans serogroup Canicola. T. pal...

  4. Immunohistochemical identification of type I procollagen in tumour cells of scirrhous adenocarcinoma of the stomach.

    OpenAIRE

    Niitsu, Y; Ito, N.; Kohda, K; Owada, M.; Morita, K.; Sato, S.; Watanabe, N.; Kohgo, Y; Urushizaki, I.

    1988-01-01

    Human gastric carcinomas were tested for their immunohistochemical reactivity with anti-type I procollagen antiserum. In all specimens of scirrhous carcinomas, staining of the tumour cells was strongly positive, while in medullary carcinomas staining of the tumour cells was generally poor. These results suggest that the tumour cells in scirrhous carcinomas produce collagen in their stroma. Images Figure 1 Figure 4 Figure 3 Figure 5

  5. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells

    OpenAIRE

    1982-01-01

    Immunofluorescence staining of cultured human umbilical vein endothelial cells has shown the presence of von Willebrand protein in the perinuclear region, in small rodlike structures through the cytoplasm, and on filaments of the extracellular matrix. Nonendothelial cells showed no staining with anti-von Willebrand protein antiserum. At the light microscope level, immunoperoxidase treatment of endothelial cells revealed the same pattern and antibody specificity as the fluorescence staining. T...

  6. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    Science.gov (United States)

    Hoehn, G T; Clark, V L

    1990-12-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. PMID:2123827

  7. Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

    Science.gov (United States)

    Martel, N; Lee, B C

    1994-02-01

    Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1. PMID:8300227

  8. Flow Cytometric Analysis of Microsporidia Belonging to the Genus Encephalitozoon

    OpenAIRE

    Moss, Delynn M.; Croppo, Gian P.; Wallace, Sara; Visvesvara, Govinda S.

    1999-01-01

    Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluores...

  9. Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding

    OpenAIRE

    Chan, June; AOKI, CHIYE; Pickel, Virginia M.

    1990-01-01

    The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5–20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization o...

  10. Production of progesterone antibodies and their use in studying reproductive functions in sheep and goats

    International Nuclear Information System (INIS)

    The characteristics of antisera raised in six rabbits by immunization with progesterone-3-0-carboxymethyloxime-BSA are described. The performance of a progesterone RIA involving the use of the best antiserum is described. This progesterone RIA, carried out in sheep and goat plasma samples collected throughout the different stages of the reproductive life cycle turned out to be a reliable method to monitor ovarian activity. (author). 8 refs, 1 fig., 3 tabs

  11. Expression of the structural gene, laf1, encoding the flagellin of the lateral flagella in Azospirillum brasilense Sp7.

    OpenAIRE

    Moens, S.; M. Schloter; Vanderleyden, J.

    1996-01-01

    The induction of the lateral flagella of Azospirillum brasilense Sp7 was studied by using a translational fusion between the laf1 promoter and gusA. The fusion was induced when cells were grown on solid media but not when they were grown in broth. The fusion was also induced by incubation of liquid-grown cells with an anti-polar flagellum polyclonal antiserum. Hindrance of polar-flagellum rotation is suggested to be the signal for this induction.

  12. GP3 is a structural component of the PRRSV type II (US) virion

    International Nuclear Information System (INIS)

    Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen to generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, 35S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20-60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.

  13. Immunologic proof of DNS irradiation damages and their repair in stationary yeast cells

    International Nuclear Information System (INIS)

    In rabbits an antiserum was produced by injecting UV-irradiated denaturated calf-thymus DNS; after inhibiting unspecific bindings, a specific serological reaction with UV-induced irradiation damages could be taken as present in this antiserum. By the ammonium sulphate precipitation as immunologic method of detection, after UV-irradiation the genesis of damages at certain sites in the DNS of different yeast lineages and their repair was observed. The elemination of UV-induced DNS damages was observed after an incubation in a nutrien medium, after photo-reactivation and after combining both therapeutic treatments. The following results were obtained: the detected DNS damage (number of induced dimeres/yeast genomes) had the same degree in the four yeast lineages. Apart from the excision-negative mutante 2094 for all yeast lineages a repair efficiency of 60% could be detected. All yeast lineages presented themselves as photographically to be reactivated; however, in all cases a DNS damage of 40 to 50% remained. The examinations for the specificity of antiserum against roentgenologically irradiated DNS led to the conclusion that the antibody population of the serum consisted mainly of immunoglobulines against unchanged DNS areas. A specific immunological reaction of only about 10% could be achieved. (orig./MG)

  14. Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver

    International Nuclear Information System (INIS)

    The cytoplasmic androgen-binding (CAB) protein of the male rate liver has been implicated to play a role in the androgen-dependent regulation of α2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SCS-polyacrylamide gel electrophoresis, the authors have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immunochemical cross-reactivity between CAB and another androgen-binding component of Mr 29K was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure

  15. Antibody coated tubes in T3 - solid phase radioimmunoassay

    International Nuclear Information System (INIS)

    The aim of this study was to develop a simple and inexpensive form of solid phase radioimmunoassay of T3 (3,5,3'-L- triiodothyronine); for the preparation of solid phase, the adsorption of anti-T3 antibodies to polystyrene tubes has been used. The polystyrene tubes were used without washing or other treatment; each tube was coated by addition of an uniform volume (175 μl) of diluted antiserum of moderately high titer. Antiserum dilution was 1:3000 and the optimal pH of buffer solution was 8.4 - 8.6. The best results were achieved with an exposure time to antiserum of 40 h at 4 deg C. The antibody - coated tubes prepared in this way were verified by using them to the radioimmunoassay of T3. The results obtained with the above mentioned solid phase in T3-RIA of three level control serums were found to be successful for setting up T3- solid phase radioimmunoassay of high precision. (authors)

  16. Prokaryotic expression of the chicken xanthine oxidase (XOD) subunit and its localization in liver and kidney.

    Science.gov (United States)

    Lin, Huayuan; Chen, Yanjun; Huang, Qiqi; Guo, Xiaoquan; Liu, Ping; Liu, Weilian; Zhang, Caiying; Cao, Huabin; Hu, Guoliang

    2016-06-01

    Xanthine oxidase (XOD) is the members of the molybdenum hydroxylase flavoprotein family and it plays a vital role in the body's purine catabolism. In this study, we cloned the XOD 37kDa subunit protein by using RT-PCR and pMD-18-T clone vector based on the total RNA extracted from chicken liver. The cloning XOD subunit protein gene was ligated into the pET-32a to construct the recombinant plasmid pET-XOD. After the pET-XOD expression vector was transformed into host cells Rosetta (DE3), the recombinant XOD subunit proteins (54.8kDa) were successfully induced by isopropy1 β-d-thiogalactoside (IPTG). Rabbit antiserums were produced by using the purification of the recombinant XOD subunit protein as antigen. The titer of the antiserum was more than 1:102,400 determined by using ELISA. The result of Western blot demonstrated that the antiserum could specifically recognize the chicken liver XOD. Immunohistochemistry and immunofluorescence showed that the XOD mainly presented in the cytoplasm of chicken hepatocytes and proximal tubular epithelial cells. Our results indicated that the XOD subunit protein polyclonal antibody prepared by this method could be used for the further researches of the biological function of the XOD in the chicken. PMID:26949113

  17. Thaumatin-like proteins are differentially expressed and localized in phloem tissues of hybrid poplar

    Directory of Open Access Journals (Sweden)

    Dafoe Nicole J

    2010-08-01

    Full Text Available Abstract Background Two thaumatin-like proteins (TLPs were previously identified in phloem exudate of hybrid poplar (Populus trichocarpa × P. deltoides using proteomics methods, and their sieve element localization confirmed by immunofluorescence. In the current study, we analyzed different tissues to further understand TLP expression and localization in poplar, and used immunogold labelling to determine intracellular localization. Results Immunofluorescence using a TLP antiserum confirmed the presence of TLP in punctate, organelle-like structures within sieve elements. On western blots, the antiserum labeled two constitutively expressed proteins with distinct expression patterns. Immunogold labelling suggested that TLPs are associated with starch granules and starch-containing plastids in sieve elements and phloem parenchyma cells. In addition, the antiserum recognized TLPs in the inner cell wall and sieve plate region of sieve elements. Conclusions TLP localization in poplar cells and tissues is complex. TLP1 is expressed predominantly in tissues with a prominent vascular system such as midveins, petioles and stems, whereas the second TLP is primarily expressed in starch-storing plastids found in young leaves and the shoot apex.

  18. Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

    International Nuclear Information System (INIS)

    An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule

  19. Characterization and application of a radioimmunoassay for reduced, carboxymethylated human luteinizing hormone α-subunit

    International Nuclear Information System (INIS)

    We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH α-subunit, with RCXM-α as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-α. A tryptic digest of RCXM α-subunit was completely reactive, while chymotryptic digestion abolished all immunoreactivity. By testing with separate tryptic fragments, the recognition site could be localized to a segment close to the amino-terminus of the peptide chain. When applied to measurement of serum and urine, an immunoreactive species, parallel to RCXM α-subunit by serial dilution, was found in concentrations of 1-2 ng/ml in serum and 3-4 ng/ml in urine. Similar levels of the immunoreactive component were found in conditions of elevated gonadotropins (e.g. pregnancy) as well as gonadotropin deficiency (panhypopituitarism and Kallmann's syndrome). After stimulation with LHRH, no rise was noted at times up to 6 h despite the fact that both LH and LH-α were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of α-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit

  20. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-06-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  1. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    International Nuclear Information System (INIS)

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in λgt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein

  2. Identification of the neurofibromatosis type 1 gene product

    International Nuclear Information System (INIS)

    The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of ∼250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to the synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested

  3. The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA

    International Nuclear Information System (INIS)

    Antiserum was prepared in rabbits against ultraviolet-irradiated DNA. Using a 125I-labeled protein A binding assay it was shown to be specific for ultraviolet-irradiated DNA, binding increasing as a function of logarithmic increase in dose. This antiserum was then used to develop a radioimmunoassay in which the competition between labeled UV-DNA and unlabeled sample DNA for antibody binding sites is monitored. Using this system the specificity of the assay could be changed depending on the nature of the labeled probe. The inability of poly(dA-dT).poly(dA-dT), as compared with poly(dA).poly(dT), to act as a competitive inhibitor established that the primary lesion recognized by the antiserum is the thymine dimer. This antigenic response did, however, depend on the presence of at least one nucleotide adjacent to the dimer. The sensitivity of the assay was optimized by using 32P-labeled plasmid DNA as competitive probe and is capable of detecting photodamage in cellular DNA at doses as low as 2.5 J m-2. (Auth.)

  4. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    International Nuclear Information System (INIS)

    The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABPPM have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABPPM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABPPM and mGOT are closely related

  5. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered

  6. Specificity of antisera produced against mitomycin C.

    Science.gov (United States)

    Fujiwara, K; Saikusa, H; Kitagawa, T; Takahashi, S; Konishi, Y

    1983-12-01

    The specificity of antisera produced in rabbits for use in mitomycin C (MMC) enzyme immunoassay has been examined employing competitive experiments using several mitomycin analogs and the chemically or biologically degraded preparations of MMC. These studies demonstrate that the antiserum distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with mitomycin A (7.8%) and B (0.78%). On the other hand, porfiromycin and acetyl MMC (Ac-MMC), which commonly possess the substituted groups (methyl and acetyl groups, respectively) at the aziridine ring, showed enhanced reactivity with the antiserum (about two times and ten times as compared to the parent MMC, respectively), suggesting that the antigen used for antibody production was the MMC acylated at the imino group of the aziridine ring. The values of the chemically or biologically degraded preparations of MMC quantified by this enzyme immunoassay were in good agreement with those of the remaining nonreacted MMC measured spectrophotometrically, thus indicating that the anti-MMC antiserum hardly cross-reacted with these degradation products. PMID:6418380

  7. Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells

    Energy Technology Data Exchange (ETDEWEB)

    Egyhazi, E.; Pigon, A. (Karolinska Institutet, Stockholm (Sweden)); Chang, Jinhong; Ghaffari, S.H.; Dreesen, T.D.; Wellman, S.E.; Case, S.T.; Olson, M.O.J. (Univ. of Mississippi Medical Center, Jackson (USA))

    1988-10-01

    Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with {sup 32}P-labeled RNA precursors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of {sup 32}P incorporation into 38 S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of {sup 32}P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of {sup 32}P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

  8. Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

    Science.gov (United States)

    Sasagawa, Ichiro; Oka, Shunya; Mikami, Masato; Yokosuka, Hiroyuki; Ishiyama, Mikio; Imai, Akane; Shimokawa, Hitoyata; Uchida, Takashi

    2016-05-01

    In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin. PMID:27139791

  9. A radioimmunoassay for equilin in equine pregnancy plasma

    International Nuclear Information System (INIS)

    It is well known that the pregnant mare produces large quantities of the ring B unsaturated steroid equilin in addition to classical oestrogens. However, the precise biogenesis of this unusual steroid remains a mystery. To facilitate a study of the interrelationship of the steroids present during equine pregnancy, a radioimmunoassay for the measurement of equilin in peripheral plasma was developed. As equilin is thought to be a product of the foeto-placental unit, such an assay may also be of use as an index of foetal well-being. Oestrone and equilin are present in similar concentrations in equine pregnancy plasma so it was important that an antiserum was produced which could differentiate between these two steroids. An antigen was synthesised in which equilin is linked to a protein carrier through the 17-position in order that the C7-C8 double bond might be fully exposed for immune recognition. This stratagem proved successful as the antiserum obtained gave a cross-reaction of only 7.3% for oestrone. The use of a radioimmunoassay incorporating this antiserum is demonstrated by measuring equilin concentrations in plasma samples taken from a mare at weekly intervals from day 60 of pregnancy through to parturition. The corresponding oestrone concentrations are also recorded and demonstrate the validity of the equilin assay in this situation

  10. Development of radioimmunoassay method for 16alpha-OH-DHEA using DHEA-17-oxime-BSA

    International Nuclear Information System (INIS)

    Microdetermination of 16α-OH-DHEA (known as a main precursor of placental estriol which is significantly increased in pregnancy) using RIA was developed and offered for clinical application. 1) Microbiological biosynthesis of 3H-labeled 16α-OH-DHEA with high specific activity was performed to correct the recovery factor in the extraction-purification process which is necessary for RIA of hormones of the steroid group. The purity was 97.4%, and the specific activity (S.a.) was 13.9 Ci/mM. 2) Applying the fact that the anti-serum against DHEA-17-oxime-BSA conjugate also presents a cross reaction of 40% with 16α-OH-DHEA, a special assay system using this anti-serum was created. Isolation of other steroids such as Δ5 Androstendiol, Pregnenolone and 17α-OH-Pregnenolone, which present cross reactions with the antiserum, was performed using thin layer chromatography. Therefore, these steroids of 0 to 300 pg could be determined with satisfactory accuracy and precision. 3) 16α-OH-DHEA was determined with the steroids, DHEA and estrogen in embryo and maternal blood. The value of 16α-OH-DHEA, which was the highest in the two umbilical arteries, was about twice as high as that of DHEA. These facts indicate that the role of the steroids as precursors of placental estrogen is important. (Ichikawa, K.)

  11. Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells

    International Nuclear Information System (INIS)

    Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with 32P-labeled RNA precursors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of 32P incorporation into 38 S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of 32P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of 32P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA

  12. 碳二亚胺法制备阿莫西林人工抗原及其鉴定%Preparation and Identification of Amoxicillin Artificial Antigen by EDC Method

    Institute of Scientific and Technical Information of China (English)

    刘庆堂; 王磊; 职爱民; 滕蔓; 胡骁飞; 孙亚宁; 宋春美; 王寅彪; 张改平

    2012-01-01

    合成、鉴定了阿莫西林(amoxicillin,AMO)人工抗原,并通过动物免疫法生产了亲和力高、特异性好的鼠源AMO多克隆抗血清.采用碳二亚胺(EDC)法将AMO分别与载体蛋白BSA和OVA偶联,合成完全免疫抗原AMO - BSA和检测抗原AMO - OVA,经紫外分光光度法和SDS -PAGE以及动物免疫进行鉴定.结果表明,偶联后的紫外吸收峰与BSA和AMO相比都发生了一定的位移,AMO - BSA在276 nm处出现最大吸收峰,BSA的泳动速度大于AMO - BSA.免疫后获得的3只小鼠多抗血清,通过间接竞争ELISA测定,效价可达1×10-4以上,1号小鼠半数抑制浓度(IC50)为573.75 ng/mL,敏感性较好.AMO完全人工抗原的合成以及鼠源多克隆抗体血清的制备,为AMO单克隆抗体的制备奠定了基础.%To synthesize the artificial antigen of AMO and obtain its mouse polyclonal antiserum, the immunogen AMO-BSA and coating antigen AMO-OVA were synthesized using EDC method and identified by ultraviolet scanning and SDS-PAGE. BALB/c mice were immunized with the synthesized antigens and the polyclonal antiserum was determined by indirect and blocking ELISA. The results showed that after conjugation, the ultraviolet absorption peak of AMO-BSA appeared at 276 nm, indicating that certain displacement occurred comparing with the ultraviolet absorption peaks of both AMO and BSA. The electropharetic mobility of BSA was observed bigger than that of AMO-BSA. Indirect ELISA showed that the antiserum titres of all the three immunized BALB/c mice were above 1 X 10~4. With the IC50 of 573. 75 ng/mL,No, 1 mouse polyclonal antiserum showed the best sensitivity. In this study, AMO-BSA and AMO-OVA were successfully synthesized and high sensitive polyclonal antiserums against AMO were prepared,which provides a basis for the preparation of monoclonal antibodies against AMO.

  13. Utilização da serologia na identificação de Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae = Using the serological technique to identify Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae

    Directory of Open Access Journals (Sweden)

    Fabiano de Mello Costa

    2009-04-01

    Full Text Available O presente trabalho teve como objetivo obter antissoro específico para Ascia monuste orseis (Latreille, 1819 com a finalidade de identificar seus predadores. Para a obtenção do antissoro, um coelho foi imunizado, via linfonódulo, com uma injeção de antígeno obtido pela maceração de lagartas do 4o e 5o instares de A. monuste orseis. Reações serológicas homólogas, realizadas por meio do método de dupla difusão em ágar, foram positivas após 21 dias da 1a inoculação do antígeno. Ovos, lagartas do 1o, 2o e 3o instares, pupas e adultos de A. monuste orseis também reagiram positivamente ao antissoro obtido.This study aimed to obtain specific antiserum for Ascia monuste orseis (Latreille, 1819, with the aim of identifying the predators associated with this insect. The preparation of specific antiserum was performed by immunizing a rabbit via a lymph nodule injection containing antigen obtained from the maceration of 4th and 5th instars of A. monuste orseis caterpillars. Serological homologous tests performed using double diffusion in agar gel were positive 21 days after the first antigen inoculation. Eggs, 1th, 2th and 3th instar caterpillars, pupae and adults of A. monuste orseis also reacted positively to the obtained antiserum.

  14. Utilização da serologia na identificação de Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae - DOI: 10.4025/actascibiolsci.v31i2.515 Using the serological technique to identify Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae - DOI: 10.4025/actascibiolsci.v31i2.515

    Directory of Open Access Journals (Sweden)

    Carlos Roberto Sousa-Silva

    2009-05-01

    Full Text Available O presente trabalho teve como objetivo obter antissoro específico paraAscia monuste orseis (Latreille, 1819 com a finalidade de identificar seus predadores. Para a obtenção do antissoro, um coelho foi imunizado, via linfonódulo, com uma injeção de antígeno obtido pela maceração de lagartas do 4o e 5o instares de A. monuste orseis. Reações serológicas homólogas, realizadas por meio do método de dupla difusão em ágar, foram positivas após 21 dias da 1a inoculação do antígeno. Ovos, lagartas do 1o, 2o e 3o instares, pupas e adultos de A. monuste orseis também reagiram positivamente ao antissoro obtido.This study aimed to obtain specific antiserum for Ascia monuste orseis (Latreille, 1819, with the aim of identifying the predators associated with this insect. The preparation of specific antiserum was performed by immunizing a rabbit via a lymph nodule injection containing antigen obtained from the maceration of 4th and 5th instars of A. monuste orseis caterpillars. Serological homologous tests performed using double diffusion in agar gel were positive 21 days after the first antigen inoculation. Eggs, 1th, 2th and 3th instar caterpillars, pupae and adults of A. monuste orseis also reacted positively to the obtained antiserum.

  15. Radioimmunological determination of oestetrol in plasma in normal and pathological pregnancies

    International Nuclear Information System (INIS)

    The radioimmunological method presented for the determination of oestetrol is suitable for measuring the concentration of this hormone in the plasma. Measuring range, accuracy and precision are comparable to those of other methods. This method will be as easy to handle as the oestriol determination method if a specific antiserum is available. The concentration of oestetrol in normal pregnancy ranges between 0.745 ng/ml in the 22nd pregnancy week and 2.501 ng/ml in the 40th p.w. when determined by means of the unspecific antiserum I; when using the specific antiserum II, it ranges between 0.267 ng/ml in the 22nd p.w. and 1.370 ng/ml in the 40th p.w., oestetrol plasma level hardly changes until the 30th p.w.; afterwards the level rises continuously till the end of gestation period. A daily rhythm was not found for oestetrol. A rapid drop in hormone concentration occurs when treated with betamethasone, β-sympathomimetics and ampicillin; however, hormon concentration begins to increase soon afterwards. Patients suffering from diabetes mellitus do not show the course of the oestetrol plasma level to deviate from the normal range. In cases of infra-uterine development insufficiency, children having a birth weigth below 25 and above 10 percentile according to Lubchenco reveal a rise in concentration corresponding to the norm. Children revealing a additional symptoms of dystrophy have an average hormone concentration lower by about one third. Patients whose children reveal a birth weight below the 10 percentile according to Lubchenco, do not show any further increase in the concentration of this hormone after the 35th p.w. (orig./MG)

  16. SALMATcor: microagglutination for Salmonella flagella serotyping.

    Science.gov (United States)

    Duarte Martínez, Francisco; Sánchez-Salazar, Luz Marina; Acuña-Calvo, María Teresa; Bolaños-Acuña, Hilda María; Dittel-Dittel, Isis; Campos-Chacón, Elena

    2010-08-01

    Salmonella is a complex bacterial group with more than 2400 serovars widely distributed in nature; they are considered zoonotic because they can infect a variety of animals and be transmitted to humans. Usually, they cause alimentary acquired diseases such as gastroenteritis, typhoid fever, and others that can lead to severe complications and death. Serotyping is useful to differentiate among Salmonella, because it shows an important correlation with their clinical and epidemiological patterns; consequently, it is of high value for public health, animal health, agriculture, and industry. To characterize all known Kauffmann-White Salmonella serovars, over 250 antisera are required. Due to this and to high prices antisera, many laboratories worldwide have limitations in establishing Salmonella surveillance. Therefore, we developed and validated a Salmonella flagella microagglutination test (SALMATcor) that significantly reduces laboratory requirements of antisera. SALMATcor is based on scaling down, by fivefold, the antigen:antiserum volumes actually required for the reference method: flagella standard tube agglutination technique (STAT). Antigen preparation, temperatures, and incubation periods remained as established for STAT. The SALMATcor was validated according to ISO/DIS 16140:1999 protocol, which included 1187 comparisons of flagella determinations conducted by SALMATcor and STAT, on 141 Salmonella isolates of 12 common serotypes and the use of antiserum recommended for STAT. SALMATcor concordance was excellent (Cohen's kappa index 0.9982), obtaining relative accuracy >99.9% and relative specificity >99.9%. Additionally, SALMATcor has been used by CNRB-INCIENSA since 2004 to respond to all 40 Salmonella proficiency testing strains, provided by World Health Organization-Global Salmonella Surveillance Network, obtaining 100% concordance on serovar identification. On the basis of the results achieved with SALMATcor and considering that it also significantly

  17. Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

    Science.gov (United States)

    Russell, V E; Klein, U; Reuveni, M; Spaeth, D D; Wolfersberger, M G; Harvey, W R

    1992-05-01

    In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources. PMID:1534830

  18. Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP)(73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.

  19. Development of fluoroimmunoassay methods for delta-9-tetrahydrocannabinol

    Energy Technology Data Exchange (ETDEWEB)

    Mason, A.P.

    1986-01-01

    Heterogeneous, competitive, labelled-ligand solid-phase primary antibody fluoroimmunoassay methods for the detection of THC in blood and plasma were proposed, and the required assay components were produced and characterized. These components included polyclonal rabbit antisera and monoclonal antibodies reactive with tetrahydrocannabinols, solid-phase immunoglobin reagents, a fluoroligand, and protein conjugates of THC for immunization and immunoassay response amplification. The stereoselective rabbit anti-THC antiserum F-444-12 was found to have a high binding titer, a high affinity (K/sub D/ = 3.4 x 10/sup -/exclamation/sup 1/ M for 5'-iodo/sup -125/I-..delta../sup 2/-THC), and high specificity versus a large number of cannabinoid compounds. Immobilization of the immunoglobulin fraction of the antiserum on hydrophilic polyacrylamide microspheres resulted in only a four fold increase in K/sub D/, and a two fold increase in the concentration of binding sites required for the production of equivalent binding titers. Specificity for small ligands was not affected, but the binding of THC-protein conjugates was reduced in potency. Two monoclonal hybridoma cell lines were produced that secrete monoclonal antibodies which bind the radioligand. The fluoroligand was synthesized from 5'-carboxy-..delta../sup 2/-THC and FITC using a diamimoethane linkage structure. While the compound had the fluorescence properties of FTIC, it was bound to the antiserum F-144-12 with a cross-reactive potency 1.4x greater than the radioligand, and 10x greater than THC.

