WorldWideScience

Sample records for antiserum

  1. Development of antiserum against bovine prolactin for radioimmunoassay

    International Nuclear Information System (INIS)

    Razdan, M.N.; Kaker, M.L.; Galhotra, M.M.

    1980-01-01

    Antiserum against bovine prolactin was raised in male rabbits and tested against crossreacting contaminants by immunodiffusion and immunoelectrophoresis. One sharp precipitin band against prolactin was observed. The antiserum showed a small faint band against bovine growth hormone. No crossreaction was observed against bovine follicle stimulating hormone, bovine luteinizing hormone, bovine thyroid stimulating hormone and normal calf serum. The crossreaction with bovine growth hormone vanished after treating the antiserum with normal calf serum. Antibody titre of the absorbed anti-prolactin serum determined by radioimmunoassay was high, which could bind 31.30% at 1:10,000 final dilution. Maximum binding was not inhibited by the addition of higher amounts of contaminants, suggesting that the absorbed antiserum was immunologically monospecific and could be used for radioimmunoassay work. (author)

  2. Production of carboxy-terminal specific antiserum against glucagon

    International Nuclear Information System (INIS)

    Liu Yibing; Han Shiquan

    1993-01-01

    To produce carboxy-terminal specific antisera against glucagon was coupled mainly via its amino terminal histidine to thyroglobulin, using the amino group reactive pentandiol at pH 7.0 for the conjugation procedure. After repeated immunization of guinea pigs and rabbits, the antisera were obtained. The titer of guinea pig antiserum against glucagon was 1:3000-1:35000 and affinity constant was 9.3 x 10 10 -11.4 x 10 10 l · mol -1 . There were no cross reaction with GIP, INS, Copeptide and gastrin. The titer of rabbit antiserum against glucagon was 1:900-1:9000 and affinity constant was 0.36 x 10 10 -3.9 x 10 10 l · mol -1 . There were no cross reaction with INS, C-peptide and gastrin. The cross reaction with GIP was 0.02%

  3. Protection of rats from experimental allergic encephalomyelitis with antiserum to guinea-pig spinal cord

    Science.gov (United States)

    Hughes, R. A. C.

    1974-01-01

    Inbred AS rats were immunized with guinea-pig spinal cord. The severity of experimental allergic encephalomyelitis (EAE) was studied in groups treated with antiserum to guinea-pig spinal cord and control groups treated with antiserum to other guinea-pig tissue. Prolonged treatment throughout the experiment strikingly reduced the severity of the clinical disease. Early treatment for the first 8 days after immunization had a similar protective effect. Late treatment starting on the 10th day after immunization did not produce significant protection. Protection was produced by antiserum from both EAE-resistant and EAE-susceptible strains. The protective effect of the serum did not correlate with the presence of haemagglutinating antibodies to basic protein or the titre of complement-fixing antibodies to galactocerebroside. These results suggest that antiserum to undefined central nervous system antigens contains an `enhancing' antibody which inhibits a relatively early stage of the immune response causing EAE. PMID:4212280

  4. 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 ...

  5. Antilymphocyte antiserum effect on incidence of spontaneous malignancy. I. Long term treated mice.

    Science.gov (United States)

    Pirofsky, B; Dawson, P J

    1976-01-01

    Long term potent immunosuppression was administered to normal 7-week-old BALB/c mice without exogenous neoplastic induction. Antilymphocyte antiserum was given biweekly for 8 weeks and azathioprine daily for 347 days. Significant immunodepression was demonstrated after 348 days of theraphy. After 2 years, all animals were sacrificed and examined for neoplasia. There was no evidence that such immunodepression increased the incidence of spontaneous malignancy. The study suggests that immunosuppressive therapy is not inately oncogenic.

  6. Immunoreactivity between venoms and commercial antiserums in four Chinese snakes and venom identification by species-specific antibody.

    Science.gov (United States)

    Gao, Jian-Fang; Wang, Jin; Qu, Yan-Fu; Ma, Xiao-Mei; Ji, Xiang

    2013-01-31

    We studied the immunoreactivity between venoms and commercial antiserums in four Chinese venomous snakes, Bungarus multicinctus, Naja atra, Deinagkistrodon acutus and Gloydius brevicaudus. Venoms from the four snakes shared common antigenic components, and most venom components expressed antigenicity in the immunological reaction between venoms and antiserums. Antiserums cross-reacted with heterologous venoms. Homologous venom and antiserum expressed the highest reaction activity in all cross-reactions. Species-specific antibodies (SSAbs) were obtained from four antiserums by immunoaffinity chromatography: the whole antiserum against each species was gradually passed through a medium system coated with heterologous venoms, and the cross-reacting components in antiserum were immunoabsorbed by the common antigens in heterologous venoms; the unbound components (i.e., SSAbs) were collected, and passed through Hitrap G protein column and concentrated. The SSAbs were found to have high specificity by western blot and enzyme-linked immunosorbent assay (ELISA). A 6-well ELISA strip coated with SSAbs was used to assign a venom sample and blood and urine samples from the envenomed rats to a given snake species. Our detections could differentiate positive and negative samples, and identify venoms of a snake species in about 35 min. The ELISA strips developed in this study are clinically useful in rapid and reliable identification of venoms from the above four snake species. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. ISOLATION OF ANTIGENIC MUTANTS OF TYPE 1 POLIOVIRUS : GROWING THE VIRUS IN THE PRESENCE OF HOMOLOGOUS ANTISERUM