  20. Anti-idiotypic antisera in man. I. Production and immunochemical characterization of anti-idiotypic antisera to human antitetanus antibodies

    International Nuclear Information System (INIS)

    Antisera were raised in rabbits against immunosorbent-purified F(ab')2 fragments of IgG antitetanus toxoid (TT) antibodies obtained from three different donors. The antisera were rendered idiotype specific by absorption with insolubilized TT-nontreactive F(ab')2. The resulting anti-idiotypic antisera precipitated more than 85% of 125I-radiolabeled F(ab')2 anti-TT and were shown to be individual specific in that they reacted only with the immunizing F(ab')2 anti-TT and not with F(ab')2 anti-TT derived from other donors. Anti-idiotypic antiserum inhibited binding of 125I-TT but not of 125I-DT to IgG derived from the donor of the immunizing F(ab')2 anti-TT, but did not inhibit 125I-TT binding to IgG derived from other donors. Finally, binding of 125I-F(ab')2 anti-TT to the anti-idiotypic antiserum was completely inhibited by IgG derived from the same donor but only partially inhibited by a great excess of TT antigen, suggesting that the anti-idiotypic antiserum is recognizing nonantigen-binding idiotypic determinants on F(ab')2 anti-TT. The data presented demonstrate that anti-idiotypic heteroantisera can be successfully raised against human antibodies to TT, indicate minimal cross-reactivity of idiotypic determinants between unrelated individuals, and suggest the presence of nonantigen binding-idiotypic determinants on the antibody molecule

  1. Critical study and applications of the radioimmunological determination of carcinoembryonic antigen

    International Nuclear Information System (INIS)

    This paper outlines our research on the development of a radioimmunological method to determine the carcinoembryonic antigen of the digestive system (ACE). The carcinoembryonic antigen is defined and situated in the framework of antigens associated with human tumours. The general principles of the radioimmunological determination are then reviewed. A detailed technical study is devoted to each of the elements involved in the reaction and to the working conditions of each method tried. A labelling procedure and a radioactive protein separation method have been worked out, guaranteeing a high specific radioactivity consistent with a good immunoreactivity. The period of effectiveness of this protein has also been determined, taking account of its deiodination. The antiserum is a very important factor in the sensitivity of the measurement. A ewe antiserum of good antibody content and volume yield was chosen, its disadvantage being the length of the determination imposed by the 48 hour preincubation time. Ammonium sulphate precipitation and double antibody techniques were used for the labelled antigen-antibody separation. In seric solution the ammonium sulphate precipitation carries down non-specifically, in the standards, a large amount of labelled antigen. This disadvantage has been offset by a method of calculation which shows the actual contribution of the labelled complex. The double antibody technique requires a special adjustment to balance quantity of second antiserum and precipitation time. The system sometimes needs an addition of serum from the animal donor of the first antibody in order to obtain an adequate separation. Where techniques are concerned, although the macro-method is suitable for determinations on perchloric extract and is still in common use we prefer to use the one described here under the name of micro-method. Finally the results obtained in experimental and clinical applications are presented

  2. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [125I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  3. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.

    OpenAIRE

    Wilson, P. O.; Barber, P. C.; Hamid, Q. A.; Power, B. F.; Dhillon, A. P.; Rode, J.; Day, I N; Thompson, R. J.; Polak, J.M. (Julia M.)

    1988-01-01

    In order to assess the potential of protein gene product (PGP) 9.5 as a marker of the nervous and neuroendocrine systems, we examined its immunolocation in human, rat and guinea-pig tissues, using a rabbit polyclonal antiserum and two new mouse monoclonal antisera, I3C4 and 3IA3. Our results demonstrate immunoreactive PGP 9.5 in neurons and nerve fibres at all levels of the central and peripheral nervous system, in many neuroendocrine cells, in part of the renal tubule, in spermatogonia and L...

  4. Histocompatibility antigens on astrocytoma cells.

    OpenAIRE

    Hirschberg, H.; Endresen, L I; Wikeby, P

    1982-01-01

    Biopsies tumour cells from astrocytoma-bearing patients were grown in primary culture for 3-5 days. Both low and high grade tumours were represented in the study. The cultured cells could be shown to express the HLA-A and -B antigens using a multispecific allo-antiserum and a rabbit anti-beta-2 microglobulin antibody. The tumour cells were negative for the HLA-DR determinants when tested with either rabbit anti-Ia-like antisera or specific anti-HLA-DR allo-antisera. They also failed to stimul...

  5. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans; Lernmark, A

    1985-01-01

    detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry...... showed specific surface immunofluorescence in 1:100-1:1000 dilutions in lymphoblastoid and blood mononucleated cells. In the latter the binding was primarily confined to monocytes and a subpopulation of lymphocytes. It is concluded that locus-specific immunological reagents to distinguish between beta...

  6. Denmark: botulism in an infant or infant botulism?

    DEFF Research Database (Denmark)

    Pærregaard, Anders; Angen, O.; Mølbak, Kare; Clausen, M.E.; Christensen, Jens Juul Dencker; Lisby, Michael; Paerregaard, A; Angen, O; Lisby, M; Mølbak, K; Clausen, M E; Christensen, J J

    2008-01-01

    A 4.5 months old, previously healthy Danish girl was admitted to a paediatric department after six days of passive behaviour and weak suck. Over the next days she became increasingly weak, developed bilateral ptosis, the muscle stretch reflexes were lost, and mydriasis with slow pupillary responses...... was noted. Botulism was suspected and confirmed by testing of patient serum in a bioassay. The condition of the patient improved following administration of botulism antiserum. The clinical picture was suggestive of intestinal (infant) botulism. However, botulism acquired from consumption of food with...

  7. Radioimmunological demonstration of DNA specific antibodies

    International Nuclear Information System (INIS)

    Using 125I chemically labelled denatured (d) and native (n) DNA, specifically binding antibodies were demonstrated in the sera of Lupus erythemathodes patients by means of the Farr technique. (NH4)2SO4 was used to separate the immunologically bound 125I-d-DNA. For 125I-n-DNA the use of a secondary antiserum for the precipitation of the primary immune complex is advantageous. The influence of antigen concentration upon the binding rate was studied. Titre determinations can be made with the proposed method. (author)

  8. Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin.

    Science.gov (United States)

    Nguyen, T M; Ellis, J M; Ginjaar, I B; van Paassen, M M; van Ommen, G J; Moorman, A F; Cartwright, A J; Morris, G E

    1990-10-15

    A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS-denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross-reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple-helical region of dystrophin (amino acids 1750-2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin-like central rod model for dystrophin. PMID:1699800

  9. Potential role of endocrine gastrin in the colonic adenoma carcinoma sequence

    OpenAIRE

    Watson, S A; Morris, T M; McWilliams, D F; Harris, J.; Evans, S.; Smith, A.; Clarke, P.A.

    2002-01-01

    The role of hyper-gastrinaemia in the incidence of colonic cancer remains to be clarified. The aim of this study was to determine whether cholecystokinin-2 (CCK-2) receptor expression predicts the sensitivity of human colonic adenomas to the proliferative effects of serum hyper-gastrinaemia. Gene expression of the classical (74 kDa) CCK-2 receptor in human colonic adenoma specimens and cell lines, was quantified by real-time PCR. Western blotting, using a CCK-2 receptor antiserum, confirmed p...

  10. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe).

    OpenAIRE

    Glasser, S W; Korfhagen, T R; Weaver, T.; Pilot-Matias, T; Fox, J L; Whitsett, J A

    1987-01-01

    Hydrophobic surfactant-associated protein of Mr 6000-14,000 was isolated from ether/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) ...

  11. [Immunofluorescent diagnosis of Entamoeba histolytica trophocoites in preserved stool specimens of patients (author's transl)].

    Science.gov (United States)

    Hess, U; Fröhlich, A

    1979-09-01

    A fixative and examination technique is described for identification of Entamoeba histolytica trophocoites in faeces with the aid of the indirect immunofluorescence technique. Magnaforms in bloody-dysenteric specimens and Minutaforms in spontaneous or saline purged specimens give equally good fluorescence. The specifity of the rabbit antiserum against axenically grown E. hist. is good: There is strong positive reaction only with trophocoites of E. hist. Weak crossreactions are encountered with trophocoites of E. coli, E. hartmanni and E. polecki. Negative reactions are encountered with trophocoites of Endolimax nana, Jodamoeba bāutschlii, and Dientamoeba fragilis. Amebic cysts, flagellates, leucocytes, epithelial cells and Blastocytis give negative reactions, too. PMID:94475

  12. Interleukin 1 of the central nervous system is produced by ameboid microglia

    OpenAIRE

    1986-01-01

    By screening specific populations of rat brain cells, we found that ameboid microglia secrete an 18 kD peptide with IL-1 biological activity. The IL-1 activity released by microglia was found to be identical to rat macrophage IL-1 on fractionation by gel filtration and high pressure liquid anion-exchange chromatography, and it was neutralized by an antiserum specific for murine IL-1. When added to astroglia grown in culture, microglial IL-1 increased the cell number of five- to sevenfold, and...

  13. The radioimmunological determination of vasopressin in urine

    International Nuclear Information System (INIS)

    This thesis describes the development of a radioimmunoassay (RIA) for antidiuretic hormone (ADH) or vasopressin, which can be used for the quantitative measurement of the urinary excretion of the hormone in man during physiological and pathological conditions. The final RIA method, using approximately 5 pg 125I-AVP diluted (1 : 50,000) antiserum 121 and charcoal-dextran separation of the antibody-bound and free fractions, is found to be specific for vasopressin and closely related substances; the sensitivity is 9 pg. The validity is demonstrated and the results of measurements of vasopressin excretion in urine from 39 normal subjects, including 4 children are presented. (Auth.)

  14. Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

    OpenAIRE

    Zorka Milićević; Vladan Bajić; Lada Živković; Jelena Kasapović; Uroš Andjelković; Biljana Spremo-Potparević

    2014-01-01

    In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most in...

  15. Production, purification, and assay of thrombopoietin. Final report, June 1, 1973--June 30, 1978

    International Nuclear Information System (INIS)

    Studies were conducted on purification and assay of thrombopoietin, the development of neutralizing antibodies to thrombopoietin, and the production of thrombopoietin by kidney cells in culture. The role of platelet-specific antiserum action in thrombocytopenia was investigated. Thrombopoietin was present in sera of thrombocytopenic mice following x radiation or injection of platelet-specific antisera. Studies with dogs showed that platelet cycles are dependent on thrombopoietin and that platelet sizes are altered inversely with platelet counts. The effects of maternal thrombocytopenia on platelet counts of pre- and postnatal mice were investigated as well as the effects of hypoxia on platelet production in mice

  16. cDNA for R-cognin: homology with a multifunctional protein.

    OpenAIRE

    Krishna Rao, A S; Hausman, R E

    1993-01-01

    Retina cognin (R-cognin) is a developmentally regulated 50-kDa protein that was isolated from chicken embryo retina cell membranes. It mediates the adhesion and reaggregation in vitro of retina cells from chicken and mouse embryos, but not of cells from other tissues, and may be involved in neuronal differentiation. We report here the cloning of a cDNA for R-cognin. A chicken embryo retina cDNA library was constructed in lambda gt11 vector and was screened with polyclonal R-cognin antiserum, ...

  17. Para-Bombay phenotype: report of a rare blood group

    OpenAIRE

    A.Yashovardhan; I.S.Chaitanya Kumar; K.V. Sreedhar Babu; B. Suresh Babu; Anju Verma; Jothi Bai, D.S.; B. Siddhartha Kumar

    2012-01-01

    The blood sample of a 54-year-old male patient who presented with signs and symptoms suggestive of anaemia was submitted to the Blood Bank for blood grouping and cross-matching. In forward grouping, no agglutination was observed with A, B and AB antisera, but agglutination was noticed with D antiserum (Group O). In reverse grouping, there was agglutination in tube labelled A and no agglutination in tubes B and O (Group B) resulting in discrepancy between forward and reverse grouping. Furth...

  18. Radioimmunoassay for salivary estriol, with use of an 125I radioligand and a solid-phase separation technique

    International Nuclear Information System (INIS)

    This simple, rapid radioimmunoassay for salivary unconjugated estriol in the third trimester of pregnancy is based on use of a Sepharose-coupled antiserum and a radioiodinated tracer, and requires only a 15-min incubation. Estriol conjugates are shown not to be present in saliva in significant concentrations; therefore a preliminary extraction is unnecessary. Determinations of unconjugated estriol in matched specimens of plasma and saliva correlate well (r . 0.95). A provisional normal range for 30 weeks to term agrees closely with those reported by others. This assay can give results quickly for the large numbers of specimens generated by weekly or daily sampling regimes

  19. USE OF MODIFIED CAMP TEST FOR PRELIMINARY NONSEROLOGIC IDENTIFICATION OF VIBRIO CHOLERAE IN STOOL SPECIMENS

    Directory of Open Access Journals (Sweden)

    Murad Lesmana

    2012-09-01

    Full Text Available Suatu modifikasi uji CAMP digunakan bersama dengan reaksi biokimiawi untuk identifikasi Vibrio cholerae pada sampel klinis. Dari 579 usap dubur penderita diare, 92 (16% memberikan hasil isolasi V. cholerae 01 biotipe El Tor dan 34 (6% V. cholerae non-01. Semua isolat V. cholerae 01 El Tor menunjukkan reaksi CAMP positif kuat dengan gambaran hemolisis sinergistik lengkap berbentuk sosis; sedangkan V. cholerae non-01 memberikan reaksi CAMP yang sempit dengan pola hemolisis menyerupai bulan sabit. Hasil uji CAMP yang dilakukan bersama dengan reaksi biokimiawi sesuai dengan metode biakan konvensional yang menyertakan tes aglutinasi dengan antiserum V. cholerae 01 untuk mengidentifikasi V. cholerae.

  20. Determination of the radioimmunologic concentration of 5-androstene-3β-17β-diole in the blood plasma of patients suffering from common psoriasis

    International Nuclear Information System (INIS)

    A new radioimmunologic method for the determination of ADIOL in the blood plasma was developed. The author produced the antiserum herself and she examined it on cross-reactions. This method resulted to be very sensitive and in the standard curve a linear range of 0.04 ng to 1.5 ng was obtained. Moreover, the ADIOL concentration was determined in the blood plasma of 16 control subjects and of 35 patients suffering from psoriasis. The result was a far-reaching correlation with the results described in literature, although they were obtained by means of differing determination procedures. (orig./MG)

  1. Isolation and purification of G immunoglobulin from guinea-pig for the production of a second antibody for radioimmunoassay

    International Nuclear Information System (INIS)

    The IgG of guinea-pig was isolated and purified by precipitation with caprylic acid and the batch absorption with DEAE-cellulose. The efficiency of the operating was verified by the determination of total proteins, during the purification stages. The purity of the end product was proved by immuno electrophoresis face to rabbit serum total antiserum of guinea-pig. it was obtained 240 mg of purity IgG to be used in the production of second specific antibody for radioimmunoassay. (C.G.C.)

  2. Specificity of immunoassays. Pt. 2

    International Nuclear Information System (INIS)

    Practical aspects of the measurement of the specificity of immunoassay are reviewed. Antibody heterogeneity in an antiserum makes a pragmatic rather than a theoretical approach necessary. A new method for the measurement of immunoassay specificity is described. This method is based on the errors caused by the cross-reacting antigens and is directly relevant to the validity of results obtained by immunoassay methods. The effect of selectively blocking the least specific antibodies in antisera raised against steroid haptens is tested. The practical consequences of these considerations are tested using steroid radioimmunoassay and enzyme-immunoassay. (orig.)

  3. Effects of Static Magnetic Field on Growth of Leptospire, Leptospira interrogans serovar canicola: Immunoreactivity and Cell Division

    CERN Document Server

    Triampo, W; Triampo, D; Wong-Ekkabut, J; Tang, I M; Triampo, Wannapong; Doungchawee, Galayanee; Triampo, Darapond; Wong-Ekkabut, Jirasak

    2004-01-01

    The effects of the exposure of the bacterium, Leptospira interrogans serovar canicola to a constant magnetic field with magnetic flux density from a permanent ferrite magnet = 140 mT were studied. Changes in Leptospira cells after their exposure to the field were determined on the basis of changes in their growth behavior and agglutination immunoreactivity with a homologous antiserum using darkfield microscopy together with visual imaging. The data showed that the exposed Leptospira cells have lower densities and lower agglutination immunoreactivity than the unexposed control group. Interestingly, some of the exposed Leptospira cells showed abnormal morphologies such as large lengths. We discussed some of the possible reasons for these observations.

  4. Radioimmunoassay for quantitation of 2',3'-didehydro-3'-deoxythymidine (D4T) in human plasma.

    OpenAIRE

    Zhou, X.J.; Chakboub, H; Ferrua, B; Moravek, J; Guedj, R; Sommadossi, J P

    1996-01-01

    2',3'-Didehydro-3'-deoxythymidine (D4T, or stavudine) has been recently approved for the treatment of AIDS. In the present study, a specific and sensitive radioimmunoassay (RIA) was developed for the quantitation of D4T in human plasma. The RIA is a double-antibody competitive binding assay which uses anti-D4T antiserum raised in rabbits as the primary antibody, a tritiated derivative of D4T as the radioactive tracer, and goat anti-rabbit immunoglobulin G as the secondary antibody. No cross-r...

  5. The Immunodominant Brugia malayi Paramyosin as a Marker of Current Infection with Wuchereria bancrofti Adult Worms

    OpenAIRE

    Langy, Sandra; Plichart, Catherine; Luquiaud, Patrick; Williams, Steven A.; Nicolas, Luc

    1998-01-01

    The full-length cDNA sequence encoding Brugia malayi L3 paramyosin has been isolated by immunoscreening a cDNA library with a mouse antiserum raised against Wuchereria bancrofti L3 infective larvae. A recombinant truncated form of paramyosin was expressed as a glutathione S-transferase fusion protein and used to evaluate humoral responses of adults from a W. bancrofti-endemic area in French Polynesia according to their parasitological status. Immunoglobulin G4 (IgG4) preferentially bound to p...

  6. Radioimmunoassay of CRF-like material in rat hypothalamus

    International Nuclear Information System (INIS)

    Corticotropin releasing factor (CRF) was recently isolated from ovine hypothalami by its ability to stimulate adrenocorticotropin (ACTH) and beta-endorphin release from dispersed rat pituitary cells. In order to study the physiology of this peptide, we have developed a sensitive and specific radioimmunoassay (RIA) for CRF. Synthetic CRF was conjugated to bovine thyroglobulin and emulsified with complete Freund's adjuvant. A suitable antiserum was obtained which showed no crossreactivity with eight naturally occurring peptides. N-Tyr-CRF was iodinated and used as tracer. With this assay, CRF-like immunoreactivity which coeluted with ovine CRF on Sephadex G50 was detected in rat hypothalami

  7. Radioimmunoassay for rhesus monkey gonadotropins

    International Nuclear Information System (INIS)

    Heterologous double-antibody radioimmunoassay methods are described for the measurement of circulating levels of rhesus monkey (Macaca mulatta) FSH and LH; the latter assay is also applicable to rhesus chorionic gonadotropin (CG) estimations. The FSH assay utilizes purified rat FSH for trace, either of two anti-human FSH antisera and a semipurified rhesus pituitary standard. The LH assay utilizes purified ovine LH for trace, an anti-human CG antiserum and the same rhesus pituitary standard. The use of these systems obviates the necessity of purifying rhesus gonadotropins which are required for the development of homologous radioimmunoassay systems. (U.S.)

  8. Multiple Serotypes of the Moderate Thermophile Thiobacillus caldus, a Limitation of Immunological Assays for Biomining Microorganisms

    OpenAIRE

    Hallberg, K B; Lindstrom, E. B.

    1996-01-01

    Phylogenetic and phenotypic analysis indicates that a moderately thermophilic isolate, C-SH12, from Australia belongs to the species Thiobacillus caldus. Antiserum generated against whole cells of T. caldus KU recognized protein antigens common to cell lysates of the three T. caldus strains KU, BC13, and C-SH12 but did not recognize whole cells of isolate C-SH12. Differences in the lipopolysaccharide (LPS) of strain C-SH12 and those of the other two T. caldus strains were found, and the anti-...

  9. Different distribution of neuromedin S and its mRNA in the rat brain: NMS peptide is present not only in the hypothalamus as the mRNA, but also in the brainstem

    OpenAIRE

    Miwa eMori; Kenji eMori; Takanori eIda; Takahiro eSato; Masayasu eKojima; Mikiya eMiyazato; Kenji eKangawa

    2012-01-01

    Neuromedin S (NMS) is a neuropeptide identified as another endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1, which have also been identified as types 1 and 2 receptors for neuromedin U structurally related to NMS. Although expression of NMS mRNA is found mainly in the brain, spleen, and testis, the distribution of its peptide has not yet been investigated. Using a newly prepared antiserum, we developed a highly sensitive radioimmunoassay for rat NMS. NMS...

  10. Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis

    International Nuclear Information System (INIS)

    Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis. (author)

  11. Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens

    OpenAIRE

    1989-01-01

    The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were no...

  12. Latex immunoassay for rapid detection of rotavirus.

    OpenAIRE

    Hughes, J. H.; Tuomari, A V; Mann, D R; Hamparian, V V

    1984-01-01

    A latex agglutination (LA) test was evaluated for the detection of human rotaviruses in stool specimens. Both antiserum and immunoglobulin G (IgG)-sensitized latex particles were used, with IgG-coated beads being more sensitive for human rotavirus antigen detection. Latex beads sensitized with anti-simian-SA-11 IgG were stable for at least 8 months when stored at 4 degrees C. The sensitivity of the test was compared with that of the Rotazyme (Abbott Laboratories, Diagnostics Div., North Chica...

  13. A multi-epitope vaccine CTB-UE relieves Helicobacter pylori-induced gastric inflammatory reaction via up-regulating microRNA-155 to inhibit Th17 response in C57/BL6 mice model

    OpenAIRE

    Lv, Xiaobo; Song, Hui; Yang, Jue; Li, Tong; Xi, Tao; Xing, Yingying

    2014-01-01

    Vaccination is an effective mean of preventing infectious diseases, including those caused by Helicobacter pylori. Th17 cell responses are critical for the pathogenesis of Helicobacter pylori infection. In view of Th17 responses to multi-epitope vaccine CTB-UE, the IL-17 production in antiserum was examined. CTB-UE immunization decreased IL-17 production, implying that Th17 responses may be inhibited. Furthermore, IL-17 aggravated GES-1 cell injury induced by H. pylori SS1; In contrast, CTB-U...

  14. Measurement of ultraviolet light-induced photolesions in mammalian DNA by microELISA

    International Nuclear Information System (INIS)

    A sensitive microELISA was developed for titration of anti-UV-DNA antisera and measurement of photolesions in DNA using UV-irradiated DNA as antigen coupled to the wells of microtitre plates. Efficient and reproducible immobilization of the antigen required precoating of the plates with poly(L-lysine). Antisera from 2 rabbits immunized with UVdsDNA recognized specifically thymine dimer associated lesions. The antiserum from one animal was used to probe photodamage in DNA. Lesions induced by an irradiation dose as small as 2.5 J/m2 could be detected. (Auth.)

  15. Intrinsic factor in human amniotic fluid as determined by radioimmunoassay

    International Nuclear Information System (INIS)

    The intrinsic factor (IF) concentration in 55 human amniotic fluid specimens was determined by radioimmunoassay (RIA). The antiserum was produced by immunizing rabbits with the cobalamin-IF complex isolated from human gastric juice. The median concentration of IF was 0.17 nmol/l and the extreme values <0.07-2.51 nmol/l. Three specimens with a clearly elevated level (0.96, 1.11 and 2.51 nmol/l) were observed. The highest value was associated with a fetal malformation, viz. obstruction of the proximal gut. There was no evident correlation between the concentration of IF in amniotic fluid and gestational age. (author)

  16. Radioimmunological determination of procollagen (type III) and procollagen peptide (type III)

    International Nuclear Information System (INIS)

    The detection of antigens circulating in the blood enables the early recognition of fibrotic processes e.g. liver cirrhosis and hepatitis. One thus uses an anti procollagen (type III) labelled with iodine 125 by the chloramine-T method, or an anti-procollagen peptide (type III) serum which is brought together with a sample of unknown contents. The separation of the antigen-antibody complex is carried out by means of a highly specific antiserum. The procollagen peptide (type III) is produced from calf skin of a foetus or from human aszites fluid. (DG)

  17. An immunohistochemical study of Flexibacter psychrophilus infection in experimentally and naturally infected rainbow trout (Oncorhynchus mykiss) fry

    DEFF Research Database (Denmark)

    Evensen, O.; Lorenzen, Ellen

    1996-01-01

    An immunohistochemical method is described for the detection of Flexibacter psychrophilus in formalin-fixed, parafiin-wax-embedded fry of rainbow trout. Rabbit antiserum as well as rainbow trout hyperimmune serum were used in the study. The distribution and tissue localization of the bacterium wa...... the lesions which are typically found during the chronic stage of the disease....... accompanied by inflammatory cell infiltrates in which polymorphonuclear inflammatory cells were shown to contain the bacterium in the cytoplasm by immunostaining. The eye changes were likewise a common finding in chronic cases with severe inflammatory changes in the retina and with numerous bacteria in...

  18. Preparation of Polyclonal Antibody of Recombinant Human Interleukin-17 and Screening of its Monoclonal Antibody Hybridoma%人白细胞介素17的多克隆抗体制备与单抗杂交瘤的筛选

    Institute of Scientific and Technical Information of China (English)

    王永红; 陈国友; 曹雪涛; 胡晋红; 苏睛

    2001-01-01

    Aim To prepare and purify of rabbit antiserum of recombinant human Interleukin-17(rhIL-17),and screen mouse positive hybridome clones of McAb by clone .Methods Antiserum was obtained by programmed immunization of rabbits with purified rhIL-17 and purified through ammonium sulfate precipitation and affinity chromatography.The spleen of immunized mouse was fused with myeloma cell line SP 2/0,and positive clones were obtained by definite dilution and cloned screening.Antibody was detected by double agar gel immune diffusion test and ELISA.Results and conclusion 2.5ml antiserum(polyclonal antibody) was purified which the purity of IgG was above 95%.5 positive clones were obtained which can secret monoclonal antibody.The antiserum and positive clones could be used in subsequent study of activity block,ELISA and affinity chromatograph purification of IL-17.%目的制备重组人IL-17的兔抗血清并纯化,克隆化筛选IL-17的阳性杂交瘤克隆。方法应用纯化的IL-17程序免疫家兔,得到兔抗人IL-17的抗血清;采用盐析法和亲和层析法对兔抗血清进行纯化。应用纯化的IL-17免疫小鼠,将其脾脏与骨髓瘤细胞SP 2/0融合,采用有限稀释法和克隆化筛选阳性杂交瘤克隆。抗体的检测分别采用琼脂糖双向扩散试验法和酶联免疫测定法(ELISA)。结果和结论纯化得到多克隆抗体2.5ml,IgG纯度大于95%,初步得到5个阳性杂交瘤克隆,可用于以后的深入研究。

  19. Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants.

    OpenAIRE

    Fukayama, M; Calderone, R A

    1991-01-01

    Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell ext...

  20. Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix

    DEFF Research Database (Denmark)

    Hogan, B L; Taylor, A; Kurkinen, M; Couchman, J R

    1982-01-01

    Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert...... interactions and are not precursors or products of each other. They contain asparagine-linked oligosaccharides, but these are not the exclusive sites of sulphate labeling. Antiserum raised against the Mr 150,000 sgp C of Reichert's membranes has been used in an immunohistochemical analysis of rat skin. In...

  1. Radioimmunoassay for the citrus bitter principle, naringin, and related flavonoid-7-O-neohesperidosides

    International Nuclear Information System (INIS)

    An immunoassay for the citrus bitter principle, naringin, and related flavonoid-7-O-neohesperidosides is reported. The assay detects ca. 2 ng of naringin and can be used to quantify this compound in the parts per billion (ppb) range in crude grapefruit juice and extracts of other plant tissues. The antiserum used is highly reactive with the 2-rhamnosyl-1-glucopyranose at the C-7 position but not with e.g. the isomeric 6-rhamnosyl-1-glucopyranose moiety and can, thus, be used to identify the stereochemistry of this disaccharide moiety at the C-7 position of flavanoids. The assay involves a directly iodinated naringin-[125I] as immunotracer. (orig.)

  2. Radioimmunoassay for the citrus bitter principle, naringin, and related flavonoid-7-O-neohesperidosides

    Energy Technology Data Exchange (ETDEWEB)

    Jourdan, P.S.; Weiler, E.W.; Mansell, R.L.

    1982-02-01

    An immunoassay for the citrus bitter principle, naringin, and related flavonoid-7-O-neohesperidosides is reported. The assay detects ca. 2 ng of naringin and can be used to quantify this compound in the parts per billion (ppb) range in crude grapefruit juice and extracts of other plant tissues. The antiserum used is highly reactive with the 2-rhamnosyl-1-glucopyranose at the C-7 position but not with e.g. the isomeric 6-rhamnosyl-1-glucopyranose moiety and can, thus, be used to identify the stereochemistry of this disaccharide moiety at the C-7 position of flavanoids. The assay involves a directly iodinated naringin-(/sup 125/I) as immunotracer.

  3. Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocellum.

    OpenAIRE

    Grépinet, O.; Chebrou, M C; Béguin, P

    1988-01-01

    An endoxylanase encoded by the xynZ gene of Clostridium thermocellum was purified from Escherichia coli harbouring a fragment of the gene cloned in pUC8. The purified enzyme showed two active bands of Mr 41,000 and 39,000, the latter one presumably derived from the former through proteolysis. The enzyme was highly active on xylan and para-nitrophenyl-beta-D-xylobioside. The major end product of xylan hydrolysis was xylobiose. With an antiserum raised against the enzyme purified from E. coli, ...

  4. The occurrence of mycoplasmas in the lungs of swine in Gran Canaria (Spain)

    DEFF Research Database (Denmark)

    Assuncao, P.; De la Fe, C.; Kokotovic, Branko; Gonzalez, O.; Poveda, J.B.

    2005-01-01

    identified as Mycoplasma species. Using different species-specific PCRs, 40, 27, 11 and 7 of the isolates were identified as M hyorhinis, M. hyopneumoniae, M. hyosynoviae and M. flocculare, respectively. Nine of the M. hyopneumoniae cultures were found to be in mixed culture with M. flocculare as...... demonstrated by PCR. By use of a M. flocculare antiserum it was possible to eliminate M. flocculare from M. hyopneumoniae mixed cultures. This study is the first report on isolation of porcine mycoplasmas at Gran Canaria (Spain)....

  5. Design of a covalently bonded glycosphingolipid microarray

    DEFF Research Database (Denmark)

    Arigi, Emma; Blixt, Klas Ola; Buschard, Karsten;

    2012-01-01

    agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G(M2)). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent...... 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut...

  6. Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli.

    OpenAIRE

    Gilmer, T M; Parsons, J T; Erikson, R L

    1982-01-01

    We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli. A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325. Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant p...

  7. Identification of multiple forms of the noncapsid parvovirus protein NCVP1 in H-1 parvovirus-infected cells.

    OpenAIRE

    Paradiso, P R

    1984-01-01

    Analysis of extracts of H-1 parvovirus-infected cells with virus-specific antiserum led to the identification of two forms of the noncapsid virus protein NCVP1. These two proteins had apparent molecular weights of 84,000 (NCVP1) and 92,000 (NCVP1') and were structurally related, based on their immunological reactivity and on peptide map analysis. Both of these proteins appeared early in the virus infection, about the same time that capsid proteins appeared. NCVP1' was a highly phosphorylated ...

  8. Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins.

    Science.gov (United States)

    Konkel, M E; Mead, D J; Cieplak, W

    1996-01-01

    A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequence of the cloned C. jejuni gene exhibits considerable similarity to peptidoglycan-associated lipoproteins from other gram-negative bacteria. PMID:8613402

  9. An Immunoassay to Evaluate Human/Environmental Exposure to the Antimicrobial Triclocarban

    OpenAIRE

    Ahn, Ki Chang; Kasagami, Takeo; Tsai, Hsing-Ju; Schebb, Nils Helge; Ogunyoku, Temitope; Gee, Shirley J.; Young, Thomas M.; Hammock, Bruce D.

    2011-01-01

    A sensitive, competitive indirect enzyme linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum #1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC50 and the detection range for TCC in buffer were 0.70 and 0.13–3.60 ng/mL, respectively. ...

  10. The light organ symbiont Vibrio fischeri possesses a homolog of the Vibrio cholerae transmembrane transcriptional activator ToxR.

    OpenAIRE

    Reich, K A; Schoolnik, G K

    1994-01-01

    A cross-hybridizing DNA fragment to Vibrio cholerae toxR was cloned from the nonpathogenic light organ symbiont Vibrio fischeri, and three proteins homologous to V. cholerae ToxR, ToxS, and HtpG were deduced from its DNA sequence. V. fischeri ToxR was found to activate a V. cholerae ToxR-regulated promoter, and an antiserum raised against the amino-terminal domain of V. cholerae ToxR cross-reacts V. fischeri ToxR.

  11. Radioimmunological and clinical studies with luteinizing hormone releasing hormone (LRH)

    International Nuclear Information System (INIS)

    Radioimmunoassay for Luteinizing Hormone Releasing Hormone (LRH) has been established, tested and applied. Optimal conditions for the performance with regards to incubation time, incubation temperature, concentration of antiserum and radiolabelled LRH have been established. The specificity of the LRH immunoassay was investigated. Problems with direct measurement of LRH in plasmas of radioimmunoassay are encountered. The LRH distribution in various tissues of the rat are investigated. By means of a system for continuous monitoring of LH and FSH in women the lowest effective dose of LRH causing a significant release of LH and FSH could be established. (Auth.)

  12. Novel /sup 125/I radioimmunoassay for the analysis of. delta. /sup 9/-tetrahydrocannabinol and its metabolites in human body fluids

    Energy Technology Data Exchange (ETDEWEB)

    Law, B.; Mason, P.A.; Moffat, A.C.; King, L.J.

    A cannabinoid radioimmunoassay (RIA) that detects some of the major ..delta../sup 9/-THC metabolites is developed and evaluated for use in forensic science. It incorporates a novel /sup 125/I radiotracer, is sensitive, reliable, relatively quick, and simple to use. The RIA uses a commercially available antiserum and detects a number of cannabinoid metabolites, including ..delta../sup 9/-THC-11-oic acid and its glucuronide conjugate in biological fluids. The method was successfully applied to the analysis of blood and urine samples submitted for forensic analysis.

  13. Reticuloendotheliosis virus: Detection of immunological relationship to mammalian type C retroviruses

    International Nuclear Information System (INIS)

    Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH2-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae

  14. Reticuloendotheliosis virus: Detection of immunological relationship to mammalian type C retroviruses. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Charman, H.P.; Gilden, R.V.; Oroszlan, S.

    1979-03-01

    Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH/sub 2/-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae.

  15. イネ水中芽生えの酸素適応過程におけるチトクロムcと11.9kDaタンパク質の増加

    OpenAIRE

    柴坂, 三根夫; 丑丸, 敬史; 大久保, 勝之; 土田, 進一; 辻, 英夫

    1994-01-01

    To examine the changes in cytocrome c content in submerged rice seedlings after exposure to air, antiserum was prepared against purified cytocrome c from rice bran. Western blottong analysis revealed that cytochrome c was detected 6 h after exposure to air, but not detected in submerged rice seedling. On a fresh weight basis, the same level of cytochrome c as that of the aerobic control was found in the 24-h-air adapted seedlings. judging from the high A408/A280 ratio (4.66),the cytochrome c ...

  16. Radioimmunoassay of phenytoin

    International Nuclear Information System (INIS)

    A radioimmunoassay procedure is described for the measurement of phenytoin (5,5-diphenylhydantoin) in serum samples. Antiserum to phenytoin was produced against phenytoin - valeric acid - bovine serum albumin conjugate. 125I labelled phenytoin - acetic acid - tyrosine methyl ester was used as a tracer. The assay covers a range of 10-500 ng/cm3 and has a sensitivity of 0.25 ng. The assay is validated by specificity tests, precision profile and recovery tests. (author) 8 refs.; 6 figs.; 2 tabs

  17. Nociceptin-induced scratching, biting and licking in mice: involvement of spinal NK1 receptors

    OpenAIRE

    Sakurada, Tsukasa; Katsuyama, Sou; Sakurada, Shinobu; Inoue, Makoto; Tan-No, Koichi; Kisara, Kensuke; Sakurada, Chikai; Ueda, Hiroshi; Sasaki, Jun

    1999-01-01

    Intrathecal (i.t.) injection of nociceptin at small doses (fmol order) elicited a behavioural response consisting of scratching, biting and licking in conscious mice. Here we have examined the involvement of substance P-containing neurons by using i.t. injection of tachykinin neurokinin (NK)1 receptor antagonists and substance P (SP) antiserum.Nociceptin-induced behavioural response was evoked significantly 5–10 min after i.t. injection and reached a maximum at 10–15 min. Dose-dependency of t...

  18. Expression and antigenicity characterization for truncated capsid protein of porcine circovirus type 2

    OpenAIRE

    Lou, Zhongzi; Li, Xuerui; Li, Zhiyong; Yin, Xiangping; Li, Baoyu; Lan, Xi; Yang, Bin; Zhang, Yun; Liu, Jixing

    2011-01-01

    Three pairs of specific primers were designed to amplify F2-1, F2-2, and XF2-2 truncated capsid protein genes of porcine circovirus type 2 (PCV-2). Amplified sequences were subcloned to pET-32a(+) vectors and expressed in Rosetta (DE3) Escherichia coli by induction of isopropy-β-D-thiogalactoside (IPTG). All of the fusion proteins had positive reactions to PCV-2 antiserum and His-XF2-2 showed the best reactivity. Proteins were used to immunize BALB/c mice to produce monoclonal antibodies (mAb...

  19. DEVELOPMENT OF A PEROXIDASE-ANTIPEROXIDASE (PAP) TECHNIQUE FOR THE IDENTIFICATION OF HAEMOPHILUS-SOMNUS IN PNEUMONIC CALF LUNGS IN DENMARK

    DEFF Research Database (Denmark)

    Tegtmeier, Conny; Jensen, N.E.; Jensen, H.E.

    A peroxidase-antiperoxidase (PAP) technique was developed for the identification of Haemophilus somnus bacteria in lung tissues of calves. Antisera raised against somatic and wall antigens of a Danish and American strain of H. somnus were produced. Experimentally infected murine tissues were used...... antigens of the Danish strain of H. somnus revealed the highest sensitivity in the PAP technique and became specific following absorption. Heterologous absorption also rendered this antiserum specific in crossed immunoelectrophoresis. Subsequently, the PAP technique was applied on formalin-fixed pneumonic...

  20. DEVELOPMENT OF A PEROXIDASE-ANTIPEROXIDASE (PAP) TECHNIQUE FOR THE IDENTIFICATION OF HAEMOPHILUS-SOMNUS IN PNEUMONIC CALF LUNGS IN DENMARK

    DEFF Research Database (Denmark)

    Tegtmeier, Conny; Jensen, N.E.; Jensen, H.E.

    1995-01-01

    A peroxidase-antiperoxidase (PAP) technique was developed for the identification of Haemophilus somnus bacteria in lung tissues of calves. Antisera raised against somatic and wall antigens of a Danish and American strain of H. somnus were produced. Experimentally infected murine tissues were used...... antigens of the Danish strain of H. somnus revealed the highest sensitivity in the PAP technique and became specific following absorption. Heterologous absorption also rendered this antiserum specific in crossed immunoelectrophoresis. Subsequently, the PAP technique was applied on formalin-fixed pneumonic...

  1. Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

    OpenAIRE

    van Alen-Boerrigter, I J; Baankreis, R; de Vos, W M

    1991-01-01

    The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Koning...

  2. A sensitive radioimmunoassay for a component of mouse casein

    International Nuclear Information System (INIS)

    Mouse casein (m.w. 22,000 daltons) has been purified by employing Sephadex G-100 and DEAE-cellulose column chromatographies. A sensitive radioimmunoassay method has been developed by using [125I]-labelled casein and antiserum elicited in rabbits after injection of glutaraldehyde-treated casein. The assay method is capable of detecting as little as 0.1 ng of casein. The use of the present radioimmunoassay method in detecting casein production in cultured mouse mammary explants has also been demonstrated

  3. Formation of slow-reacting substance by guinea pig immunoglobulins.

    Science.gov (United States)

    Jancar, S.; Akimura, O. K.; Dias da Silva, W.

    1976-01-01

    The capacity of guinea pig antibodies to mediate the antigen-induced release of slow-reacting substance (SRS) in the rat peritoneal cavity is restricted to IgG2 and, to a lesser extent, to IgG1 populations of immunoglobulin. IgM and homocytotropic antibody of the reaginic type lacked this activity. The process was partially blocked by previous decomplementation of the rats, was not affected by previous reduction of the circulating leukocytes, and was partially suppressed by previous depletion of circulating platelets with an antiserum to rat platelets. PMID:11696

  4. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

    OpenAIRE

    Stålbrand, H; Saloheimo, A; Vehmaanperä, J; HENRISSAT, B.; Penttilä, M

    1995-01-01

    beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanas...

  5. Solid-phase radioimmunoassay for the detection of antibodies to collagens

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay using 125I-protein A is described for the detection of antibodies to collagens of different types. The optimal conditions for the adsorption of collagen onto polystyrene microplates, then the incubations with the antiserum and finally with the 125I-protein A have been evaluated. The technique was applied successfully to antisera raised in rabbit, goat, guinea pig and mouse against human type I, II, III, IV, V and bovine type I, II, 1α2α3α, X1-X7 collagens

  6. EXPRESSION OF THE TUMOR-ASSOCIATED PROTEINS IN THE OVARIAN CANCER

    OpenAIRE

    T. S. Bobrova; K. I. Zhordania; Yu. V. Chuyev

    2014-01-01

    With the help of monoclonal antibodies (MCA-1, MCA-3, G1 class immunoglobulins) and polyspecific antiserum (As) by means of immunoblotting (IB) the increase of the expression of the tumor-associated proteins (TAP) in the samples of the tumor tissues (in 94, 55 and 38% respectively), metastatic lymph nodes — LN(m+) and sera of the patients with ovarian cancer (OC) (in 82, 77 and ~100% of the cases, respectively), was detected. The important feature of As was the reactivity with the samples of ...

  7. Molecular characterization of tospoviruses associated with ringspot disease in bell pepper from different districts of Himachal Pradesh.

    Science.gov (United States)

    Sharma, Anshul; Kulshrestha, Saurabh

    2016-06-01

    Bell pepper (Capsicum annuum L.), an important cash crop for the farmers of Himachal Pradesh was found to be affected with tospovirus like disease. An extensive survey was conducted in the bell pepper grown areas in the five districts of Himachal Pradesh to identify and characterize the causative agent. Hence, 60 symptomatic bell pepper plants exhibiting characteristics symptoms were collected from Solan, Sirmaur, Hamirpur, Kangra and Bilaspur districts. Out of 60 samples, 53 samples were found to be positive by DAS-ELISA with tospovirus group specific antiserum. To confirm the presence of tospovirus, DAC-ELISA was performed using GBNV/CaCV polyclonal antiserum and DAS-ELISA with two monoclonal antibodies i.e. TSWV, GRSV. All the 53 samples were found negative for TSWV and GRSV and positive for GBNV/CaCV. Further, eleven infected isolates from both poly-house and open field conditions were selected for characterization at molecular level. RT-PCR was performed with N gene specific primers for TSWV, GBNV and CaCV. The eleven samples selected for molecular identification were further found to be negative for TSWV and positive for CaCV using RT-PCR. One of the samples from district Sirmaur was found to be positive for mixed infection of GBNV and CaCV. N gene phylogenetic analysis of CaCV/GBNV provided important information about the movement and evolution of tospoviruses in Himachal Pradesh. PMID:27366771

  8. Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells

    International Nuclear Information System (INIS)

    The protein (p59/sup rel/) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel. To obtain polyclonal antibodies directed against a larger number of p59/sup rel/ epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-Δ-rel) precipitated p59/sup rel/ from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with peptide antisera. The authors used this new antiserum to localize p59/sup rel/ in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59/sup rel/ was found in the cytoplasmic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies, it was shown that immune precipitates formed with one of the three p59/sup rel/ peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59/sup rel/ or a protein closely associated with it

  9. Identification and characterization of the pseudorabies virus UL43 protein

    International Nuclear Information System (INIS)

    Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants

  10. Radioimmunoassay of 16α-OH-DHA and its sulfate

    International Nuclear Information System (INIS)

    16α-OH-DHA-3-succinate BSA was synthesized and used it as an antigen. This antigen was injected serially into New Zealand white buck rabbits to obtain the antiserum. When it was used in RIA, this antiserum reacted well with both 16α-OH-DHA and its sulfate, and only slightly cross reacted with DHA and its sulfate. The coefficient of variation of the intra assay was 18.64% for 16α-OH-DHA and 21.05% for 16α-OH-DHA-sulfate. The C.V. of the inter assay was 25.5 for 16α-OH-DHA and 28.05% for 16α-OH-DHA-sulfate. Maternal peripheral blood at term registered 3.36 +- 1.91 ng/ml 16α-OH-DHA and 870 +- 220 ng/ml, 16α-OH-DHA-sulfate. The umbilical artery concentration was 7,81 +- 5.52 ng/ml and the umbilical venous concentration was 13.29 +- 10.96 ng/ml for 16α-OH-DHA. The 16α-OH-DHA-sulfate concentrations were 4465.71 +- 2203.05 ng/ml and 3232.85 +- 1322.34 ng/ml respectively. (Evans, J.)

  11. Immunoradiometric assay for the determination of E. coli proteins in recombinant dna derived human growth hormone produced at IPEN-CNEN/SP

    International Nuclear Information System (INIS)

    An immunoradiometric assay (IRMA) for the determination of multiple antigens was set up in order to quantify E. coli (ECP) in lots of purified recombinant human growth hormone (rec-hGH). SDS-PAGE and Western Blotting techniques were carried out, in parallel, to confirm the results obtained by IRMA and to provide more information about the contaminants. Anti-ECP antibodies were obtained by rabbit immunization with ECP, which were submitted to the same purification process utilized for rec-hGH with the exception of the last step. A strain-process-specific assay was thus set up. The antiserum obtained was purified through an affinity column prepared with the same ECP used for immunization, this provided an highly sensitive assay (0,03 ng ECP/mL). This IRMA was shown to be specific, not presenting any cross reaction with hGH and studies carried out on precision, accuracy and linearity of response with dilution confirmed its validity as one of the fundamental purity tests for rec-hGH produced at IPEN-CNEN/SP, whose principles can be easily extended to the analysis of other similar products. These studies have also shown that the utilization of an affinity column, prepared with the described anti-ECP antiserum was very effective, providing rec-hGH lots with less then 10 parts per million (0,001%) of contaminating proteins. (author). 45 refs., 15 figs., 11 tabs

  12. Binding of progesterone to cell surfaces of human granulosa-lutein cells.

    Science.gov (United States)

    Younglai, Edward V; Wu, Yanjing; Foster, Warren G; Lobb, Derek K; Price, Thomas M

    2006-09-01

    Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14-70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action. PMID:16905308

  13. Rearrangement of S-100 immunoreactive Langerhans' cells in human psoriatic skin treated with peptide T.

    Science.gov (United States)

    Wang, L; Hilliges, M; Talme, T; Marcusson, J A; Wetterberg, L; Johansson, O

    1995-01-01

    Dendritic cells marked by protein S-100 (S-100) antiserum in the suprabasal layers of the epidermis have previously been identified to be Langerhans' cells. In this study, S-100 immunoreactive cells have been investigated in psoriatic lesioned skin during and after peptide T treatment. Peptide T is an octapeptide with affinity for the CD4 receptor. Nine patients were intravenously infused with peptide T, 2 mg in 500 ml saline per day for 28 days. Sections from involved skin before, every week during, and after the treatment were processed by indirect immunofluorescence using S-100 antiserum. Before the treatment the epidermal Langerhans' cells were numerically decreased or even completely gone in the involved skin of psoriasis as compared to skin from normal healthy controls, while the dermal dendritic cells instead were increased and gathered in cell clusters around vascular structures. Four of the nine patients had histopathological improvements after the peptide T treatment, and, in those cases, the dendritic cells in the dermis were reduced in number, and the Langerhans' cells in the epidermis were numerically increased as well as even reversed to normal position and morphology. These changes in the distribution and density of Langerhans' cells represent their rearrangement during the course of psoriasis and/or the remission after peptide T treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7727353

  14. Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule-Recombinant L7/L12 Ribosomal Protein of Brucella suis.

    Science.gov (United States)

    Du, Zhi-Qiang; Li, Xin; Wang, Jian-Ying

    2016-08-01

    Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock's reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene's expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein. PMID:27075455

  15. A radioimmunoassay for human ACTH

    International Nuclear Information System (INIS)

    A radioimmunoassay method for human adrenocorticotropic hormone (ACTH) is described. The antiserum was produced in a guinea pig by multiple injections of a total of 1 mg of porcine ACTH adsorbed with CM-cellulose and mixed with complete Freund's adjuvant. The antiserum used for this study at a final dilution of 1:500,000 was obtained from a guinea pig after 10 immunizations. A highly purified native ACTH (Li, α sub(h)sup(1-39) ACTH) was used as an assay standard and a tracer hormone. Separation of free and bound hormone was achieved by dextran coated charcoal. Extraction of ACTH from plasma samples was performed by Donald's method modified with silicic acid. The antibody appeared to be directed against the C-terminal portion of the hormone molecule and showed no significant reaction with other pituitary hormones (GH, TSH, LH, FSH, Hpr, Oxytocin, Arginine-and Lysine-Vasopressin). Plasma ACTH levels of 5 healthy subjects at nine o'clock averaged 32 +- 8.5 pg/ml (M +- SD). Plasma ACTH concentrations in a case of Sheehan's syndrome and of an untreated adrenogenital syndrome at nine o'clock were less than 20 and 194 pg/ml, respectively. A case of Cushing's syndrome accompanied with bilateral nodular hyperplasia of the adrenal cortex showed diminished diurnal variation and normal levels of plasma ACTH. In contrast, elevated plasma ACTH levels and lack of diurnal rhythm of ACTH secretion were observed in a suspected case of Cushing's disease. (auth.)

  16. Antibodies in Cerebrospinal Fluid of Some Alzheimer Disease Patients Recognize Cholinergic Neurons in the Rat Central Nervous System

    Science.gov (United States)

    McRae-Degueurce, Amanda; Booj, Serney; Haglid, Kenneth; Rosengren, Lars; Karlsson, Jan Erik; Karlsson, Ingvar; Wallin, Anders; Svennerholm, Lars; Gottfries, Carl-Gerhard; Dahlstrom, Annica

    1987-12-01

    The etiology of Alzheimer disease is unclear. However, immunological aberrations have been suggested to be critical factors in the pathogenesis of this neurodegenerative disease. This study was carried out to investigate if cerebrospinal fluid (CSF) from Alzheimer disease patients contains antibodies that recognize specific neuronal populations in the rat central nervous system. The results indicate that in a subgroup of patients this is indeed the case. The antibodies reported in this study have the following properties: (i) they recognize neuronal populations and components in the medial septum and spinal motor neurons in rats perfused with a mixture that fixes small neurotransmitter molecules; (ii) adsorption of the patient CSF with staphylococcal protein A-Sepharose and using a polyclonal antiserum against human IgG3 indicates that the immunocytochemical reaction in these brain regions is mainly due to the subclass IgG3; and (iii) the CSF immunocytochemical reaction is blocked by preincubation of the sections with a rabbit anti-acetylcholine antiserum. These results provide evidence that antibodies in the CSF of some, but not all, Alzheimer disease patients recognize acetylcholine-like epitopes in cholinergic neurons in the rat central nervous system.

  17. Antibodies directed to the gram-negative bacterium Neisseria gonorrhoeae cross-react with the 60 kDa heat shock protein and lead to impaired neurite outgrowth in NTera2/D1 cells.

    Science.gov (United States)

    Reuss, B; Asif, A R

    2014-09-01

    Children of mothers with prenatal gonococcal infections are of increased risk to develop schizophrenic psychosis in later life. The present study hypothesizes an autoimmune mechanism for this, investigating interactions of a commercial rabbit antiserum directed to Neisseria gonorrhoeae (α-NG) with human NTera2/D1 cells, an established in vitro model for human neuronal differentiation. Immunocytochemistry demonstrated α-NG to label antigens on an intracellular organelle, which by Western blot analysis showed a molecular weight shortly below 72 kDa. An antiserum directed to Neisseria meningitidis (α-NM) reacts with an antigen shortly below 95 kDa, confirming antibody specificity of these interactions. Two-dimensional gel electrophoresis and partial Western transfer, allowed to localize an α-NG reactive protein spot which was identified by LC-Q-TOF MS/MS analysis as mitochondrial heat shock protein Hsp60. This was confirmed by Western blot analysis of α-NG immunoreactivity with a commercial Hsp60 protein sample, with which α-NM failed to interact. Finally, analysis of neurite outgrowth in retinoic acid-stimulated differentiating NTera2-D1 cells, demonstrates that α-NG but not α-NM treatment reduces neurite length. These results demonstrate that α-NG can interact with Hsp60 in vitro, whereas pathogenetic relevance of this interaction for psychotic symptomatology remains to be clarified. PMID:24577885

  18. Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

    Institute of Scientific and Technical Information of China (English)

    Yu-ying YANG; Ai-jian QIN; Xiong-yan LIANG; Shu-mei TONG

    2008-01-01

    The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC strain).

  19. Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

    Science.gov (United States)

    Orihuela, C. J.; Mills, J.; Robb, C. W.; Wilson, C. J.; Watson, D. A.; Niesel, D. W.

    2001-01-01

    Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

  20. Cloning and characterization of nanos gene in silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Guoli Zhao; Keping Chen; Qin Yao; Weihua Wang

    2008-01-01

    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Droso phila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding se quence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.

  1. Cloning and characterization of nanos gene in silkworm Bombyx mori.

    Science.gov (United States)

    Zhao, Guoli; Chen, Keping; Yao, Qin; Wang, Weihua

    2008-02-01

    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells. PMID:18407054

  2. Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

    International Nuclear Information System (INIS)

    We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of [35S]methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of 125I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice

  3. Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Strand, M.

    1987-10-01

    We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of (/sup 35/S)methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of /sup 125/I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.

  4. Comparative assessment of immunization procedures for development of anti proinsulin antisera for radioimmunoassay

    International Nuclear Information System (INIS)

    Two schedules of immunization were employed for developing anti proinsulin antisera for radioimmunoassay. Biosynthetic human proinsulin-h P I (Elli Lilly. US), was injected subcutaneously in guinea pigs in multiple sites. In the first schedule were used 50 u g of h P I and the booster injections were administered 4 weeks after the primary immunization and then at 3-week intervals. In the second schedule was used 250 u g of h P I and boosters were done 7, 9 and 18 weeks later. As the antisera were not sufficiently specific for h P I they were purified and assessed for kinetic of precipitation and avidity. Both immunization schedules gave comparable responses. The antisera generated by the use of 50 u g of h P I presented higher cross-reactivity with insulin while the reactivity with cpeptide was of the same order in both antiserum groups. The avidity was very variable in the two groups and the three most sensitive antisera required 24 h at 4o C for achieving maximum binding with the 125 I-h P I. However, only one antiserum (from the first group) was suitable for the radioimmunoassay. This study emphasizes the difficulties of making valid comparisons between different immunization procedures, especially in the cases when highest avidity is required. (author)

  5. A soluble-phase proinsulin radioimmunoassay and its use in diagnosis of hypoglycaemia

    International Nuclear Information System (INIS)

    A soluble-phase proinsulin assay has been developed which does not require solid-phase antibody-binding. A human proinsulin standard curve is prepared in insulin-free and proinsulin-free plasma for comparison with unknown plasma samples. Proinsulin and insulin are bound with excess anti-insulin antiserum, and free C-peptide is removed by charcoal adsorption. The supernatant is then assayed using a routine C-peptide radioimmunoassay which utilises anti-C-peptide antiserum. The sensitivity of the assay (2 standard deviations above zero) is 9 pmol/L using 200 μL plasma sample. The assay is free from insulin cross-reactivity up to 100 mU/L and C-peptide up to 2000 pmol/L. Between-assay CV is 13% at 100 pmol/L. The assay has been used in subjects with hypoglycaemia of various aetiologies and has shown that a raised plasma proinsulin in the presence of hypoglycaemia can occur in sulphonylurea-induced and reactive hypoglycaemia as well as in insulinomas. After hyperglycaemic clamps at 7.5, 10 and 15 mmol/L glucose, type II diabetics both on and off sulphonylurea, were found to have lower proinsulin concentrations compared with normal subjects, commensurate with the diabetics' lower insulin responses. (author)

  6. Soluble-phase proinsulin radioimmunoassay and its use in diagnosis of hypoglycaemia

    Energy Technology Data Exchange (ETDEWEB)

    Naylor, B.A.; Matthews, D.R.; Turner, R.C.

    1987-07-01

    A soluble-phase proinsulin assay has been developed which does not require solid-phase antibody-binding. A human proinsulin standard curve is prepared in insulin-free and proinsulin-free plasma for comparison with unknown plasma samples. Proinsulin and insulin are bound with excess anti-insulin antiserum, and free C-peptide is removed by charcoal adsorption. The supernatant is then assayed using a routine C-peptide radioimmunoassay which utilises anti-C-peptide antiserum. The sensitivity of the assay (2 standard deviations above zero) is 9 pmol/L using 200 ..mu..L plasma sample. The assay is free from insulin cross-reactivity up to 100 mU/L and C-peptide up to 2000 pmol/L. Between-assay CV is 13% at 100 pmol/L. The assay has been used in subjects with hypoglycaemia of various aetiologies and has shown that a raised plasma proinsulin in the presence of hypoglycaemia can occur in sulphonylurea-induced and reactive hypoglycaemia as well as in insulinomas. After hyperglycaemic clamps at 7.5, 10 and 15 mmol/L glucose, type II diabetics both on and off sulphonylurea, were found to have lower proinsulin concentrations compared with normal subjects, commensurate with the diabetics' lower insulin responses.

  7. Measurement of insulin in human sera using a new RIA kit. 1

    International Nuclear Information System (INIS)

    A sensitive and versatile radioimmunoassay (RIA) for insulin was established using human insulin standard, a specific guinea pig anti-insulin antiserum and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine T method. Tracer preparations were used for as long as 6 weeks after iodination. The standard curve ranges from 0.044 to 1.2 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.4 and 0.9 nmol/l. The average recovery of human insulin added to plasma or serum samples was 100.2 ± 2.0% (n = 38) and 100.1 ± 1.9% (n = 42), respectively. In addition to human insulin, procine, canine, rabbit and bovine insulin can also be determined but not rat or mouse insulin. The cross-reactivity of the antiserum with porcine proinsulin was found to be 40 % on the molar basis. The range of mean fasting plasma insulin concentrations in healthy subjects and under various pathological conditions were estimated. (author)

  8. The repair of cyclobutane dimers and PYR(6-4) PYO adducts in normal and repair-deficient mammalian cells

    International Nuclear Information System (INIS)

    Antiserum raised against DNA irradiated with a high dose of far UV light was found to have at least two antibody populations. A competitive assay in which the labelled antigen (/sup 32/P-labelled poly (dA):poly (dT)) was irradiated with far UV light (254nm) was shown to be specific for Pyr (6-4) Pyo adducts. Labelled antigen irradiated with near UV light (320nm) competed specifically for cyclobutane dimers. This assay had the same specificity as one consisting of labelled antigen irradiated with far UV light in competition for antiserum raised against DNA irradiated with near UV light in the presence of acetophenone. The authors conclude from these results that their previous data for removal of antibody-binding sites demonstrated repair of Pyr(6-4)Pyo adducts in normal, but not UV-hypersensitive, mammalian cell lines. Using the cyclobutane dimer-specific assay they have been able to correlate the removal of antibody-binding sites with repair kinetics previously published for T4 endonuclease V sensitive sites in mouse, hamster, normal human and Xeroderma pigmentosum cells

  9. A radioimmunoassay for lignin in plant cell walls

    International Nuclear Information System (INIS)

    Lignin detection and determination in herbaceous tissue requires selective, specific assays which are not currently available. A radioimmunoassay (RIA) was developed to study lignin metabolism in these tissues. A β-aryl ether lignin model compound was synthesized, linked to keyhole limpet hemocyanin using a water-soluble carbodiimide, and injected into rabbits. The highest titer of the antiserum obtained was 34 ηg/mL of model derivatized BSA. An in vitro system was developed to characterize the RIA. The model compound was linked to amino activated polyacrylamide beads to mimic lignin in the cell walls. 125I Radiolabelled protein A was used to detect IgG antibody binding. The RIA was shown in the in vitro system to exhibit saturable binding. The amount of antibody bound decreased when the serum was diluted. Immunoelectrophoresis and competitive binding experiments confirmed that both aromatic rings of the lignin model compound had been antigenic. Chlorogenic acid, a phenolic known to be present in plant cells, did not compete for antibody binding. The RIA was used to measure lignin in milled plant samples and barley seedlings. Antiserum binding to wheat cell walls and stressed barley segments was higher than preimmune serum binding. Antibody binding to stressed barley tissue decreased following NaClO2 delignification. The RIA was found to be less sensitive than expected, so several avenues for improving the method are discussed

  10. Monoclonal L-citrulline immunostaining reveals nitric oxide-producing vestibular neurons

    Science.gov (United States)

    Holstein, G. R.; Friedrich, V. L. Jr; Martinelli, G. P.

    2001-01-01

    Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

  11. Fundamental studies on the development of C-peptide radioimmunoassay kit

    International Nuclear Information System (INIS)

    We have studied the development of the C-peptide radioimmunoassay kit which is usable in the pancreatic function test with satisfactory results. The C-peptide antiserum was prepared by immunizing rabbits with synthetic human connecting peptide. The antiserum revealed no cross reaction with any C-peptides other than human C-peptide, porcine insulin and gastrointestinal hormone, and showed high specificity to human C-peptide. We adopted the double antibody method in B,F separation, and chose 40C, 48 hrs. for 1st. incubation and 40C, 24 hrs. for 2nd. incubation. On this kit, the assay range was 0.5 ng/ml-30 ng/ml, the recovery rate was 98.4%-107.8% in the recovery test, the coefficient of variance was 6.2% in the intra assay and was 7.6% in the inter assay. We think this kit is sufficiently usable to assay C-peptide in blood. (auth.)

  12. Relationship between PLAP and high-risk pregnancy

    International Nuclear Information System (INIS)

    PLAP was isolated and purified from human placenta and the antiserum was obtained by immunizing the rabbits. A radioimmunoassay of PLAP (PLAP RIA) was established by labelling the antigen using the chloramine-T method. Its sensitivity was 1.54 μg/L, the recovery rate was between 96.7% and 105.2%, the intra- and inter-assay CV were 8.94% and 9.43%, respectively, the antiserum provided a linear response from 2 to 1000 μg/L. The assay has no cross-reactivity with liver AP. Serum level of PLAP were measured by PLAP RIA in 649 cases of normal pregnancy and 164 cases of high-risk pregnancy. The results indicated that the PLAP level increased proportionally with the advance of gestational age (r = 0.9843). In 33 cases of pregnancy induced hypertension and 21 cases of intrauterine fetal growth retardation, the PLAP were at significantly low level. In 7 cases of neonatal asphyxia and 26 cases of fetal distress, the PLAP level in the mother's serum were also low. In 53 cases of intrahepatic cholestasis of pregnancy, the PLAP level were similar to those of normal pregnancy. This study illustrated that PLAP RIA can play an important role in evaluation of placental function and fetal prognosis for cases of high-risk pregnancy

  13. Borrelia burgdorferi strain-specific Osp C-mediated immunity in mice.

    Science.gov (United States)

    Bockenstedt, L K; Hodzic, E; Feng, S; Bourrel, K W; de Silva, A; Montgomery, R R; Fikrig, E; Radolf, J D; Barthold, S W

    1997-11-01

    Antibodies to the outer surface proteins (Osps) A, B, and C of the spirochete Borrelia burgdorferi can prevent infection in animal models of Lyme borreliosis. We have previously demonstrated that immune serum from mice infected with B. burgdorferi N40 can also prevent challenge infection and induce disease regression in infected mice. The antigens targeted by protective and disease-modulating antibodies are presently unknown, but they do not include Osp A or Osp B. Because Osp C antibodies are present in immune mouse serum, we investigated the ability of hyperimmune serum to recombinant Osp C (N40) to protect mice against challenge infection with N40 spirochetes. In both active and passive immunization studies, Osp C (N40) antiserum failed to protect mice from challenge infection with cultured organisms. Mice actively immunized with recombinant Osp C (N40) were susceptible to tick-borne challenge infection, and nymphal ticks remained infected after feeding on Osp C-hyperimmunized mice. In contrast, similar immunization studies performed with Osp C (PKo) antiserum prevented challenge infection of mice with a clone of PKo spirochetes pathogenic for mice. Both Osp C (N40) and Osp C (PKo) antisera showed minimal in vitro borreliacidal activity, and immunofluorescence studies localized Osp C beneath the outer membrane of both N40 and PKo spirochetes. We conclude that Osp C antibody-mediated immunity is strain specific and propose that differences in Osp C surface expression by spirochetes in vivo may account for strain-specific immunity. PMID:9353047

  14. Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit

    Science.gov (United States)

    Chen, B. Y.; Janes, H. W.

    1997-01-01

    ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.

  15. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    International Nuclear Information System (INIS)

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary λgt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/β-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of 125I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene

  16. Characterization of serum immunoglobulin in channa striata (BLOCH) and kinetics of its response to Aeromonas hydrophila antigen.

    Science.gov (United States)

    Rauta, P R; Mohanty, J; Garnayak, S K; Sahoo, P K

    2013-01-01

    The immunoglobulin (Ig) from the serum of Channa striata was isolated by gel electroelution and characterized further to understand its nature and subsequent applications in studying the immune response. The purity of the sample was confirmed with the presence of a single band on native gradient PAGE and the molecular weight of ∼897 kDa was determined from the gel. In SDS-PAGE, C. striata Ig was reduced to produce two bands corresponding to H (heavy) (∼72 kDa) and L (light) (∼27 kDa) chain subunits. Polyclonal antiserum against the purified Ig was raised in a rabbit and adsorbed with 10% liver tissue homogenate of C. striata to enhance its specificity. By an indirect ELISA standardized using the adsorbed rabbit antiserum, the normal serum Ig concentration in C. striata was estimated to be 3.48 mg/mL. Further, a kinetic study of specific immunoglobulin response to formalin-killed Aeromonas hydrophila antigen was undertaken using another indirect ELISA, which showed a significant increase in serum immunoglobulin titer from day 2 onwards and reached its peak at day 14. Subsequently, the Ig titer was dropped from day 21 onwards till the completion of the experiment at day 42, although it was at a significantly higher level than the control. PMID:23656248

  17. EFFECT OF VASOPRESSIN ON DELAYED NEURONAL DAMAGE IN HIPPOCAMPUS FOLLOWING CEREBRAL ISCHEMIA AND REPERFUSION IN GERBILS

    Institute of Scientific and Technical Information of China (English)

    刘新峰; 金泳清; 陈光辉

    1996-01-01

    Mongolian gerbils were used as delayed neuronal damage (DND) animal models.At the end of 15 minute cerebral ischermia and at various reperfusion time ranging from 1 to 96 hours,the content of water and arginine vasopressin (AVP) in the CA1 sector of hippocampus were measured by the specific gravity method and radioimmunoassy.Furthermore,we also examined the effect of intracerebroventricular (ICV) injection of AVP,AVP antiserum on calcium,Na+,K+-ATP ase activity in the CA1 sector after ischemia and 96 hour reperfusion.The results showed that AVP Contents of CA1 sector of hippocampus during 6 to 96 hour recirculation,and the water content of CA1 sector during 24 to 96 hour were significantly and continuously increased.After ICV injection of AVP,the water content and calcium in CA1 sector of hippocampus at cerebral ischemia and 96 hour recirculation further increased,and the Na+,K+-AT-tion of AVP antiserum,the water contenr and calcium in CA1 sector were significantly decreased as compared with that of control.These suggested that AVP was involved in the pathopysiologic process of DND in hippocampus following cerbral ischemia and reprfusion.Its mechanism might be through the change of intracellular action mediated by specific AVP receptor to lead to Ca inos over-load of neuron and inhibit the Na+,K+-ATPase activity,thereby to exacerbate the DND in hippocampus.

  18. Partial biological and molecular characterization of a Cucumber mosaic virus isolate naturally infecting Cucumis melo in Iran.

    Science.gov (United States)

    Rasoulpour, Rasoul; Afsharifar, Alireza; Izadpanah, Keramat

    2016-06-01

    Melon seedlings showing systemic chlorotic spots and mosaic symptoms were collected in central part of Iran, and a virus was isolated from diseased plants by mechanical inoculation. The virus systemically infected the most inoculated test plants by inducing mosaic symptoms, while, in the members of Fabaceae family and Chenopodium quinoa induced local lesions. Agar gel diffusion test using a polyclonal antiserum against a squash Cucumber mosaic virus (CMV) isolate showed the presence of CMV in the mechanically inoculated plants (designated CMV-Me). The virus was purified by polyethylene glycol precipitation and differential centrifugation. A polyclonal antiserum was produced against the virus that reacted specifically with virus antigen in ELISA and agar gel diffusion tests. The virus was molecularly characterized by PCR amplification of the full length of the coat protein gene using cucumovirus genus specific primer pair CPTALL-3/CPTALL-5 and sequence analysis of the resulting product. No RNA satellite was detected using the primer pair CMVsat3H/sat5T7P. Phylogenetic analysis based on the coat protein amino acid sequences showed that CMV-Me belongs to Subgroup IB. These results may be helpful in melon breeding programs, focusing on plant resistance to plant viruses including CMV. PMID:27366772

  19. Magnetoreception: activated cryptochrome 1a concurs with magnetic orientation in birds

    Science.gov (United States)

    Nießner, Christine; Denzau, Susanne; Stapput, Katrin; Ahmad, Margaret; Peichl, Leo; Wiltschko, Wolfgang; Wiltschko, Roswitha

    2013-01-01

    The radical pair model proposes that the avian magnetic compass is based on radical pair processes in the eye, with cryptochrome, a flavoprotein, suggested as receptor molecule. Cryptochrome 1a (Cry1a) is localized at the discs of the outer segments of the UV/violet cones of European robins and chickens. Here, we show the activation characteristics of a bird cryptochrome in vivo under natural conditions. We exposed chickens for 30 min to different light regimes and analysed the amount of Cry1a labelled with an antiserum against an epitope at the C-terminus of this protein. The staining after exposure to sunlight and to darkness indicated that the antiserum labels only an illuminated, activated form of Cry1a. Exposure to narrow-bandwidth lights of various wavelengths revealed activated Cry1a at UV, blue and turquoise light. With green and yellow, the amount of activated Cry1a was reduced, and with red, as in the dark, no activated Cry1a was labelled. Activated Cry1a is thus found at all those wavelengths at which birds can orient using their magnetic inclination compass, supporting the role of Cry1a as receptor molecule. The observation that activated Cry1a and well-oriented behaviour occur at 565 nm green light, a wavelength not absorbed by the fully oxidized form of cryptochrome, suggests that a state other than the previously suggested Trp•/FAD• radical pair formed during photoreduction is crucial for detecting magnetic directions. PMID:23966619

  20. Morphological Analysis for Neuron-Like Cells in the Vomeronasal Organ of Human Fetuses at the Middle of Gestation.

    Science.gov (United States)

    Takami, Shigeru; Yukimatsu, Maiko; Matsumura, George; Horie, Sawa; Nishiyama, Fumiaki

    2016-01-01

    The vomeronasal organ (VNO) of 5-month-old fetuses was examined immunohistochemically by the use of an antiserum to protein gene product 9.5 (PGP). The purpose was to identify if the human fetal VNO is lined by neuroepithelium. The PGP antiserum labeled abundant cells within the vomeronasal epithelium (VE), nerve fiber bundles in its lamina propria, and cells associated with these bundles. PGP-immunoreactive (ir) vomeronasal epithelial cells were classified into three subtypes. Type I cells, about 44% of the total cells observed, did not have any processes and tended to be located in the basal layer of the VE. Type II cells, about 37% had a single apical process that projected toward the lumen, ending at the epithelial surface. Type III cells sent a prominent process mainly toward the basement membrane, and occupied about 19% of the total cells observed. In the lamina propria, a considerable number of PGP-ir cells was observed. Some of them were present in nerve fiber bundles and contained processes parallel to the bundles. In addition, PGP-ir nerve fiber bundles and cells associated with them were even present in the portion of the nasal septal mucosa that was very close to the brain. The present results strongly suggested that the VE in human fetuses at mid-gestation is a neuroepithelium and that the VE may produce migrating cells toward the brain. PMID:26565893

  1. Monoclonal nicotine-specific antibodies reduce nicotine distribution to brain in rats: dose- and affinity-response relationships.

    Science.gov (United States)

    Keyler, D E; Roiko, S A; Benlhabib, E; LeSage, M G; St Peter, J V; Stewart, S; Fuller, S; Le, C T; Pentel, P R

    2005-07-01

    Vaccination against nicotine is being studied as a potential treatment for nicotine dependence. Some of the limitations of vaccination, such as variability in antibody titer and affinity, might be overcome by instead using passive immunization with nicotine-specific monoclonal antibodies. The effects of antibodies on nicotine distribution to brain were studied using nicotine-specific monoclonal antibodies (NICmAbs) with K(d) values ranging from 60 to 250 nM and a high-affinity polyclonal rabbit antiserum (K(d) = 1.6 nM). Pretreatment with NICmAbs substantially increased the binding of nicotine in serum after a single nicotine dose, reduced the unbound nicotine concentration in serum, and reduced the distribution of nicotine to brain. Efficacy was directly related to antibody affinity for nicotine. Efficacy of the highest affinity NICmAb, NICmAb311, was dose-related, with the highest dose reducing nicotine distribution to brain by 78%. NICmAb311 decreased nicotine clearance by 90% and prolonged the terminal half-life of nicotine by 120%. At equivalent doses, NICmAb311 was less effective than the higher affinity rabbit antiserum but comparable efficacy could be achieved by increasing the NICmAb311 dose. These data suggest that passive immunization with nicotine-specific monoclonal antibodies substantially alters nicotine pharmacokinetics in a manner similar to that previously reported for vaccination against nicotine. Antibody efficacy is a function of both dose and affinity for nicotine. PMID:15843487

  2. Purification and partial characterization of English sole (Pleuronectes vetulus) vitellogenin.

    Science.gov (United States)

    Roubal, W T; Lomax, D P; Willis, M L; Johnson, L L

    1997-11-01

    Vitellogenin (VTG) was purified by double-step chromatography from plasma of male English sole treated with 17 beta-estradiol. The intact protein appeared to exist as a dimer in two forms of approximately 300 and 320 kDa and had an isoelectric point of 6.63. In SDS-PAGE, it was reduced to a single monomer of approximately 130 kDa. In immunoblotting, the protein showed cross-reactivity with coho salmon VTG antiserum. Native PAGE (sample not treated with the reducing agent mercaptoethanol) and immunoblotting of plasma from control and estradiol-treated male sole and gravid female sole demonstrated that the putative English sole VTG was normally female specific and estradiol inducible in males. It was immunocytochemically localized in liver and ovary of English sole, rock sole and starry flounder, using polyclonal antiserum to the purified protein from the estradiol-treated male English sole. The protein was characterized as a phospholipoglycoprotein by native PAGE, staining the gels for phosphorus with methyl green, for lipid with Sudan black B and for carbohydrate by an improved periodic-acid Fuchsin sulfite method. The amino acid composition of the putative VTG was generally similar to that of VTGs from other teleosts, with the non-polar amino acids alanine, valine, leucine and isoleucine accounting for one-third of the total amino acids present. However, English sole vitellogenin contained a higher proportion of leucine and a lower proportion of glycine than most other teleost vitellogenins isolated to date. PMID:9467873

  3. Protective role of antibodies induced by Brucella melitensis B115 against B. melitensis and Brucella abortus infections in mice.

    Science.gov (United States)

    Adone, Rosanna; Francia, Massimiliano; Pistoia, Claudia; Petrucci, Paola; Pesciaroli, Michele; Pasquali, Paolo

    2012-06-01

    It has been demonstrated that antibodies specific for O-PS antigen of Brucella smooth strains are involved in the protective immunity of brucellosis. Since the rough strain Brucella melitensis B115 was able to protect mice against wild Brucella strains brucellosis despite the lack of anti-OPS antibodies, in this study we evaluated the biological significance of antibodies induced by this strain, directed to antigens other than O-PS, passively tranferred to untreated mice prior to infection with Brucella abortus 2308 and B. melitensis 16M virulent strains. The protective ability of specific antisera collected from mice vaccinated with B. melitensis B115, B. abortus RB51 and B. abortus S19 strains was compared. The results indicated that antibodies induced by B115 were able to confer a satisfactory protection, especially against B. abortus 2308, similar to that conferred by the antiserum S19, while the RB51 antiserum was ineffective. These findings suggest that antibodies induced by B115 could act as opsonins as well as antibodies anti-O-PS, thus triggering more efficient internalization and degradation of bacteria within phagocytes. This is the first study assessing the efficacy of antibodies directed to antigens other than O-PS in the course of brucellosis infection. PMID:22521283

  4. Role of aVb3 integrin in embryo implantation in the mouse

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Integrin, a heterodimeric adhesive molecule composed of a and b subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the "implantation window" stage, aVb3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that aVb3 integrin was clearly expressed in the mouse blastocyst. Injection of aVb3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, aVb3 integrin antisera at 1:100 and 1:200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of aVb3 integrin antiserum was dosage/dilution-dependent. Thus, aVb3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at "implantation" stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.

  5. Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A

    Institute of Scientific and Technical Information of China (English)

    Si-Wu Fu; Ya-Li Zhang; Dian-Yuan Zhou

    2004-01-01

    AIM: To establish an ELISA kit using monoclonal antibodiesagainst Clostridium difficile ( C. difficile) toxin A.METHODS: An indirect sandwich ElISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 fiat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficiletoxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif infantis, 5 strains of V. cholera, 2 strains ofS. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL.CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

  6. NEWCASTLE DISEASE VIRUS IN THE INTESTINAL CONTENTS OF BROILERS AND LAYERS

    Directory of Open Access Journals (Sweden)

    S. Ullah, M. Ashfaque, S.U. Rahman, M. Akhtar and A. Rehman

    2004-01-01

    Full Text Available Two hundred intestines pieces (100 each of broilers and layers of about 8 cm length were collected from the poultry sale shops in Faisalabad city. These pieces were opened, scratched and vigorously shaken into sterilized normal saline, the suspension was centrifuged and supernatants were subjected to spot haemagglutination with 2% chicken RBC’s. Out of 200 samples, 95% samples of layers and 75% of the broilers showed positive spot haemagglutination. Micro haemagglutination inhibition with Newcastle disease (ND antiserum revealed, 85 and 66 samples positive in layers and broilers respectively. A total of 10% samples of the layers and 9% of the broilers were not inhibited by ND antiserum suggesting other HA viruses. A total of 20 samples were used to isolate the virus in embryonated eggs (allantoic route. These isolates were confirmed as NDV by haemagglutination inhibition test. Five isolates were tested for intracerebral pathogenicity index (ICPI in day old chicks. The ICPI values obtained were 0.28, 0.31, 0.37, 0.38 and 0.46. The isolates were found to be lentogenic.

  7. Novel antennal lobe substructures revealed in the small hive beetle Aethina tumida.

    Science.gov (United States)

    Kollmann, Martin; Rupenthal, Anna Lena; Neumann, Peter; Huetteroth, Wolf; Schachtner, Joachim

    2016-03-01

    The small hive beetle, Aethina tumida, is an emerging pest of social bee colonies. A. tumida shows a specialized life style for which olfaction seems to play a crucial role. To better understand the olfactory system of the beetle, we used immunohistochemistry and 3-D reconstruction to analyze brain structures, especially the paired antennal lobes (AL), which represent the first integration centers for odor information in the insect brain. The basic neuroarchitecture of the A. tumida brain compares well to the typical beetle and insect brain. In comparison to other insects, the AL are relatively large in relationship to other brain areas, suggesting that olfaction is of major importance for the beetle. The AL of both sexes contain about 70 olfactory glomeruli with no obvious size differences of the glomeruli between sexes. Similar to all other insects including beetles, immunostaining with an antiserum against serotonin revealed a large cell that projects from one AL to the contralateral AL to densely innervate all glomeruli. Immunostaining with an antiserum against tachykinin-related peptides (TKRP) revealed hitherto unknown structures in the AL. Small TKRP-immunoreactive spherical substructures are in both sexes evenly distributed within all glomeruli. The source for these immunoreactive islets is very likely a group of about 80 local AL interneurons. We offer two hypotheses on the function of such structures. PMID:26496732

  8. Studies on porcine pancreatic elastase activity, 1

    International Nuclear Information System (INIS)

    An improved method of radioimmunoassay was devised to offer a successful formula for determining blood concentration of elastase. With porcine pancreatic elastase as the antigen, rabbits were immunized to obtain antiserum. Iodinated elastase labeled by the chloramine-T procedure using 131I (or 125I) had a specific activity of 200 - 300 mCi/mg. The double antibody method was used for BF separation. While the usual method of radioimmunoassay was not always successful in obtaining accurate serum concentration of elastase, the use of diisopropyl fluorophosphate (DFP) was able to eliminate the disturbing influence of intra-serous inhibitors, α1-AT and α2-M, eventually producing satisfactory results. With the use of DFP, the elastase standard curve and the porcine serum dilution curve had a statistically significant correlation; precision and recovery were both satisfactory; cross-reactivity of the antiserum with trypsin and chymotrypsin was less than 0.001%. The minimal detectable concentration of elastase was 5 ng/ml, and the range of normal fasting porcine serum level was 70 - 100 ng/ml. (author)

  9. Preclinical evaluation of three polyspecific antivenoms against the venom of Echis ocellatus: Neutralization of toxic activities and antivenomics.

    Science.gov (United States)

    Calvete, Juan J; Arias, Ana Silvia; Rodríguez, Yania; Quesada-Bernat, Sarai; Sánchez, Laura V; Chippaux, Jean Philippe; Pla, Davinia; Gutiérrez, José María

    2016-09-01

    Snakebite envenoming has a heavy burden in the public health in sub-Saharan Africa. The viperid species Echis ocellatus (carpet viper or saw-scaled viper) is the medically most important snake in the savannahs of western sub-Saharan Africa. Several antivenoms are being distributed and used in this region for the treatment of envenomings by E. ocellatus, but the preclinical efficacy of some of these antivenoms has not been assessed. The present study evaluated the preclinical efficacy against E. ocellatus venom of three polyspecific antivenoms: (a) Snake Venom Antiserum (Pan Africa), manufactured by Premium Serums and Vaccines (India); (b) Snake Venom Antiserum (Africa), manufactured by VINS Bioproducts (India); and (c) Antivipmyn(®) Africa, manufactured by Instituto Bioclon (Mexico). Antivenomics analysis revealed the ability of the three antivenoms to immunocapture the majority of components of the venoms of E. ocellatus from Cameroon, Nigeria and Mali, although their maximal immunocapturing capability varied. Bioclon and Premium Serums antivenoms were effective in the neutralization of lethal, hemorrhagic and in vitro coagulant activities of the venom of E. ocellatus from Cameroon, albeit with different potencies. VINS antivenom neutralized hemorrhagic activity of this venom, but failed to neutralize lethality at the highest antivenom dose tested, and had a low neutralizing efficacy against in vitro coagulant effect. PMID:27377229

  10. Identification of the blood-borne somatotroph-differentiating factor during chicken embryonic development.

    Science.gov (United States)

    Morpurgo, B; Dean, C E; Porter, T E

    1997-11-01

    Somatotrophs become a significant population by day 16 of chicken embryonic development. We have previously demonstrated that an earlier induction of GH cell differentiation is possible with the addition of day 16 embryonic serum to cultures of day 12 pituitary cells, an age when somatotrophs are rare. The present study was designed to identify the blood-borne signal(s) responsible for the serum activity, using reverse hemolytic plaque assays to identify individual GH-secreting cells. The activity was found to be a heat-stable, ether-soluble compound(s) that is bound or inhibited by a trypsin-sensitive protein. The extent of GH cell differentiation was greater (P estradiol, corticosterone, and progesterone. However, the estradiol receptor antagonist, tamoxifen, while abolishing the effect of estradiol, had no effect on the induction of differentiation by day 16 serum. In contrast, RU486, a specific glucocorticoid receptor antagonist in chickens, blocked the stimulatory effects of corticosterone, progesterone, and day 16 serum on somatotroph differentiation. We next tested whether the active compound in day 16 embryonic serum was corticosterone, the predominant glucocorticoid in chickens. Incubation of day 16 serum with corticosterone antiserum, but not control antiserum, suppressed day 16 serum-induced GH cell differentiation. Therefore, we conclude that corticosterone is the blood-borne signal capable of stimulating somatotroph differentiation in vitro. The present findings together with previous reports indicate that somatotroph differentiation during embryonic development may result from an increase in circulating glucocorticoid concentrations. PMID:9348174

  11. Effect of radiation on normal hematopoiesis and on viral induced cancers of the hematopoietic system. Technical progress report, August 1, 1974--May 1, 1975. [Mice, x radiation

    Energy Technology Data Exchange (ETDEWEB)

    Okunewick, J.P.

    1975-01-01

    Studies carried out during the above period on viral leukemia have conclusively shown that the pluripotent hematopoietic colony forming stem cell (CFU-S) is a target cell for the leukemia virus. Treatment of this cell population with antiserum prepared in syngeneic mice against the disease resulted in inactivation of up to 50 percent of the CFU-S obtained from the spleens of viral leukemic mice. At the same time, normal serum had no effect on these cells, nor did the antiserum have any effect on normal CFU-S. Data indicated that a considerable time delay, on the order of a week, preceded the expression of the viral antigen in the leukemic CFU-S, but that it could be seen at all times after that up to the terminal point of the disease. We examined the effect of the virus on DNA synthesis (S-phase cells) in the CFU-S immediately after virus injection. The results showed that a doubling of the number of cells in S could be seen as early as four hours after introduction of the virus into the animal. Studies with ethidium bromide, an inhibitor of viral reverse transcriptase, were found to be in agreement with this observation. When given to viral leukemic animals in combination with fractionated exposure to x-ray, the data suggested that ethidium bromide did act to extend survival somewhat, but not much over that seen through the use of x-ray alone.

  12. Production of antibodies against secretin and their use for radioimmunoassay of secretin

    International Nuclear Information System (INIS)

    Synthetic thyroglobulin was bonded to bovine albunia and thyroglobulin according to the principle of the carbodiimide condensation reaction. 16 rabbits were immunized with these conjugates and with unconjugated secretin. Secretin labelling with 125I was carried out by the chloramin-T method. The tracer has a specific activity of 15.45 mCi/mg. A secretin RIA was developed using the double antibody method. The sensitivity of the system could be raised by variation of the specific activity of the tracer and optimisation of the incubation parameters. Antisera were compared. The titers of secretin/bovine albumine conjugate antisera were similar to the antisera against secretin thyroglobulin conjugate. The sensitivity of the standard curves was higher for secretin/bovine albumin conjugate antisera than for thyroglobulin conjugate antisera. Two antisera were tested for specificity. The detection threshold of antiserum S 5 IX was 12,43 pmol/l while the 50% intercept was at 55.45 pmol/l. This antiserum is particularly suitable for a secretin RIA. (orig./MS)

  13. Characterization of a multimeric polypeptide complex on the surface of thymus-derived cells in the Mexican axolotl.

    Science.gov (United States)

    Kerfourn, F; Guillet, F; Charlemagne, J; Tournefier, A

    1993-10-01

    We previously raised a rabbit antiserum (L12) against a 38 kD polypeptide which is expressed on the surface of thymocytes and peripheral T cells of an Urodele Amphibian, the Mexican axolotl (Ambystoma mexicanum). Here we show that L12 antibodies immunoprecipitate several labelled molecules from surface iodinated axolotl spleen cells, including the 38 kD molecule, but also two polypeptides of 43 and 22 kD which are covalently linked to other elements. Another rabbit antiserum (L10) was raised against detergent-solubilized axolotl thymocyte membranes and shown to recognize the majority of thymocytes and about half of the splenocytes in immunofluorescence. In Western blotting, L10 antibodies recognized a limited number of surface polypeptides in thymocyte and splenocyte lysates, including 43, 38, and 22 kD elements. Immune complexes formed between L10 antibodies and solubilized splenocyte membranes were used to immunize BALB/c mice intrasplenically in the aim of raising MoAbs specific for axolotl T cells. Monoclonal antibody 87.16 was shown to stain in immunofluorescence 26.7% of thymocytes and 26.8% of spleen cells. This MoAb recognized a 43 kD polypeptide that can covalently associate on the T-cell surface with several other molecules to form a multimeric complex. PMID:8211000

  14. Evaluation of radioimmunoassays using mono- and polyclonal antibodies for the quantitation of human chorionic gonadotropin in early pregnancy

    International Nuclear Information System (INIS)

    Five commercial kits for the quantitative determination of human chorionic gonadotropin (hCG) were evaluated for use in an In Vitro Fertilization program. We focused on practicability, precision, sensitivity and the early detection of pregnancy. The IRE method uses purified antiserum against complete hCG and requires overnight incubation. Precision is poor but hCG results agree closely with the other assays. Three other kits use a β-subunit specific antiserum (Amersham, Becton-Dickinson, Clinical Assays). All these assays have acceptable precision especially in their mid-range. The Amersham assay has the shortest assay time, while the Clinical Assays method requires the least manipulation. The Becton Dickinson assay is precise, less sensitive and has the highest frequency of slightly elevated hCG levels in males and non pregnant females. The Hybritech kit is an immunoradiometric assay based on two monoclonal antibodies. It was tested with a two-step incubation procedure needing a longer assay time than the β-subunit specific assays but it has the best precision at low hCG levels and is the most sensitive test with the least interference in samples obtained from males and non-pregnant females. For the early detection of pregnancy all assays performed equally well

  15. Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands.

    Science.gov (United States)

    Miller, Patti J; Afonso, Claudio L; Spackman, Erica; Scott, Melissa A; Pedersen, Janice C; Senne, Dennis A; Brown, Justin D; Fuller, Chad M; Uhart, Marcela M; Karesh, William B; Brown, Ian H; Alexander, Dennis J; Swayne, David E

    2010-11-01

    The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons. PMID:20702635

  16. Radioimmunoassay for serum 11-deoxy-17-ketosteroids

    International Nuclear Information System (INIS)

    A simplified method for evaluating serum 11-deoxy-17-ketosteroids (11-deoxy-17-KS) equivalent to dehydroepiandrosterone sulfate (DHEAS) has been developed without solvolysis and chromatography. Blood serum or plasma (5 mu l) was added to 1 ml of ethanol, mixed, and centrifuged, and 10 or 20 mu l of the supernatant was evaporated to dryness and incubated with anti-11-deoxy-17-KS antiserum obtained by immunizing a rabbit with DHEA-3.O. CO-BSA which was prepared from DHEA-3.O.COCl and containing DHEAS-7α3H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEAS-7α3H. The accuracy, precision and sensitivity were satisfactory. The blank values could not be differentiated from zero. As the antiserum reacted not only on DHEAS but also on androsterone sulfate and etiocholanolone sulfate, serum 11-deoxy-17KS obtained by the radioimmunoassay expressed nearly the sum of 100 percent of DHEAS, 45 percent of androsterone sulfate and 35 percent of etiocholanolone sulfate in the serum. A good correlation was found between serum 11-deoxy-17-KS and DHEAS obtained by the radioimmunoassay described in a previous paper. The present radioimmunoassay is the simplest method for the evaluation of the concentrations of C19 steroids in the serum. (auth)

  17. Specificity of direct radioimmunoassays of unconjugated estrone and estradiol-17β in plasma

    International Nuclear Information System (INIS)

    Procedures for radioimmunoassay of estrone and estradiol-17β in plasma, which do not include a chromatographic step or even plasma extractions are now described in the literature and are recommended in most commercially available kits. Besides leading often to a systematic overestimation of estrogen concentrations, such techniques are liable to give results which are volume-dependent. Since this defect might be due to the type of antiserum used or the mode of separation of free and antibodybound steroid, the influence of plasma volume was studied, either with a modification of Castanier and Scholler method - i.e. suppression of Sephadex LH-20 chromatography -, using antisera of different types, or with estrogen kits. The separation step was a toluene extraction in the modified method, and dextran-coated charcoal or polyethylene glycol in procedures of commercial kits. The volume effect was always found, with varying degrees, according to the plasma sample. It was most marked in plasmas of hyperlipidemic patients. The addition of fatty acids, at an upper physiological dose, led to a modification of the standard curve, with over-estimation at low levels and under-estimation at high levels, resulting in calculated estrogen concentrations depending on volume. The use of highly specific antiserum as determined by cross-reaction studies with authentic steroids does not necessarily imply the possibility of performing direct assays which ought to pass the test of plasma volume effect before being accepted. (orig.)

  18. Polyclonal B-cell activation by Neisseria meningitidis capsular polysaccharides elicit antibodies protective against Trypanosoma cruzi infection in vitro.

    Science.gov (United States)

    Oliveira, T G; Milani, S R; Travassos, L R

    1996-01-01

    A hyperimmune rabbit antiserum against group C Neisseria meningitidis agglutinated and lysed Trypanosoma cruzi metacyclic trypomastigotes in a complement-mediated reaction. Immunization of rabbits with the purified polysaccharide C from N. meningitidis and of human volunteers with the AC-polysaccharide vaccine against meningitis also resulted in antibody production cross-reactive with T. cruzi infective forms. The rabbit antibodies bound to parasites, lysed metacyclic forms, and recognized several components from lysates of cell-derived trypomastigotes. The sera from six human volunteers reacted with cell-cultured trypomastigotes in vitro, lysed these forms, and recognized glycoconjugates migrating diffusely on the top of immunoblots. One serum also reacted with the isolated mucin-like glycoconjugate carrying the Ssp-3 epitope from cell-derived trypomastigotes, but treatment with sialidase did not abolish this reactivity. The anti-AC human antiserum also protected against HeLa cell infection and markedly decreased the number of parasites liberated after cell burst. The polyclonal response that resulted from human immunization with N. meningitidis polysaccharides A and C comprised trypanolytic antibodies that recognized nonsialylated epitopes expressed on infective forms of the parasite. It is suggested that human AC vaccination could be potentially helpful as an adjuvant to a specific immunotherapy of Chagas disease, developed with native or recombinant antigens of the parasite. PMID:8811466

  19. A commercial human protamine-2 antibody used in several studies to detect mouse protamine-2 recognizes mouse transition protein-2 but not protamine-2.

    Science.gov (United States)

    Eckhardt, Matthias; Wang-Eckhardt, Lihua

    2015-11-01

    The exchange of histones for transition proteins (TNPs) and finally protamines is an essential process during spermatogenesis that enables the strong condensation of chromatin during sperm formation. Research on this process obviously depends on the availability of specific antibodies recognizing these nuclear proteins. A commercial antibody generated against human protamine-2 (PRM2) has been described to cross-react with mouse PRM2 and in fact has been used in several studies to detect mouse PRM2. Some inconsistent results obtained with this goat-derived antibody prompted us to re-examine its specificity. In immunofluorescence experiments with epididymal sperm, only a low percentage of sperm nuclei were stained by this antibody, whereas a mouse monoclonal anti- PRM2 antibody stained most sperm, as expected. Western blot analysis of basic nuclear proteins from spermatids and sperm separated by acid urea (AU) gel electrophoresis revealed that the goat anti- PRM2 antiserum binds to mouse TNP2 but not mouse PRM2. Epitope mapping using glutathione-S-transferase-fusion proteins with peptide sequences conserved in human PRM2 and mouse TNP2 identified the tetrapeptide arginyl-lysyl-arginyl-threonine as an epitope of the goat anti- PRM2 antiserum. Our findings underline the importance of using AU gel electrophoresis to confirm specificities of antibodies directed against basic nuclear proteins, which are not well separated, and may show abnormal migration behaviour, in SDS-polyacrylamide gel electrophoresis. PMID:26268249

  20. Robert Feulgen Prize Lecture. Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease.

    Science.gov (United States)

    Green, C R; Severs, N J

    1993-02-01

    In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131-142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal

  1. Production, isolation, and characterization of rabbit anti-idiotypic antibodies directed against human antithyrotrophin receptor antibodies.

    Science.gov (United States)

    Baker, J R; Lukes, Y G; Burman, K D

    1984-01-01

    Previous studies have shown that anti-idiotypic antibodies can be developed in vivo through animal immunization with idiotype, and that these antibodies can be isolated from other anti-immunoglobulin antibodies by affinity purification. These techniques have relied on large amounts of idiotype, which were produced either by hyperimmunization or by monoclonal antibodies, to serve as the affinity adsorbent. In the present study, we produced anti-idiotypic antibodies to human anti-thyroid-stimulating hormone (TSH) receptor antibodies by first injecting rabbits with (TSH receptor purified) IgG from Graves' patients. The resulting antiserum was then adsorbed with Sepharose-coupled TSH in an attempt to specifically bind and isolate the anti-idiotype. The antibody obtained from this process was shown to bind specifically to TSH receptor-binding antibodies from Graves' patients, and this binding could be inhibited by 56% with the addition of 10(-4) M TSH but not by HCG (10(-2) M). The anti-idiotype also bound to TSH, and this binding could be specifically inhibited by receptor-purified Graves' IgG (60% inhibition at 10 micrograms/ml IgG), but not by IgG from normal subjects (no inhibition at 50 micrograms/ml IgG). In a TSH receptor binding assay, the anti-idiotype could inhibit TSH receptor binding in Graves' sera at a 1,000-fold lower concentration than could anti-kappa/lambda antiserum; the anti-idiotypic antiserum also inhibited in vitro TSH-mediated adenylate cyclase stimulation at an IgG concentration of 5 micrograms/ml, while heterologous anti-TSH antisera and normal IgG at similar concentrations had no effect. Finally, despite being generated against a single patient's TSH receptor binding antibody, the anti-idiotype was able to block TSH receptor binding in the serum of six other Graves' patients, thus suggesting that there may be conformational conservation in the antigen that is recognized by different individuals' TSH receptor-binding immunoglobulins. PMID

  2. Solid phase radioimmunoassay for plasma testosterone using a plastic microtiter tray

    International Nuclear Information System (INIS)

    In order to simplify radioimmunoassay for plasma testosterone and to measure many samples at the same time, a method of solid phase radioimmunoassay utilizing a plastic disposable microtiter tray (DMT) by which chromatography can be omitted was investigated. Other steroids except for 5α-dihydrotestosterone (5α-DHT) had a low degree of cross reactivity with the antiserum. Five α-DHT which could be measured together with testosterone in this assay was not a problem clinically because of its strong androgenic activity. The best standard curve was obtained when the antiserum was diluted to 1:1000. The sensitivity of this assay was 10 pg-tube. The maximal adsorption of antibody to plastic DMT was observed when the pH of the antiserum was within the range of 6.5-9.5 and the precoating time was 24 hr at room temperature. The best pH of the incubation buffer was 0.8, and the antigen-antibody reaction became a plateau when the incubation exceeded 6 hrs. The water blank in this assay was 4.6 +- 2.1 pg/tube. The recovery of testosterone (50, 100, 200 pg) when added to 0.1 ml female plasma was 99 +- 6.8%. Coefficients of variation within assay and between assays were below 11.2% and 20.0%, respectively. Correlation between this method and the dextran-coated charcoal method was fairly good (r=0.938). Plasma testosterone levels in 10 normal males and 12 normal females were 616 +- 202 (mean +- SD) ng/dl and 66 +- 29 (mean +- SD) ng/dl, respectively. The levels were low in patients with hypopituitarism, hypogonadism and acromegaly. They were normal in patients with Cushing's syndrome due to adrenal hyperplasia and adenoma, but they were high in a patient with adrenal carcinoma. In a patient with testicular feminization, the level was 632 ng/dl. This increased after the administration of HCG, and decreased to 127.5 ng/dl after castration. (auth.)

  3. The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques

    Directory of Open Access Journals (Sweden)

    Burnside Kellie L

    2009-11-01

    Full Text Available Abstract Background ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV, is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1 and three species of macaques (RFHVMm, RFHVMn and RFHVMf, and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively. We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus and MneRV2 (pig-tail, with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses

  4. Expression of Arcanobacterium pyogenes pyolysin in Escherichia coil and the hemolytic activity identification%化脓隐秘杆菌溶血素蛋白的原核表达及其溶血活性鉴定

    Institute of Scientific and Technical Information of China (English)

    孟祥莉; 母晓宇; 刘晓丹; 徐凝; 王璞; 高明春; 张文龙; 王君伟

    2013-01-01

    为研究化脓隐秘杆菌致病机制及其病原学诊断方法,本研究克隆了编码化脓隐秘杆菌溶血素蛋白的plo基因,并构建重组表达质粒pET-plo,转化大肠杆菌Rosetta (DE3)感受态细胞中诱导表达.SDS-PAGE检测结果显示,表达的重组蛋白约为62 ku,western blot分析表明表达的重组蛋白可以与鼠抗化脓隐秘杆菌血清发生反应.采用重组蛋白免疫新西兰白兔制备的多克隆抗体效价达到1∶128 000,western blot和琼脂双扩散试验表明制备的多克隆抗体能够与天然PLO蛋白发生反应.溶血试验表明重组蛋白能够溶解红细胞,制备的多克隆抗体能有效中和重组蛋白的溶血活性.%Pyolysin (PLO),the hemolytic exotoxin expressed by Arcanobacterium pyogenes,is a major virulence factor in infections by A.pyogenes.The gene encoding PLO was amplified by PCR from A.pyogenes genome and cloned into the pET-30a for expression in E.coli Rosetta.The expressed protein was approximately 62 ku,mainly in form of inclusion body.The recombinant PLO protein (rPLO) was recognized by mouse antiserum against A.pyogenes.In addition,the polyclonal antibody against rPLO was prepared in rabbits and purified.The titre of antiserum was 1:128,000 detected by ELISA,which reacted with native PLO of A.pyogenes in western blot and agar gel diffusion test.Hemolytic assays show that rPLO possessed hemolytic activity which was completely neutralized by the antiserum.these results provide necessary basic for pathogenesis investigation and development of diagnostic method of A.pyogenes infection.

  5. Cloning, sequencing, characterisation and implications for vaccine design of the novel dihydrolipoyl acetyltransferase of Neisseria meningitidis.

    Science.gov (United States)

    Ala' Aldeen, D A; Westphal, A H; De Kok, A; Weston, V; Atta, M S; Baldwin, T J; Bartley, J; Borriello, S P

    1996-12-01

    A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity

  6. [A rare cause of compartment syndrome of the forearm and hand following snake bite injury].

    Science.gov (United States)

    Schnecker, K

    1990-06-01

    With the intention to commit suicide a 25 year old patient was bitten by his own rattle snake. At the time of the admission the skin of the right forearm was dark, a hemorrhagic necrotizing colour, and the patient was in shock. He was immediately taken to the intensive care unit and the shock symptoms were treated there. Parasthesias in the area of the nervus medianus were also noticed. The treatment included an antiserum and the release of the tourniquet which caused a further increase of the swelling of the forearm. The lesion led to a hemorrhagic necrotizing inflammation. The surgical incision of the loge of Guyon, the carpal channel, the forearm and proximal of the lacertus fibrosus was persuaded. The circulation improved immediately and after three weeks the nerval function had recovered. The skin defect was covered 14 days after the first operation with meshgraft. PMID:2382320

  7. Immunoreactive transforming growth factor alpha and epidermal growth factor in oral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Therkildsen, M H; Poulsen, Steen Seier; Bretlau, P

    1993-01-01

    Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected with a...... monoclonal mouse antibody and EGF with polyclonal rabbit antiserum. Thirty-five of the tumours were positive for TGF-alpha and 26 of the tumours for EGF. None of the poorly differentiated tumours was positive for EGF, but they all were for TGF-alpha. In sections including normal differentiated oral mucosa......, the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells...

  8. Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

    DEFF Research Database (Denmark)

    Gibson, W T; Couchman, J R; Badley, R A; Saunders, H J; Smith, C G

    1983-01-01

    The aim of this study was to investigate whether epidermal cells can synthesise fibronectin and whether the distribution of this glycoprotein is related to the adhesion and cytoskeletal organisation of these cells. The production of fibronectin by newborn rat epidermal cells was shown by indirect...... immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact...... revealed evidence of a relationship between external fibronectin and internal structure in epidermal cells. Immunofluorescence showed that tonofilaments (keratin) and microtubules were present as fibrillar arrays but were not related to fibronectin distribution. Vimentin and desmin were absent. Actin was...

  9. Nerve Growth Factor Inhibits Sympathetic Neurons' Response to an Injury Cytokine

    Science.gov (United States)

    Shadiack, Annette M.; Vaccariello, Stacey A.; Sun, Yi; Zigmond, Richard E.

    1998-06-01

    Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

  10. Immunocharacteristics of oestrogen and androgen target cells in the anterior pituitary gland of the chick as embryo demonstrated by a combined method of autoradiography and immunohistochemistry

    International Nuclear Information System (INIS)

    The distribution of oestrogen and androgen target cells in the anterior pituitary gland of the chick embryo on days 10, 12 and 15 of incubation was studied 1 h after the injection of tritium-labelled steroid hormone using the thaw-mount autoradiographic technique. Oestradiol target cells were localized in the caudal zone that corresponds to the so-called 'caudal lobe', while androgen target cells were found throughout the rostral and caudal lobes of the anterior gland. With a combined autoradiography and immunohistochemistry technique, most of the oestrogen target cells showed immunoreactivity to turkey LH antiserum but not to adrenocorticotrophin (1-24) and β-thyrotrophin antisera. In contrast, androgen target cells did not show positive immunoreactivity to the three antisera used. The results suggested a direct and early involvement of oestrogens but not of androgens in the feedback regulation of pituitary gonadotrophin secretion in the chick embryo. (U.K.)

  11. Simple immunochemical method for measuring DNA repair rate in u.v.-irradiated bacteria

    International Nuclear Information System (INIS)

    A straightforward immunochemical method is reported to determine the repair rate of DNA in u.v. (ultraviolet)-irradiated bacteria based on use of antibodies specifically interacting with u.v.-irradiated DNA. The method involves quantitative precipitation of labeled 3H-DNA on nitrocellulose filters in the presence of specific antiserum. The method is justified by the evaluation of thymine dimers excision in various strains of E. coli K12. It was found that the dimers are almost completely eliminated in cells of wild strain during postradiation incubation after u.v.-irradiation with 400 ergs/mm2. In the cells of u.v.-sensitive uvr A6 mutant the dimers are practically not affected, whereas in those of uvr 502 strain excision of the dimers does take place, though more slowly than in wild strain bacteria. The reported approach gives results in good accord with those obtained by others chromatographically. (author)

  12. Homologous radioimmunoassay of human prolactin

    International Nuclear Information System (INIS)

    Gelfiltration on Sephadex G-75 showed a heterogenity of prolactin in serum of patients with prolactinoma and in culture medium of a prolactinoma. Serum of patients with prolactinoma and culture medium of a prolactinoma were examined as possible sources of prolactin by gel filtration and ion exchange chromatography. Polyacrylamide electrophoresis revealed both preparations as contaminated by other proteins. Nevertheless prolactin isolated form culture medium of a prolactinoma is good enough as a tracer in our radioimmunoassay because contaminating proteins in this preparation do not inferfere in our system. An hPRL antiserum created in a rabbit against a crude fraction of human serum of a patient with prolactinoma was tested by titration, saturation studies, and ion exchange chromatography. In comparison with lactoperoxidase-iodinated prolactin Chloramine T iodinated prolactin showed higher loss of immunochemical properity, however higher specific activity. Specifity and precision in our radioimmunoassay system were described and the conditions of optimal sensitivity in our assay were evaluated. (orig.)

  13. Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; LI Tie-jun; ZHU Xiao-xia; XU Li-na; LIU Feng-quan; HU Bai-shi; JIANG Ying-hua; CAO Bin

    2007-01-01

    The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.

  14. Examination of potato virus X proteins synthesized in infected tobacco plants.

    Science.gov (United States)

    Price, M

    1992-09-01

    Nucleotide sequence analysis of potato virus X (PVX) genomic RNA predicts five open reading frames (ORFs). Previous analysis of total RNAs from PVX-infected leaf tissue suggested that six subgenomic RNAs are synthesized during infection. However, the proteins encoded by the genomic RNA, the subgenomic RNAs, or the predicted ORFs have not been identified in vivo. To characterize the coding properties of the viral RNA, particularly to determine whether the five predicted ORFs function in vivo, total protein extracts prepared from PVX-infected leaf tissue were analyzed by using antibodies raised against virus-specific synthetic peptides and against the virus capsid protein. Dot blot analyses showed that these antibodies reacted to PVX-infected extracts, indicating in vivo expression of the five predicted ORFs. In addition, Western blot (immunoblot) analysis of the extracts showed that ORF 1, 2, 3, and 4 peptide antisera and coat protein antiserum detect predominantly a single protein. PMID:1501297

  15. Radioimmunoassay for wheat gliadin to assess the suitability of gluten free foods for patients with coeliac disease

    Energy Technology Data Exchange (ETDEWEB)

    Ciclitira, P.J.; Ellis, H.J. (United Medical School of Guy' s and St. Thomas' Hospital, London (UK)); Evans, D.J. (Hammersmith Hospital, London (UK)); Lennox, E.S. (Medical Research Council, Cambridge (UK))

    1985-03-01

    Coeliac disease is a clinical condition characterised by malabsorption secondary to abnormalities of the small intestine. The condition is known to be exacerbated by wheat gliadin, rye, barley and possibly oats. The only assays that are available for testing for the presence of wheat gluten in foods are double diffusion against rabbit anti-gliadin antiserum and measurement of Kjeldahl nitrogen in products derived from wheat flour. We have developed a radioimmunoassay for wheat gliadin with a detection limit of 1 ng. Nominally gluten free foods based on wheat starch have been shown to contain up to 1.9 x 10/sup -2/% wheat gliadin. Bread made from Nutregen wheat starch which has now been withdrawn contains 6.4 mg gliadin per standard 30 g slice. A radioimmunoassay for wheat gliadin could be used to define standards for the suitability of gluten free products based on wheat starch for patients with coeliac disease.

  16. Radioimmunoassay for human pancreatic amylase: comparison of human serum amylase by measurement of enzymatic activity and by radioimmunoassay

    International Nuclear Information System (INIS)

    A radioimmunoassay (RIA) for human pancreatic amylase has been developed for the determination of human serum amylase content. The assay was shown to be sensitive (7 ng/mI), reproducible and specific, but human pancreatic amylase and salivary amylase could not be distinguished by the antiserum used. In normal subjects, the mean concentration of amylase determined by the RIA was found to be 122.1 ng/ml (range: 55-250 ng/ml). A good correlation was observed between the concentration of amylase and its enzymatic activity in normal subjects. In some instances with high amylase activity, however, the rise in enzymatic activity was not accompanied by increasing amount of amylase content. (Auth.)

  17. Preparation of a highly purified antibody and a solid-state immunoassay for porcine pancreatic α-amylase

    International Nuclear Information System (INIS)

    Porcine pancreas and parotid cell suspensions provide model systems in which to study the mechanism of induction of a specific protein, α-amylase, by hormones acting via cAMP. A highly purified antibody against porcine pancreatic α-amylase was prepared using affinity chromatography of the total IgG fraction derived from rabbit anti-α-amylase antiserum and was used to develop a radioimmunoassay for α-amylase. The antigen--antibody complex was separated from unbound α-amylase using either glutaraldehyde gelled α-amylase or a second antibody technique; a linear standard curve was achieved over a 100- to 1000-fold range of α-amylase concentration. Tissue homogenates did not interfere with this assay, and assayed levels of α-amylase in porcine pancreas were linear using 10 to 200 μg of homogenate. No levels or very low levels of α-amylase were detected in control tissues. (U.S.)

  18. Highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D. (Hebrew Univ., Jerusalem (Israel). Hadassah Medical School)

    1982-08-27

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10/sup 6/ RBC.

  19. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet Gs protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al+3 and F- agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF-4 strongly inhibited 3H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  20. The protozoa dinoflagellate Oxyrrhis marina contains selenoproteins and the relevant translation apparatus

    International Nuclear Information System (INIS)

    In the phylogenetic tree, selenoproteins and the corresponding translation machinery are found in Archaea, Eubacteria, and animals, but not in fungi and higher plants. As very little is known about Protozoa, we searched for the presence of selenoproteins in the primitive dinoflagellate Oxyrrhis marina, belonging to the Protoctista kingdom. Four selenoproteins could be obtained from O. marina cells cultured in the presence of 75Se. Using O. marina or bovine liver cytosolic extracts, we could serylate and selenylate in vitro total O. marina tRNAs. Moreover, the existence of a tRNASec could be deduced from in vivo experiments. Lastly, an anti-serum against the specialized mammalian translation elongation factor mSelB reacted with a protein of 48-kDa molecular mass. Altogether, our data showed that O. marina contains selenoproteins and suggests that the corresponding translation machinery is related to that found in animals

  1. Kinetic study of antibody adhesion on a silicon wafer by laser reflectometry

    Science.gov (United States)

    Riquelme, Bibiana D.; Valverde, Juana R.; Rasia, Rodolfo J.

    2003-05-01

    Antibody adhesion kinetic in real time has been studied by laser reflectometry technique. An ellipsometer is used to measure the light intensity reflected by a silicon wafer. Light intensity reflected by the wafer presents a minimum at the pseudo-Brewster angle. Then, the reflectance increases as the antibodies (monoclonal anti- AB) adhere on interface. Mathematical analysis of reflectance curves versus time verifies that the antibody adhesion at the interface follows Langmuir kinetics (Prog. Biomed. Opt. Imaging 1(5) (2000) 19) for low antibody concentrations. Parameters obtained allow to carry out a detailed study of the antibody adsorption and the antigen-antibody interaction. This conduces to development of an optical immunosensor for detection and quantification of soluble antigens, and a novel method for commercial antiserum quality control. This technique does not require labeled antibodies, being also independent of cellular factors. Also, this technique is quicker and sensible than the conventional immunohematology methods.

  2. The amyloid in familial amyloid cardiomyopathy of Danish origin is related to pre-albumin.

    Science.gov (United States)

    Husby, G; Ranløv, P J; Sletten, K; Marhaug, G

    1985-04-01

    Amyloid obtained from the myocardium of a patient (Han) with familial amyloid cardiomyopathy of Danish origin was studied. Gel filtration and electrophoresis of purified and denatured amyloid fibrils Han revealed various fractions ranging in mol. wt from 40,000 to 8,000 daltons. Amyloid Han and fractions reacted with an antiserum against amyloid Han showing a reaction of identity with each other; partial identity between Han and human pre-albumin was observed, while no reaction was seen with AA or AL proteins. Cardiac tissue sections from Han showed reactivity with antisera to amyloid Han, pre-albumin and protein AP, but not with anti-AA or anti-AL in indirect immunofluorescence. Amino acid composition and sequence studies of a protein fraction of amyloid Han with mol. wt 15,000 daltons confirmed the structural relationship with pre-albumin. PMID:3924450

  3. Postirradiation malignant fibrous histiocytoma expressing cytokeratin. Implications for the immunodiagnosis of sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Weiss, S.W.; Bratthauer, G.L.; Morris, P.A.

    1988-07-01

    A malignant fibrous histiocytoma of the sacrum complicating the course of radiation therapy for endometrial carcinoma is presented. Although the tumor fulfilled the clinical, radiologic, and histologic criteria for a postirradiation malignant fibrous histiocytoma of bone, it also expressed cytokeratin. That this immunoreactivity reflected keratin synthesis by the tumor and not an unusual pattern of cross-reactivity with another intermediate filament such as vimentin is strongly suggested by the reproducibility of the immunoreactivity utilizing both polyclonal and monoclonal antibodies and extinction of the immunoreactivity following absorption of the primary antiserum with keratin proteins. This is the first reported instance of keratin expression by a malignant fibrous histiocytoma; it indicates that sarcomas apart from synovial sarcoma and epithelioid sarcoma may sometimes express this protein.

  4. Postirradiation malignant fibrous histiocytoma expressing cytokeratin. Implications for the immunodiagnosis of sarcomas

    International Nuclear Information System (INIS)

    A malignant fibrous histiocytoma of the sacrum complicating the course of radiation therapy for endometrial carcinoma is presented. Although the tumor fulfilled the clinical, radiologic, and histologic criteria for a postirradiation malignant fibrous histiocytoma of bone, it also expressed cytokeratin. That this immunoreactivity reflected keratin synthesis by the tumor and not an unusual pattern of cross-reactivity with another intermediate filament such as vimentin is strongly suggested by the reproducibility of the immunoreactivity utilizing both polyclonal and monoclonal antibodies and extinction of the immunoreactivity following absorption of the primary antiserum with keratin proteins. This is the first reported instance of keratin expression by a malignant fibrous histiocytoma; it indicates that sarcomas apart from synovial sarcoma and epithelioid sarcoma may sometimes express this protein

  5. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  6. The importance of radioimmunological assay of placental function in the gynaekological practice

    International Nuclear Information System (INIS)

    The determination of human placental lactogen (HPL) has a primary importance in the early diagnostics of placental insufficiency and indirectly, of foetal endangerment. For the evaluation of HPL immune methods based on haemagglutination inhibition, immune diffusion methods and recently a much more sensitive radioimmune procedure are used. The principle of the latter is based on the linkage between a standard labelled with 125I and the specific antiserum. Eliminating the time-consuming preparative work a Phadebaskit (Pharmacia) was used for the radioimmunological assay. The HPL-values from various literary sources implied the author's method, deviated considerably from one another. Experimental data showed that the serum HPL-levels of the radioimmunological assay satisfactorily changed as a function of gestation and gave a good correlation with the haemagglutination method. (D.E.)

  7. Increased muscle glucose uptake during contractions

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Galbo, H; Richter, E A

    1984-01-01

    We reinvestigated the prevailing concept that muscle contractions only elicit increased muscle glucose uptake in the presence of a so-called "permissive" concentration of insulin (Berger et al., Biochem. J. 146: 231-238, 1975; Vranic and Berger, Diabetes 28: 147-163, 1979). Hindquarters from rats...... in severe ketoacidosis were perfused with a perfusate containing insulin antiserum. After 60 min perfusion, electrical stimulation increased glucose uptake of the contracting muscles fivefold. Also, subsequent contractions increased glucose uptake in hindquarters from nondiabetic rats perfused for 1...... Berger et al., 3-O-methylglucose uptake increased during contractions and glucose uptake was negative at rest and zero during contractions. An increase in muscle transport and uptake of glucose during contractions does not require the presence of insulin. Furthermore, glucose transport in contracting...

  8. Characterization of a radioimmunoassay for ovine FSH utilizing an anti-bovine FSH serum

    International Nuclear Information System (INIS)

    In the radioimmunoassay for ovine FSH, utilizing a bovine FSH antiserum and 125I-labelled bovine FSH tracer, the sensitivity was shown to be 5 ng NIH-FSH-S6/ml and cross-reactivities of other ovine pituitary glyco-protein and protein hormones (LH,TSH, growth hormone and prolactin) were less than 1.0%. Both pituitary and serum FSH of the sheep cross-reacted in a manner parallel with that of standard NIH-FSH-S6. Serum FSH concentrations in 4 ewes at various times after the onset of oestrous peaked more or less coincidently with the preovulatory increase of serum LH. The serum levels of FSH and LH did not correlate proportionally but fluctuated considerably in individual animals. The radioimmunoassay system appeared to be sensitive and specific for measuring serum concentrations of FSH in rams and ewes in different physiological states. (author)

  9. Studies on radioallergosorbent test (RAST)

    International Nuclear Information System (INIS)

    The radioallergosorbent test (RAST) is one of the solid phase radioimmunoassays. The authors first studied Ceska's method, a RAST with a paper disc instead of a solid phase. The patients with nasal allergic disorders were selected. The following items were used as antigens: Acarus, House Dust, Sugi, American Sugi timber, Alternaria, Candida, Penicillium and Protargol. The RAST was very reproducible with an average error of 5% according to our results. Treatments to decrease sensitivity did not influence RAST values within a nine-month period. The purity of the 125I-labeled anti-serum was considered to be most important in the success of the RAST. The RAST was useful for all of these tests for allergy to Acarus, House Dust, Sugi pollen, Ragweed pollen, Typha angustate pollen, Alnus japonica pollen, Alternaria, Candida and Penicillium. Applications of American Sugi timber and Protargol to the allergic tests have not been studied. (Tsukamoto, Y.)

  10. The radioimmunoassay for type III procollagen peptide. A technical note

    International Nuclear Information System (INIS)

    We have studied the immunoreactivity of human sera and synovial fluids in a radioimmunoassay which utilizes an anti-bovine procollagen type III antiserum and the bovine type III procollagen peptide as standard and tracer. We have noticed a complete cross section reaction between bovine col 1-3 and human synovial fluid, which thus contains substance(s) having the same antigenic determinant(s) as col 1-3. On the contrary, we have demonstrated non parallelism between col 1-3 and human serum, whether the serum was obtained from normal subjects or patients suffering from various diseases. These results suggest that immunoreactive substances related to col 1-3 and detected in sera are at least partially different from those detected in synovial fluids

  11. Homologous radioimmunoassay for guinea pig corticosteroid-binding globulin

    International Nuclear Information System (INIS)

    A rapid, specific, and sensitive (requiring only 20 fmole of antigen equivalent to 0.007) μl of serum) radioimmunoassay (RIA) was developed for the measurement of guinea pig corticosteroid-binding globulin (CBG). CBG was purified to homogeneity from guinea pig serum by affinity chromatography and used for immunization, as the standard and as the radiolabeled trace in the RIA. The antiserum to CBG was raised in rabbits. It was judged specific by immunoelectrophoresis and by comparison of RIA values with steroid-binding assay profiles obtained on serum separated on the basis of size and ion-exchange properties. The results of the radioimmunoassays agree with those of a steroid-binding assay run on identical samples. The sensitivity of the assay allows detection of CBG in serial serum samples, other biologic fluids such as milk, and cell culture supernatants

  12. Process for preparation of a solid-phase radioimmunoassay support and use thereof

    International Nuclear Information System (INIS)

    A process is described for the preparation of a support useful in radioimmunoassay chromatographic columns. The process involves the preparation of a chromatographic gel capable of selectively retaining one or more components contained in an antigen-antibody-containing solution. The gel is bound to the appropriate antiserum, then freeze-dried, pulverized and compressed into a tablet. The tablet support swells upon contact with an antigen-antibody-containing solution to conform to the shape of the columns. An example of the application of this support in the radioimmunoassay of thyroid-stimulating hormone is described. This type of support is also particularly useful in second antibody solid phase radioimmunoassays since there is no limit to the size of the antigen to which this technology may be applied. (U.K.)

  13. Brain antigens: components of subfractions from human grey matter

    Science.gov (United States)

    Rajam, P. C.; Bogoch, S.

    1966-01-01

    1. Using chromatography on DEAE-cellulose, a neutral, low ionic strength extract of human grey matter has been separated into fractions of proteins with basic and progressively acidic groups. 2. The reactions of each group with rabbit antiserum against the original extract, in double-diffusion tests, suggest the presence of a minimum total of thirteen distinct antigens between them. These results are supported by immunoelectrophoretic findings, which indicate the basic group to contain five, and the progressively acidic group seven to eight distinct antigens. These antigens do not appear to be human serum proteins. 3. Antigens belonging to the BE class (resistant to boiling and relatively soluble in ethanol) are present among the progressively acidic proteins, and possibly among the basic proteins also. ImagesFIG. 2 PMID:4958737

  14. Highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

    International Nuclear Information System (INIS)

    A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process. An antiserum was prepared from rabbits immunized with α-human ANP (α-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.2 pg/tube of synthetic α-hANP utilized as authentic standard. Recovery of α-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 health subjects, the plasma ANP concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography

  15. A distinct tospovirus causing necrotic streak on Alstroemeria sp. in Colombia.

    Science.gov (United States)

    Hassani-Mehraban, Afshin; Botermans, Marleen; Verhoeven, J Th J; Meekes, Ellis; Saaijer, Janneke; Peters, Dick; Goldbach, Rob; Kormelink, Richard

    2010-03-01

    A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5' untranslated region and a part of the intergenic region 3' of the N gene, was cloned and sequenced. The deduced N protein sequence showed highest amino acid identity (82%) to that of TCSV, indicating that the virus represents a new tospovirus species, for which the name Alstroemeria necrotic streak virus (ANSV) is coined. Phylogenetic analysis based on the N protein sequence revealed that this Alstroemeria-infecting tospovirus clustered with tospoviruses from the American continent. Frankliniella occidentalis was identified as potential vector species for ANSV. PMID:20151164

  16. Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event

    DEFF Research Database (Denmark)

    Danielsen, E M

    1992-01-01

    enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a...... posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate...... interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding....

  17. Enhanced synthesis and secretion of apolipoprotein E from sciatic nerves of streptozotocin-induced diabetic rats after injury

    International Nuclear Information System (INIS)

    To elucidate the pathogenesis of diabetic neuropathy, synthesis and secretion of apolipoprotein E (apo E) from sciatic nerves after injury was studied in normal and streptozotocin-induced diabetic rats. Seven, 14, 28, 45 and 59 days after making crush injury on sciatic nerves with concomitant administration of streptozotocin (50 mg/kg body weight), the nerves were taken out and incubated with [35S]methionine. The [35S]labeled apo E was precipitated with specific antiserum. The amounts of apo E secreted into medium by nerves of diabetic rats were 7 times greater than those of non-diabetic rats 7 days after injury. This enhanced secretion of apo E was relatively selective for this protein, since the ratio of the immunoprecipitable apo E to the TCA preciptitable protein in the medium increased in diabetic rats. Intriguing possibility deduced from these results is that the secretion of apo E is involved in the development of diabetic neuropathy

  18. Characterization of high-pressure-treated egg albumen.

    Science.gov (United States)

    Iametti, S; Donnizzelli, E; Pittia, P; Rovere, P P; Squarcina, N; Bonomi, F

    1999-09-01

    Addition of NaCl or sucrose to egg albumen prior to high-pressure treatment (up to 10 min at 800 MPa) prevented insolubilization or gel formation after pressure treatment. As a consequence of protein unfolding, the treated albumen had increased viscosity but retained its foaming and heat-gelling properties. Susceptibility of egg albumen proteins to hydrolysis by trypsin increased dramatically after pressure treatment. The S-form of ovalbumin, the presence of which is an index of egg aging, was not found in any of the pressure-treated samples, which also did not display evidence for covalent protein aggregation. However, recognition of ovalbumin by an anti-ovalbumin antiserum was reduced to 40% of that of untreated sample. PMID:10552693

  19. Plasmodium falciparum: characterization of toxin-associated proteins and identification of a hemoglobin containing parasite cytokine stimulator

    DEFF Research Database (Denmark)

    Kristensen, G; Jakobsen, P H

    1996-01-01

    ]-methionine and immunoprecipitated the labeled antigens with an antiserum against IMP which blocks malaria parasite-induced TNF production. We detected four proteins associated with IMP when the immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography. To evaluate the capacity of different P....... falciparum antigens to induce cytokine production we separated a mixture of exoantigens by SDS-PAGE gels. Antigen fractions of 43-71 kDa and of a low molecular mass of <20 kDa contained the dominant inducers of TNF alpha interleukin 1 alpha, and interleukin 6 production from human mononuclear cells. The low......Previous studies have indicated the inositol monophosphate (IMP) is a component of the malaria parasite toxin that induces cytokines such as tumour necrosis factor (TNF). To further characterize the toxin we have labeled Plasmodium falciparum in vitro cultures with [14C]inositol or [35S...

  20. Measurement of plasma canine C peptide by radioimmunoassay

    International Nuclear Information System (INIS)

    A sensitive radioimmunoassay for canine C peptide (CCP) was established using synthetic CCP, a specific antiserum, and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine T method. Tracer preparations have been used for 6 weeks after iodination. The standard curve ranges from 0.028 to 3.0 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.3 and 0.8 nmol/l. The average recovery of CCP added to plasma samples was 100.6% (n = 9). Canine insulin, porcine proinsulin, bovine proinsulin, and human C peptide exhibited no cross-reactivity. The mean fasting plasma CCP concentration was 0.089 +- 0.021 nmol/l in normal dogs and -0.005 +- 0.007 nmol/l (mean +- SEM) in diabetic dogs, respectively. (author)

  1. Antibody against Porcine Parvovirus VP2 Protein Differentiated This Virus from Other Viruses

    Directory of Open Access Journals (Sweden)

    Weijuan Zhu§, Yudong Ren§1, Guangxing Li, Dingding Su2, Qing Yang3 and Xiaofeng Ren*

    2012-06-01

    Full Text Available Viral protein 2 (VP2 of porcine parvovirus (PPV is the major viral structural protein and responsible for eliciting neutralizing antibodies in immunized animals. In this study, the gene encoding VP2 of PPV was amplified by PCR. The VP2 gene was then cloned into the prokaryotic expression vector, pET-32a followed by expression in Escherichia coli Rosetta. The VP2 protein expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit polyclonal antiserum was generated using the recombinant VP2 protein. The optimal titer of the anti-VP2 antibody was determined by ELISA. The anti-VP2 antibody was able to distinguish PPV from other viruses in ELISA.

  2. The synthesis of a small library of prospective growth hormone secretagogues

    Directory of Open Access Journals (Sweden)

    JELENA JOKSIMOVIC

    1999-10-01

    Full Text Available Employing tools of combinatorial chemistry, an original methodological approach has been developed and applied for the design and synthesis of a small library of peptide-like compounds, prospective growth hormone (GH secretagogues. For this purpose seven building blocks of tBoc- and Fmoc-protected amino acids was used. In this way, a small, tripeptoid library on polyethylene glycol monomethyl ether 5000 (PEG 5000 as a soluble support was obtained. The library was screened by a new, simple system, based on polyclonal rabbit antiserum raised against "GH secretagogue pharmacophore" of a known growth hormone secretagogue GHRP-6 (Hexarelin® and the most promising GH secretagogue candidate was selected.

  3. Iodine-125 radioimmunoassay for the direct detection of benzodiazepines in blood and urine

    Energy Technology Data Exchange (ETDEWEB)

    Goddard, C.P.; Stead, A.H.; Mason, P.A.; Law, B.; Moffat, A.C.; McBrien, M.; Cosby, S.

    1986-05-01

    A radioimmunoassay (RIA) for the direct detection of benzodiazepines in blood and urine is described. It is based on a commercially available antiserum and an easily synthesised radio-iodinated derivative of clonazepam that allows the use of relatively simple gamma-counting procedures. The assay can detect low therapeutic levels of all of the benzodiazepines currently available in the UK in 50-..mu..l samples of blood and urine (1-50 ng ml/sup -1/, depending on the drug); no prior sample preparation is required. It is inexpensive, rapid, simple to perform and is broadly specific for the benzodiazepine class of drugs. The assay offers a most suitable means of screening large numbers of samples of forensic interest for the presence of the benzodiazepines.

  4. An iodine-125 radioimmunoassay for the direct detection of benzodiazepines in blood and urine

    International Nuclear Information System (INIS)

    A radioimmunoassay (RIA) for the direct detection of benzodiazepines in blood and urine is described. It is based on a commercially available antiserum and an easily synthesised radio-iodinated derivative of clonazepam that allows the use of relatively simple gamma-counting procedures. The assay can detect low therapeutic levels of all of the benzodiazepines currently available in the UK in 50-μl samples of blood and urine (1-50 ng ml-1, depending on the drug); no prior sample preparation is required. It is inexpensive, rapid, simple to perform and is broadly specific for the benzodiazepine class of drugs. The assay offers a most suitable means of screening large numbers of samples of forensic interest for the presence of the benzodiazepines. (author)

  5. Immunoreactive luteinizing hormone-releasing hormone in the seminal plasma and human semen parameters

    International Nuclear Information System (INIS)

    A luteinizing hormone-releasing hormone (LH-RH)-like substance has been detected in human seminal plasma by a radioimmunoassay (RIA) with a highly specific anti-LH-RH antiserum. The seminal samples - not only the plasma itself but also the sample extracted by an acid/alcohol method - showed satisfactory displacement curves in our RIA system. The relationship between fertility and the LH-RH values in the seminal plasma was studied by comparing the peptide levels with sperm concentration and motility. By these two parameters, 103 samples were divided into four groups. In the low-concentration groups (oligozoospermic patients), the hormonal concentrations differed significantly between those specimens demonstrating good and poor motility. These data suggest that this immunoreactive LH-RH may play a role in human spermatogenesis

  6. Comparação da eficiência dos caldos de enriquecimento seletivo no isolamento de Salmonella Dublin Comparison of the efficiency of selective enrichment broths for Salmonella Dublin isolation

    Directory of Open Access Journals (Sweden)

    D.G. Silva

    2008-06-01

    Full Text Available The aim of this study was to compare three different selective enrichment broths: Rappaport-Vassiliadis (RV, selenite cystine (SC and Muller-Kauffmann tetrathionate (MKT for Salmonella Dublin isolation from faecal samples of calf experimentally infected. The bacteriological procedure involved pre-enrichment stages in Hajna-GN broth (only for the samples inoculated in RV broth, selective enrichment, culture in modified brilliant green agar (BGA, presumptive biochemistry tests (using triple-sugar-iron agar and lysine-agar and slide agglutination test with poli-O and poli-H Salmonella antiserum. The effects of enrichment temperatures using RV broth were also evaluated (37ºC and 42ºC. SC broth was significantly more efficient in the isolation of Salmonella Dublin (P<0,05, whereas RV broth incubated at 42ºC had a lower efficiency in the microbiological isolation.

  7. Anti-enrofloxacin Antibody Production by Using Enrofloxacin-screened HSA as an Immunogen

    Institute of Scientific and Technical Information of China (English)

    LIU Chune; LIN Hong; CAO Limin; JIANG Jie

    2005-01-01

    A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating enrofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA experiments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration range studied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sulphamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specific antibody responses in BALB/C mice against EF.

  8. Oxytocin/vasopressin-like immunoreactivity is present in the nervous system of hydra

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Dierickx, K; Boer, G J

    1982-01-01

    Nerve cells have been found in hydra, which react with antisera to oxytocin, vasopressin and mesotocin. These nerve cells have a high density in the ectoderm of basal disk and tentacles and lower density in the ectoderm of peduncle, gastric region and hypostome. A very small number of nerve cells...... occur also in the endoderm of foot, gastric region and hypostome. By using a technique for simultaneous visualisation of nerve cells reacting with antisera to oxytocin and vasopressin, it can be shown that these nerve cells belong to a single population. In agreement with this, the staining of the nerve...... cells can be abolished by absorbing each antiserum with either oxytocin, vasopressin, [Lys8]vasopressin, vasotocin, mesotocin or isotocin, indicating that the antigenic determinant of hydra cross-reacts with those antibody subpopulations, which recognize common portions (sequence 1-2, 5-7, 9) of the...

  9. Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

    International Nuclear Information System (INIS)

    UV (ultraviolet) damaged DNA and its repair of various cultured animal cells were observed by radioimmunoassay using anti-serum against the UV irradiation induced heat-degenerated DNA. There is some difference among the cells of used animals according to their DNA repairabilities. The cells were divided into four groups according to the existence or strength of their repairabilities. 1) excision repair type: cells of men and chimpanzees. 2) photoreactivation type: cells derived from Tachydromus tachydromoides and chicks. 3) photoreactivation with excision repair: cells of rats, kangaroos and mosquitos. 4) non-excision repair type: cells of mice, Meriones and rats. Animal cells have plural types of repair. Main types of repair will differ according to the kind of animals. (Ichikawa, K.)

  10. Evaluation of anti-pneumococcal capsular antibodies as adjunctive therapy in experimental pneumococcal meningitis

    DEFF Research Database (Denmark)

    Brandt, Christian; Frimodt-Moller, N; Lundgren, Jens Dilling;

    2006-01-01

    OBJECTIVE: Bacteraemia concomitant with meningitis has been shown to greatly affect outcome. Consequently, the efficacy of serotype-specific anti-pneumococcal antiserum (APAS) was investigated in a rat model of pneumococcal meningitis. METHODS: Rats were infected with Streptococcus pneumoniae...... serotype 3. All rats received ceftriaxone starting 26 h post-infection. APAS was administered either at the time of infection or 26 h post-infection and effects were compared with rats treated with antibiotics only. RESULTS AND CONCLUSION: A significant clinical benefit was found when APAS was given at the...... time of infection whereas no effect was found when administered 26 h after infection. This work indicates that the clinical value of using APAS in pneumococcal meningitis may be limited...

  11. 64 kDa protein is a candidate for a thyrotropin-releasing hormone receptor in prolactin-producing rat pituitary tumor cells (GH4C1 cells)

    International Nuclear Information System (INIS)

    A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [3H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [3H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [3H]TRH, the 64 kDa protein cannot be demonstrated by [3H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells

  12. Development of a double-antibody radioimmunoassay for the detection of methotrexate in the serum

    International Nuclear Information System (INIS)

    This double-antibody radioimmunoassay (RIA) designed to detect methotrexate (MTX) in the serum is a precise, rapid and cost-saving quantitative procedure, which meets all the requirements regarding accuracy and reproducibility. Its main components are anti-methotrexate as antiserum, 3H-MTX as tracer substance and a second antibody as separating agent. The measuring technique using tritium is based on a method involving removal of the supernate by suction after separation of the antibody, dissolution of the precipitate in NaCl and addition of the scintillator. As opposed to separation techniques based on carbon the radioactivity to be determined is contained in the precipitate, not in the supernate. The procedure permits the required amount of scintillator and the associated radioactive waste to be reduced by 90%. A further decisive advantage is its low limit of detection. (TRV)

  13. Methotrexate concentrations in biological fluids: Comparison of results obtained by radioimmunoassay and direct ligand binding radioassay

    International Nuclear Information System (INIS)

    A sensitive (sensitivity 2.2 x 10-9 mol/l) and specific (practically no cross-reaction with circulating folates) radioimmunoassay for the determination of methotrexate concentrations in biological fluids is described and compared with a commercial competitive protein binding assay. Antiserum with high titer was produced in rabbits immunized with MTX-human serum albumin conjugate. Fitness for use in pharmacokinetic drug level determinations was shown in three patients, who received both low doses and high dose therapy combined with citrovorum factor rescue. An excellent correlation was found between plasma and urine MTX concentrations obtained by RIA and competitive protein binding assay. A two-compartment pharmacokinetic model was found adequately describing the serum decay curves, but there was a great interindividual variability in the calculated pharmacokinetic parameters. (author)

  14. Preparation of 125iodine-labelled methotrexate and its use in a magnetisable particle solid-phase radioimmunoassay

    International Nuclear Information System (INIS)

    Two techniques for the iodination of methotrexate are described, involving covalent linkage to the drug of 125I-labelled N-succinimidyl-3-(4-hydroxyphenyl) propionate (Bolton and Hunter reagent), or to 125I-labelled histamine. A rapid highly specific radioimmunoassay for methotrexate was developed, employing a specific antiserum covalently linked to magnetisable particles, and 125I-labelled methotrexate as tracer. Incubation time for the assays were 60 and 10 min for the Bolton and Hunter reagent-linked methotrexate and 125I-labelled histamine-linked methotrexate respectively. Separation of bound from free antigen was achieved by a rapid magnetic separation system. Results obtained for serum samples correlated closely with those using an enzymatic (dihydrofolate reductase) competitive protein binding assay for methotrexate. A major advantage of the assay is its potential for processing large numbers of samples rapidly, making it highly suitable for routine clinical use. (Auth.)

  15. Distribution of two basement membrane proteoglycans through hair follicle development and the hair growth cycle in the rat

    DEFF Research Database (Denmark)

    Couchman, J R; King, J L; McCarthy, K J

    1990-01-01

    The distribution of two distinct populations of basement membrane proteoglycans has been monitored through hair growth development in the rat embryo and subsequent hair growth cycle. An antiserum against a small heparan sulfate proteoglycan uniformly stained the dermal-epidermal junction of...... embryonic rats throughout the period of hair follicle formation. On the other hand, monoclonal antibodies recognizing a basement membrane-specific chondroitin sulfate proteoglycan only weakly stained 16-d embryo dermal-epidermal junction, but strong staining was associated with hair follicle buds as they...... developed. Through the hair growth cycle, it was found that the heparan sulfate proteoglycan persisted around the follicles, while the chondroitin sulfate proteoglycan decreased in amount through catagen until it was undetectable at the base and dermal papilla of the telogen follicle. As anagen commenced...

  16. Gastrin-releasing peptide in the porcine pancreas

    DEFF Research Database (Denmark)

    Holst, J J; Poulsen, Steen Seier

    1987-01-01

    to consist of one main form, namely the 27-amino acid peptide originally extracted from porcine stomach, and small amounts of a C-terminal fragment identical with the C-terminal 10-amino acid peptide. Gastrin-releasing peptide-like immunoreactivity released from the isolated perfused porcine pancreas during...... electrical vagal stimulation was shown by gel filtration to consist of the same two forms. By use of immunocytochemical techniques employing an antiserum directed against its N terminus, GRP was localized to varicose nerve fibers in close association with the exocrine tissue of the porcine pancreas...... in particular. Some fibers were found penetrating into pancreatic islets also. Immunoreactive nerve cell bodies as well as fibers were found within intrapancreatic ganglia. The potency of GRP in stimulating exocrine as well as endocrine secretion from the porcine pancreas, its presence in close contact...

  17. Vasoactive intestinal polypeptide (VIP) in the pig pancreas

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1984-01-01

    Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion...... of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas...... with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas....

  18. Biological characterisation of Sarcocystis neurona isolated from a Southern sea otter (Enhydra lutris nereis)

    Science.gov (United States)

    Lindsay, D.S.; Thomas, N.J.; Dubey, J.P.

    2000-01-01

    Sarcocystis neurona was isolated from the brain of a juvenile, male southern sea otter (Enhydra lutris nereis) suffering from CNS disease. Schizonts and merozoites in tissue sections of the otter's brain reacted with anti-S. neurona antiserum immunohistochemically. Development in cell culture was by endopolyogeny and mature schizonts were first observed at 3 days postinoculation. PCR of merozoite DNA using primer pairs JNB33/JNB54 and restriction enzyme digestion of the 1100 bp product with Dra I indicated the organism was S. neurona. Four of four interferon-γ gene knockout mice inoculated with merozoites developed S. neurona-associated encephalitis. Antibodies to S. neurona but not Sarcocystis falcatula, Toxoplasma gondii, or Neospora caninum were present in the serum of inoculated mice. This is the first isolation of S. neurona from the brain of a non-equine host.

  19. Impact of tumour necrosis factor-alpha and interferon-gamma on tetrahydrobiopterin synthesis in murine fibroblasts and macrophages.

    Science.gov (United States)

    Werner, E R; Werner-Felmayer, G; Fuchs, D; Hausen, A; Reibnegger, G; Yim, J J; Wachter, H

    1991-01-01

    Tumour necrosis factor-alpha causes an up to 30-fold induction of GTP cyclohydrolase I (EC 3.5.4.16) activity in murine dermal fibroblasts in a dose-dependent manner. Owing to the high constitutive activities of 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), this potentiates biosynthesis of tetrahydrobiopterin. Murine macrophages already contain high activities of GTP cyclohydrolase I when unstimulated, and this is further augmented up to 4-fold by tumour necrosis factor-alpha/interferon-gamma. In Western blots an antiserum to murine liver GTP cyclohydrolase I does not stain cell extracts with high enzyme activities, suggesting that the cytokine induced peripheral form of GTP cyclohydrolase I might differ from the liver form. Images Fig. 2. PMID:1764035

  20. Comparative Serological and Random Amplified Polymorphic DNA Typing for Bordetella avium Isolates in China

    Directory of Open Access Journals (Sweden)

    Ping-Ping Yang, Rong-De Ma1, Xue Zhao and Rui-Liang Zhu*

    2012-10-01

    Full Text Available To study the similarity among Bordetella avium isolates in China, antigens and diagnostic antiserum of 22 B. avium isolates were prepared for serotyping, and a set of 20 commercially available primers was screened out to identify suitable primers for random amplified polymorphic DNA fingerprinting (RAPD analysis in this study. Twenty-two B. avium isolates were divided into two serovars (A and B based on their reaction in the plate-agglutination test. Four primers R1, R2, R4 and R10 resulted in informative fingerprints and were used to evaluate the B. avium isolates. Based on their RAPD patterns, a dendrogram allowed the separation of the B. avium isolates into six genetic similarity clusters. However, no direct correlation was observed between serotypes and RAPD typing among the isolates.

  1. Modification of the theophylline radioimmunoassay

    International Nuclear Information System (INIS)

    The routine utilization of a commercially available radioimmunoassay (RIA) for theophylline (GammaDab), although reliable, is currently prohibited by the high cost of the reagents. In an effort to reduce these costs we have diluted the [125I]theophylline tracer and theophylline antiserum reagents by one-half, contrary to the manufacturer's recommendations. We have demonstrated that an excellent correlation exists (r . 0.968) between our modified RIA method and a conventional high-performance liquid chromatographic technique, despite reagent dilution. Accordingly, our reagent costs have been reduced by half. We conclude that the GammaDab kit reagents can be diluted twofold and still provide an accurate determination of serum theophylline. We must also emphasize that any further alteration(s) of this theophylline RIA procedure would require a thorough evaluation before its routine use could be substantiated

  2. [A case report of pleural involvement in primary macroglobulinemia].

    Science.gov (United States)

    Noguchi, M; Yamaguchi, A; Tsuboi, E; Watanabe, T; Narui, K; Yoshimura, K; Chonabayashi, N; Nakata, K; Kanbayashi, H; Endo, Y

    1990-01-01

    A case of primary macroglobulinemia with pleural and gastric involvement was presented. A 48-year-old female was admitted with productive cough. On physical examination neither lymphoadenopathy nor hepatosplenomegaly were found. In addition, no bleeding tendency nor disturbance of the visual acuity were detected. Her chest roentgenogram showed a moderate amount of pleural effusion in the left pleural cavity without infiltration in the lung fields and no evidence of swollen hilar or mediastinal lymphnodes. A monoclonal M-band of to IgM-kappa type was observed in her serum and the pleural effusion. The diffuse ulcerative lesion in the gastric mucosa was detected by gastrofiberscopy. The lymphoid cells taken from the pleural effusion and the gastric mucosa stained positively with fluorescein-conjugated antiserum to u or the kappa chain. Pleural effusion and gastric infiltration of lymphoid cells improved remarkably following ACOP therapy. PMID:2113145

  3. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Institute of Scientific and Technical Information of China (English)

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA

    2006-01-01

    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  4. Radioimmunoassay of human plasma trypsin

    International Nuclear Information System (INIS)

    A radioimmunoassay has been developed for the determination of human trypsin in plasma. It allows the measurement of trypsin concentration in spite of the presence of plasma or pancreatic inhibitors. The human trypsin used as a standard as well as for labelling was isolated from pancreatic tissue and purified by affinity chromatography. The antiserum was obtained from guinea-pigs immunized with partially purified human trypsin. In the radioimmunoassay, the values of trypsin in serial dilutions of plasma were parallel to those of the standard curves. The assay was shown to be reproducible, sensitive and specific. However, the two antisera used did not distinguish between the enzyme and its proenzyme. In normal subjects, plasma values were found to be around 400 ng/ml. They were 10-40 times higher in patients with acute pancreatitis. The method appears to be much more specific for the diagnosis of acute pancreatitis than the current determinations of amylase and lipase activity

  5. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Tan, B H; Fu, J; Sugrue, R J; Yap, E H; Chan, Y C; Tan, Y H

    1996-02-15

    The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA. PMID:8607261

  6. Development of a radioimmunoassay for formoterol

    Energy Technology Data Exchange (ETDEWEB)

    Yokoi, K.; Murase, K.; Shiobara, Y.

    1983-10-01

    The development of a radioimmunoassay (RIA) for the ..beta../sub 2/-stimulant formoterol is described. The sensitivity of the method is 0.1 ng/ml in plasma and urine, when a l-ml sample is used. The cross-reactivity of the antiserum with formoterol glucuronide was 30%. Since formoterol is metabolized extensively to formoterol glucuronide in rats, dogs and human, extraction with ethyl ether prior to the radioimmunoassay was carried out. Satisfactory agreement was obtained for levels of formoterol in plasma and urine when they were determined by RIA and gas chromatography-mass spectrometry. The concentration of formoterol was determined in dog plasma and human urine after oral administration of formoterol fumarate to dogs (61 mcg/kg) and humans (40 mcg).

  7. Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

    International Nuclear Information System (INIS)

    Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny

  8. Immunoassay of ergotamine and dihydroergotamine using a common /sup 3/H-labelled ligand as tracer for specific antibody and means to overcome experienced pitfalls

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthaler, J.; Munzer, H.; Voges, R.; Andres, H.; Gull, P.; Bolliger, G.

    1984-01-01

    A highly sensitive radioimmunoassay for the determination of ergotamine and dihydroergotamine is described. The limit of detection is about 9 pg/mL blood plasma for both compounds. The specificity of the gamma-globulin, which was prepared from rabbit antiserum, was investigated in the presence of compounds synthesized as possible metabolites. It was found that the tricyclic peptide moiety common to both molecules is an essential structural feature for binding to the gamma-globulin. From dilution experiments with the radioactively labelled compound it followed that ergotamine and to a lesser extent also its dihydro derivative are adsorbed on various tube wall materials using known buffer solutions. A practically insuperable obstacle is rearrangement of ergotamine under the experimental conditions, forming a stereoisomer by inversion at the C-8 position. The equilibrium of ergotamine in equilibrium ergotaminine found in human plasma remains stable under the incubation conditions of the radioimmunoassay.

  9. A "new" primed lymphocyte typing (PLT) defined DP-antigen associated with a private HLA--DR antigen

    DEFF Research Database (Denmark)

    Morling, N; Jakobsen, B K; Platz, P;

    1980-01-01

    We have recently described a "new" private HLA-DR antigen, DR"LTM", which has a frequency of approximately 0.6% in Danes. Primed Lymphocyte Typing (PLT) cells directed towards DR"LTM"-associated determinants were generated in vitro by haplotype primings in two unrelated families with DR...... total agreement between the results obtained by HLA-DR typing with the antiserum "LTM" and by PLT-typing with these two haplotype primed PLT-cells. None of the DP"LTM"-positive individuals carried more than one of the antigens HLA-Dw/-DRw/DP1-8 and the local specificity D/DP"H". Accordingly, this "new......" PLT-defined antigen, DP"LTM", most probably belongs to the series of HLA-D/DR-associated DP-antigens previously described....

  10. Cerebrospinal fluid beta-endorphin radioimmunoassay: Methodological problems

    International Nuclear Information System (INIS)

    This note describes the technical details of a radioimmunoassay for #betta#-endorphin (#betta#-EP). An antiserum raised in rabbits against synthetic #betta#-EP measures a ''#betta#-EP-like'' immunoreactivity (#betta#-EP and #betta#-LPH). Thus, filtration chromatography is used to separate both peptides. #betta#-EP is the 31 amino-acid C-terminal end of #betta#-LPH, with potent opiate-like activity. The measurement of CSF #betta#-EP in man may advance the understanding of numerous brain functions especially in pain problems. This determination is difficult since #betta#-LPH contains the immunological determinants of both #betta#-LPH and #betta#-EP. The measurement of #betta#-EP in human CSF has been approached by using gel filtration of CSF extracts followed by a RIA employing an anti-#betta#-EP antibody which cross reacts with #betta#-LPH. (author)

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

    Science.gov (United States)

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

    1999-03-01

    Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range. PMID:10217635

  12. Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Nielsen, Jens; Bille-Hansen, Vivi

    The first case of porcine reproductive and respiratory syndrome (PRRS) in Denmark was diagnosed in March 1992 by the detection of specific antibodies against PRRS virus in serum samples originating from sows in a herd located on the island of Als. Subsequently, PRRS virus was isolated from a 200...... infection was demonstrated by the re-isolation of PRRS virus from approximately 45% of the piglets from the experimentally infected gilts. However, the experimental infection produced no significant reproductive disorders or other clinical signs. At autopsy, histopathological examination revealed slight......-sow farrow-to-finish herd with clinical signs consistent with PRRS. The virus was isolated by inoculation of pleural fluid from a stillborn piglet onto porcine pulmonary alveolar macrophages. The isolate was identified as PRRS virus by staining with a specific antiserum. By electron microscopy, the...

  13. Effects of gamma radiation on crotoxin (toxin of Crotalus durissus terrificus venom)

    International Nuclear Information System (INIS)

    The crotoxin in a great neurotoxin found on Crotalus durissus terrificus venom. This protein was isolated using molecular exclusion cromatrography with Sephadex G-75 and irradiated in a source of 60Co GAMMACELL in the concentration of 2 mg/ml 0.85% NaCl with dose rate of 1.19 x 103 Gy/hr. The doses used were 250, 500, 1000 and 2000 Gy. It was determinated for this samples, the proteic concentration, the diffusion immunoassay using crotalic antiserum and eletrophoresis (SDS-PAGE). The results showed some changes on the irradiated toxin. Preliminary results with doses of radiation of 100, 250, 500 and 1000 Gy showed that the letal dose 50% (LD50) in mice increase greatly with the increase of radiation's dose. (author)

  14. Differential expression of 2':3'-cyclic nucleotide 3'-phosphodiesterase in cultured central, peripheral, and extraneural cells.

    Science.gov (United States)

    Sprinkle, T J; McMorris, F A; Yoshino, J; DeVries, G H

    1985-07-01

    The relative levels of the central nervous system myelin marker enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C6 and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme. PMID:2995854

  15. Detection of porcine endogenous retrovirus (PERV) using highly specific antisera against Gag and Env

    International Nuclear Information System (INIS)

    Porcine endogenous retroviruses (PERV) are considered an obstacle to the safe use of cells, tissues, and organs from pigs in the course of xenotransplantation. Thus, the detection of viral proteins and of a potential PERV infection is of major interest. Recently, we have published the generation of a highly specific antiserum directed against the nucleocapsid (p10) of PERV (Xenotransplantation 7 (2000), 221). Here we present new peptide-antisera specific to the capsid protein (p30) and the surface molecule of PERV class B (SU, gp70(B)) as well as the transmembrane moiety of the envelope protein (TM, p15E) of PERV which showed functionality in several immunological assays, such as immunoblots, immunofluorescence, and immunogold staining. Thus, these antisera can be used as tools for the identification of viral proteins in basic research as well as clinical trials

  16. Characterization of Elongation Factor Tu of Mycoplasma ovipneumoniae

    Directory of Open Access Journals (Sweden)

    Xuan Zhang, Yue-feng Chu, Ping Zhao, Peng-cheng Gao, Ying He, Nu Wang and Zhong-xin Lu*

    2013-11-01

    Full Text Available Mycoplasma ovipneumoniae is considered as an important pathogen of small ruminants, but its antigenic proteins are not well known so far. In this study, we cloned the EF-Tu gene of M. ovipneumoniae and analyzed the molecular features of the gene and its coding protein for the first time. The gene was then expressed in E.coli and the antigenicity of the coding protein was evaluated as well. The EF-Tu gene of M. ovipneumoniae is 1209 bp in length, encodes 402 amino acids, and shares the highest DNA sequence identity of 87.5% and deduced amino acid sequence identity of 97.8% with those of M. hyopneumoniae, respectively. The recombinant EF-Tu protein can react with the polyclonal antiserum of M. ovipneumoniae and can induce humoral immune responses in mice, which indicated that the EF-Tu may be used as a candidate protein in developing the technologies to control the disease.

  17. Efficacy of rabies immunoglobulins in an experimental post-exposure prophylaxis rodent model.

    Science.gov (United States)

    Servat, Alexandre; Lutsch, Charles; Delore, Valentine; Lang, Jean; Veitch, Keith; Cliquet, Florence

    2003-12-12

    In a recently published Syrian hamster animal challenge study [Vaccine 19 (2001) 2273], a highly purified, heat-treated equine rabies immunoglobulin (pERIG HT, Favirab) did not elicit satisfactory protection. The efficacies of this batch, a second stage pERIG HT batch and reference RIG preparations (Imorab, Imogam Rage pasteurised, Berna antiserum) were compared in mice challenged with either Ariana canine field strain or CVS strain. Survival rates against Ariana challenge with the second pERIG HT batch were indistinguishable from those of other licensed preparations (83-90% survival), but the deficient batch did not provide satisfactory protection (53%). These data confirm the inadequate response to a first stage pERIG HT batch, but a current batch provides equivalent protection to that afforded by licensed HRIG and ERIG preparations. PMID:14615152

  18. Basement membrane changes in breast cancer detected by immunohistochemical staining for laminin

    DEFF Research Database (Denmark)

    Albrechtsen, R; Nielsen, M; Wewer, U;

    1981-01-01

    micrometastases were present, these cells also stained strongly for laminin. In nonmalignant breast tissues, the epithelial cells of the duct were positive for laminin, but the staining was weaker than in the carcinomas. Pretreatment of the fixed tissue sections with trypsin markedly enhanced the staining of......The distribution of the basement membrane glycoprotein laminin was studied by the immunoperoxidase technique in benign and malignant human breast tissue and in axillary lymph nodes from patients with breast cancer. An antiserum prepared against rat laminin was used. The specificity of this...... molecular weights of 400,000 and 200,000 of rat laminin in sodium dodecyl sulfate:polyacrylamide gel electrophoresis. The neoplastic cells in malignant breast tissues showed strong cytoplasmic staining for laminin, and a positive reaction was aslo found in lymph node metastases. In some cases in which only...

  19. Quantitative determination of urinary tetrahydroaldosterone by a radioimmunological method. Contribution to the study of its excretion

    International Nuclear Information System (INIS)

    The aim here is to propose a method of urinary tetrahydroaldosterone determination based on radioimmunology, the advantages of which distinguish this technique from the many others already available. The work is divided into three parts: - the first, of more basic interest, gives an overall view of the biological and biochemical properties of tetrahydroaldosterone, the understanding of its analysis and the interpretation of the results via that of its metabolism; - part two considers aspects of the radioimmunological method, first in general then applied to tetrahydroaldosterone determination, and the elements needed for its use: reference standard, immunogen and antiserum preparation. Its value is governed by two fundamental sets of requirements those concerning the conditions of validity of any analysis technique and those relating to radioimmunology; - part three discusses in practical terms the determination of more than 470 pathological cases. Results already obtained by other authors on certain aspects of corticoadrenal pathology are examined for comparative purposes

  20. Rapid concentration and dialysis of proteins with single hollow fibers: possible applications in analysis of protein secretion by isolated cells and steroid radioimmunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Rommerts, F.F.G.; Clotscher, W.F.; Van der Molen, H.J.

    1977-10-01

    Single hollow fibers were used in specially made cells for fast concentration and dialysis of solutions containing macromolecules. Volumes on the order of 5 ml of diluted protein solutions could be concentrated to 50--100 ..mu..l or less within 7 min with a protein recovery of 60--80%. More than 99% of the molecules with a molecular weight less than 500 could be removed in less than 1 hr. A possible application of the rapid dialysis method for the mechanization of radioimmunoassays is indicated. It was shown that in the radioimmunoassay of steriods the unbound steroids could be removed after incubation with antiserum, within 10 min and without a change in volume.

  1. Production of Anti-triiodothyronine sulfate antibody for radioimmunoassay applications

    International Nuclear Information System (INIS)

    Triiodothyronine sulfate (T3S) may be an obligatory intermediate metabolic of the metabolism of thyroid gland hormones invertebrates in peripheral during the process of deiodination of the inactive form of the thyroid gland hormones, thyroxine(T4), into the active form triiodothyronine (1,2). Construction of a reliable procedure for the estimation of T3S accurately in blood serum will be of great importance for medical, biochemical and physiological investigations. In this work we developed a robust method for the production of anti-triiodothyronine sulfate polyclonal antiserum with good specifications using a derivatized immuno gen and a modified immunization process and a sensitive radioimmunoassay system was designed and developed

  2. An enzyme immunoassay for plasma betamethasone

    International Nuclear Information System (INIS)

    A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-beta-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using 3H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol as 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay

  3. An enzyme immunoassay for plasma betamethasone

    Energy Technology Data Exchange (ETDEWEB)

    Kominami, G.; Yamauchi, A.; Ishihara, S.; Kono, M.

    1981-03-01

    A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-beta-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using /sup 3/H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol as 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.

  4. Two cases of viper bite: still an important health problem

    Directory of Open Access Journals (Sweden)

    Adrija Hajra

    2016-04-01

    Full Text Available Viper venoms act mainly as hemotoxic. Manifestations of snakebites depend on specific toxins that constitute the venom. The local and systemic snake bite related symptoms are directly linked to the toxicity of the venom. Edema, ecchymoses, hematoma, and gangrenous lesions are reported to occur as local symptoms. Systemic symptoms may include fever, nausea, vomiting, delirium, jaundice, circulatory collapse, convulsions, and coma. Death from secondary infections, neurotoxicity, disseminated intravascular coagulation (DIC, intracranial hemorrhage, and acute renal failure are the well-known facts. For reduction of morbidity and mortality, it is important that antiserum is administered at the appropriate dose as early as possible after snake bite. There are several case reports about various complications of viperid bite. Here we are discussing two cases of viper bite. These cases are unique because of the extensive tissue necrosis. One of them succumbed to septicemia after acute pancreatitis. [Int J Res Med Sci 2016; 4(4.000: 1274-1277

  5. A radioimmunoassay for serum medroxyprogesterone acetate.

    Science.gov (United States)

    Shrimanker, K; Saxena, B N; Fotherby, K

    1978-04-01

    When injected intramuscularly in a dose of 150 mg, Depo Provera, a microcrystalline suspension of medroxyprogesterone acetate (MPA), will provide a contraceptive effect for at least 3 months. This paper describes a sensitive radioimmunoassay for MPA which has been used in the author's laboratory for the past 2 years. MPA was converted to MPA-3-CMO and the oxime was conjugated with bovine serum albumin (BSA) by the mixed anahydride method. 4 rabbits were immunized with the antiserum. A high titre of MPA antibodies was detected 6 months after immunization. Serum from the rabbit with the highest titre of antibodies to MPA was subjected to radioimmunoassay. 7 days after the intramuscular injection of 150 mg Depo-Provera, serum levels of MPA were found in the range of 1750 to 9000 pg/ml. By 75 days, the levels had decreased to 680-2600 pg/ml. The method was found to have adequate accuracy, precision and sensitivity. PMID:661315

  6. A radioimmunoassay for virus antibody using binding of 125I-labelled protein A

    International Nuclear Information System (INIS)

    An assay for virus antibodies using protein A from Staphylococcus aureus is described. Type B and type C RNA tumour viruses adsorbed on to polystyrene microtitre plate wells were incubated with antiserum and then with 125I-labelled protein A (I-pA) and bound radioactivity was determined. Technical details such as labelling, antigen concentration, storage of I-pA are reported. The specificity of the reaction was investigated in detail by competition experiments with purified unbound homologous viruses. This assay also proved to be sensitive for demonstration of autogenous immunity to both type B and type C RNA tumour viruses. A study using antisera against purified core and envelope virus proteins of mammary tumour and leukaemia viruses suggested that the reaction mainly involves surface antigens of the intact virions. (author)

  7. Immobilization of viral antigens on filter paper for a [125I] staphylococcal protein A immunoassay

    International Nuclear Information System (INIS)

    A new technique is described for the rapid detection and quantitation of herpes simplex virus (HSV) antigens and antiviral antibodies. It involves immobilization of HSV antigens on filter paper discs and subsequent analysis by 125I-labelled staphylococcal protein A (SPA) radioimmunoassay. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. It is rapid, simple, sensitive and specific, and requires only small volumes of antiserum and few target cells. The results may be readily and objectively quantitated. This technique permits the simultaneous assay of a large number of specimens in less than 1h. Its sensitivity is considerably greater than that of other currently used immunologic techniques, and it is amenable to automation. These characteristics suggest that this [125 I]SPA immunofiltration technique may be applicable to the rapid diagnosis of viral infections. (Auth.)

  8. Establishment of colloid gold immunity chromatography assay for cardiac troponin I (cTnI)

    International Nuclear Information System (INIS)

    Objective: To establish the colloid gold Immunity chromatography assay for cardiac troponin I. Methods: To purify cTnI from human cardiac muscle and immunize rabbit with it. cTnI antibody of rabbit anti-human cardiac muscle has been prepared and colloid gold immunity chromatography assay was established by using immunity chromatography technology. Results: Anti-serum titles of cTnI were 1:100000, Ka=2.38 x 109 L/mol; Methodological index: Sensitivity: 5 ng/ml; Specificity: cTnI is no cross-reaction with cTnT, cTnC and CK-MB. conclusion: The assay is highly specific, quick and simple. It can be widely used for the early diagnosis of AMI and scientific research. (authors)

  9. Enhanced expression of beta2-microglobulin and HLA antigens on human lymphoid cells by interferon

    DEFF Research Database (Denmark)

    Heron, I; Hokland, M; Berg, K

    1979-01-01

    Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 hr in the presence or absence of human interferon. The relative quantities of HLA antigens and beta(2)-microglobulin on the cultured cells were determined by quantitative...... immunofluorescence (fluorescence-activated cell sorter) and by the capacity of cells to absorb out cytotoxic antibodies against the relevant antigens. Interferons of different origin and purities enhanced the expression of HLA antigens and beta(2)-microglobulins, whereas membrane immunoglobulins and antigens...... recognized by antiserum raised against human brain and T cells were the same on interferon-treated and control cells. Similar interferon effects were observed on an Epstein-Barrvirus-negative Burkitt lymphoma cell line. The enhanced expression of histocompatibility antigen subsequent to intereferon treatment...

  10. Purification and characterization of an organ specific haemorrhagic toxin from Vipera russelli russelli (Russell's viper) venom.

    Science.gov (United States)

    Kole, L; Chakrabarty, D; Datta, K; Bhattacharyya, D

    2000-04-01

    A haemorrhagic toxin (VRR-12) from Vipera russelli russelli (Russell's viper) venom has been purified by ion-exchange chromatography on CM-Sephadex C-50 followed by size-exclusion HPLC to electrophoretically homogeneous state. It is a 12 kDa single polypeptide having 1 mole of Zn+2 ion. This toxin induces intense intestinal haemorrhage and to a lesser extent skeletal muscle haemorrhage in mice. It does not show detectable proteolytic and esterolytic activity with selected substrates under specified conditions, haemolytic and phospholipase activity. When VRR-12, preincubated with bivalent antiserum against Saw-scaled and Russell's viper venom or EDTA was injected, haemorrhagic activity was not reduced, on the other hand preincubation with phenylmethyl sulphonyl fluoride reduced the activity markedly. Biodistribution studies with 125I VRR-12 show that haemorrhagic manifestation by this toxin is not a direct function of the fraction of the totally administered toxin distributed to that tissue. PMID:10983422

  11. Solid-phase radioimmunoassay for vitamin B12 in serum, with use of radioiodinated tyrosine methyl ester of vitamin B12

    International Nuclear Information System (INIS)

    Although radioassays for vitamin B12 with use of any of several binding proteins have been available for many years, a radioimmunoassay for B12 has not been reported. We describe here such a radioimmunoassay, incorporating, for the first time, a radioiodinated tyrosine methyl ester of B12 as the radioactive tracer. Polypropylene tubes are coated with antiserum raised in a rabbit against B12/bovine serum albumin to simplify the separation of bound and free radioactivity. Factors affecting the preparation of coated tubes are described. The assay is accurate, sensitive, precise, and specific for vitamin B12. Accuracy of the assay is unaffected by the presence of denatured protein. The advantages of this radioimmunoassay over conventional radioassays are discussed

  12. Antibody responses induced by Japanese whole inactivated vaccines against equine influenza virus (H3N8) belonging to Florida sublineage clade2.

    Science.gov (United States)

    Yamanaka, Takashi; Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Kondo, Takashi; Matsumura, Tomio

    2011-04-01

    In 2010, the World Organisation for Animal Health recommended the inclusion of a Florida sublineage clade2 strain of equine influenza virus (H3N8), which is represented by A/equine/Richmond/1/07 (Richmond07), in equine influenza vaccines. Here, we evaluate the antigenic differences between Japanese vaccine strains and Richmond07 by performing hemagglutination inhibition (HI) assays. Ferret antiserum raised to A/equine/La Plata/93 (La Plata93), which is a Japanese vaccine strain, reacted with Richmond07 at a similar titer to La Plata93. Moreover, two hundred racehorses exhibited similar geometric mean HI antibody titers against La Plata93 and Richmond07 (73.1 and 80.8, respectively). Therefore, we can expect the antibody induced by the current Japanese vaccines to provide some protection against Richmond07-like viruses. PMID:21099188

  13. Study and development of a radioimmunoassay of antidiuretic hormone sensitive at 10/sup -12/M

    Energy Technology Data Exchange (ETDEWEB)

    Caillens, H.; Rousselet, F. (Faculte des Sciences Pharmaceutiques, (France)); Paillard, F. (Hopital Tenon, Paris (France))

    1982-07-01

    A radioimmunoassay of antidiuretic hormone is described. The antiserum was obtained by immunization of rabbits with lysine vasopressin conjugated to hemocyanine. The specificity of the antibody was selective and directed against the pentapeptide ring of the vasopressin molecule: oxytocin showed no cross-reactivity at 10/sup -9/M. The labelled hormone (/sup 125/I-AVP) prepared using the chloramine-T method had a high specific activity (1860 Ci/mmol). Incubation was performed in an equilibrium system. Comparative studies of different separation methods of bounds and free /sup 125/I-AVP showed that the sensitivity and the precision of the standard curve were better using charcoal dextran. The limit of detection of the assay was 1,6 pg per ml.

  14. Studies on the Feeding Habits of Lutzomyia (Lutzomyia longipalpis (Lutz & Neiva, 1912 (Diptera: Psychodidae: Phlebotominae Populations from Endemic Areas of American Visceral Leishmaniasis in Northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Margarete Martins dos Santos Afonso

    2012-01-01

    Full Text Available The aim of this study was to identify potential blood feeding sources of L. (L. longipalpis specimens from populations in Northeastern Brazil, endemic areas of American Visceral Leishmaniasis (AVL and its correlation with the transmission of L. (L. i. chagasi. The ELISA technique was applied using bird, dog, goat, opossum, equine, feline, human, sheep, and rodent antisera to analyze 609 females, resulting in an overall positivity of 60%. In all municipalities, females showed higher positivity for bird followed by dog antiserum and sand fly specimens were also positive for equine, feline, human, sheep, goat, opossum, and rodent antisera. The finding for 17 combinations of two or three types of blood in some females corroborates the opportunistic habit of this sand fly species. The results demonstrating the association between L. (L. longipalpis and opossum suggest the need for further evaluation of the real role of this synanthropic mammal in the eco-epidemiology of AVL.

  15. Preliminarily functional analysis of a cloned novel human gene ADAM29

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ADAM is a family of type I integral membrane proteins which are characterized by sharing a disintegrin and metalloprotease domain and involved in many important physiological processes such as fertilization, neurogenesis and inflammatory response. A novel human ADAM gene--ADAM29, which was cloned in our laboratory, is exclusively expressed in human testis and contains a potential fusion domain. A full-length cDNA of ADAM29 was obtained by using multiple-step PCR. Phylogenetic tree of known mammalian ADAMs specifically expressed in testis was reconstructed. Polyclonal antiserum was raised by immunizing the rabbits with sub-peptide of ADAM29 (Leu268-Asp374) as immunogen. The result of immunohistochemical test on human testis showed that ADAM29 is expressed in different stages of spermatogenesis and in interstitial cells. ADAM29 may play a certain role in the signal transduction during the maturation of testis-associated cells.

  16. First experiences with commercial RIA kits for prostatic acid phosphatase (PAP)

    International Nuclear Information System (INIS)

    Five commercial PAP RIA kits were intercompared by common RIA quality control criteria. All RIAs performed basically well although some differences existed in respect to concentration range, specific and non-specific binding, 50%-intercept, sensitivity and measurements of serum PAP in male and female controls. The latter finding may have been due to differences in antigen purity, antiserum specificity and composition of the assay medium employed. Good correlation was found between PAP determination by RIA and by enzyme assay. First measurements of PAP in patients treated for prostatic carcinoma being performed for orientation purposes are demonstrated. The PAP RIA has been introduced into our routine diagnostic and follow-up of prostatic carcinoma. (orig.)

  17. The Role of Mosquitoes in the Diet of Adult Dragon and Damselflies (Odonata).

    Science.gov (United States)

    Pfitzner, Wolf Peter; Beck, Matthias; Weitzel, Thomas; Becker, Norbert

    2015-06-01

    The flood plains of the Upper Rhine Valley provide excellent conditions for the proliferation of mosquitoes as well as for the development of dragon and damselflies. It could be assumed that mosquitoes belong to the diet of the Odonata and that the latter could be harmed by the reduction of the mosquito population with the purpose of diminishing the massive nuisance for the people living there. A total of 41 adult dragonflies and damselflies were examined by immunoblot for remnants of mosquitoes in their guts. A rabbit antiserum against Aedes vexans proteins was used for the immunoblot. Only 3 Aeshna cyanea and 1 Platycnemis pennipes could be shown to have fed on mosquitoes. In specimens of the genus Sympetrum no mosquitoes were detected. It seems very doubtful that mosquitoes are an essential part of the Odonata diet. PMID:26181697

  18. Gamma radiation effects on crotoxin (toxin of crotalus durissus terrificus venom)

    International Nuclear Information System (INIS)

    The crotoxin is a great neurotoxin found on Crotalus durissus terrificus venom. This protein was isolated using molecular exclusion cromatography with Sephadex G-75 and irradiated in a source of 60 Co GAMMA-CELL in the concentration of 2 mg/ml 0.85% NaCl with dose rate of 1.19 x 103 Gy/hr. The doses used were 250, 500, 1000 and 2000 Gy. It was determinated for this samples, the proteic concentration, the diffusion immunoassay using crotalic antiserum and eletrophoresis (SDS-PAGE). The results showed some changes on the irradiated toxin. Preliminary results with doses of radiation of 100, 250, 500 and 1000 Gy showed that the letal dose 50% (LD50) in mice increase greatly with the increase of radiation's dose. (author)

  19. Cyclic nucleotides and calcium ions in activation of mouse B lymphocyte motility activated by anti-immunoglobulin serum

    International Nuclear Information System (INIS)

    This paper studies the role of cyclic nucleotides and Ca++ ions in activation of B lymphocytes by antiserum against immunoglobulins, with particular reference to induction of motility of these cells by serum, on the basis of the concept of the central role of cyclic nucleotides and calcium in processes of cellular proliferation and motility. Experiments were carried out on 75 male C57BL/6 mice weighing 20-25 g. After decapitation and homogenization of the spleen, lymphocytes were obtained. Radioimmunoassay was used to determine the levels of cAMP and cGMP in the supernatant. The viability of the cells was estimated at all stages on the basis of staining with 0.1% trypan blue. The mechanism of mobilization of intracellular calcium described makes B lymphocytes independent of extracellular Ca++ in the processes of activation of B lymphocytes by immunogenic and other stimuli

  20. Immunoassay of human TSH using dried blood samples

    International Nuclear Information System (INIS)

    A sensitive, semi-quantitative radioimmunoassay method to screen for elevations of TSH concentration in blood is described. The method requires two 0.32 cm dots of dried blood-impregnated filter paper (equivalent to 3 μl plasma) and a 3-day incubation. Separation of bound and free is obtained using polyethylene glycol. The method can recognize TSH concentrations as low as 22 μU/ml using a highly sensitive antiserum developed by one of us (AFP). TSH in dried cord and newborn blood from four infants with congenital hypothyroidism was clearly higher than in normal infants. The method is suitable for use in a newborn screening program to confirm the suspicion of primary hypothyroidism in specimens with low T4 concentrations