    OpenAIRE

    HASHIMOTO, Nobuo

    1980-01-01

    The antigenic mutants, which were significantly different intra-typically from the parental Mahoney-1709 type 1 poliovirus, were repeatedly isolated after a number of serial virus passages in the presence of homologous anti-Mahoney sera. The levels of the antigenic variation of those mutant viruses varied, depending on the individual antiserum employed for mutant selection and the passage numbers with same antiserum. The susceptibility to certain serum inhibitors of the antigenic mutants vari...

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

    International Nuclear Information System (INIS)

    Silva, S.R. da.

    1993-01-01

    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

  9. 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

  10. Inhibition of dye-coupling in Patella (mollusca) embryos by microinjection of antiserum against Nephrops (arthropoda) gap junctions

    NARCIS (Netherlands)

    Serras, F.; Buultjens, T.E.J.; Finbow, M.E.

    1988-01-01

    Antiserum raised against Nephrops gap junctions was injected into single cells of the 2-, 4-, 8-, 16-, and 32-cell stage of the Patella vulgata embryos. The pattern of junctional communication by iontophoresis of Lucifer Yellow CH was tested at the 32-cell stage. The results show that the normal

  11. The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli O 157.

    Science.gov (United States)

    Kleanthous, H; Fry, N K; Smith, H R; Gross, R J; Rowe, B

    1988-10-01

    Using DNA probes specific for the genes encoding Vero cytotoxins 1 and 2 in hybridization experiments on faecal samples, Vero cytotoxin-producing Escherichia coli (VTEC) of serogroup O 157 were detected in 21 of 63 cases of haemorrhagic colitis, 9 of 31 cases of non-bloody diarrhoea and 14 of 68 cases of haemolytic uraemic syndrome. Compared with these results sorbitol-MacConkey agar in conjunction with a specific O 157 antiserum gave a sensitivity of 62% in haemorrhagic colitis, 56% in non-bloody diarrhoea and 57% in haemolytic uraemic syndrome. The specificity of this method was 100% in all three groups. This demonstrates that sorbitol-MacConkey agar is a useful screening method for the detection of VTEC of serogroup O 157 when used in conjunction with a specific homologous antiserum. However, this method does not detect VTEC belonging to other serogroups and such strains were found, particularly in cases of haemolytic uraemic syndrome.

  12. Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G; Choudhary, Neelima; Routh, Patty A; Cholon, Deborah M; Hobbs, Marcia M; Almond, Glen W; Orndorff, Paul E; Elkins, Christopher

    2011-08-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

  13. Passive Immunization with a Polyclonal Antiserum to the Hemoglobin Receptor of Haemophilus ducreyi Confers Protection against a Homologous Challenge in the Experimental Swine Model of Chancroid▿

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G.; Choudhary, Neelima; Routh, Patty A.; Cholon, Deborah M.; Hobbs, Marcia M.; Almond, Glen W.; Orndorff, Paul E.; Elkins, Christopher

    2011-01-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme. PMID:21646451

  14. Conjugated oestrogen during the menstrual cycle measured by a direct radioimmunoassay with an antiserum prepared against oestradiol-17-glucosiduronate-(C-6)-BSA conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Honjo, H.; Otsubo, K.; Yasuda, J.; Kitawaki, J.; Okada, H. (Department of Obstetrics and Gynaecology, Kyoto Prefectural University of Medicine, Kyoto); Ohkubo, T.; Nambara, T. (Pharmaceutical Institute, Tohoku University, Sendai, Japan)

    1984-01-01

    Early morning and 24 h urine samples and serum were collected daily throughout the menstrual cycle in women. Urinary oestradiol-17-glucosiduronate (E/sub 2/-17G) was measured with a direct radioimmunoassay whose antiserum has been prepared against E/sub 2/-17-G-(C-6)-bovine serum albumin conjugate and was very specific. On the average, E/sub 2/-17-G in early morning and 24 h urine samples showed a prominent peak one day before the peak of urinary LH. The time relationship between these urinary E/sub 2/-17-G and serum E/sub 2/ levels and peaks was also investigated.

  15. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice.

    Directory of Open Access Journals (Sweden)

    Toru Takeo

    Full Text Available Superovulation is a reproductive technique generally used to produce genetically engineered mice. Superovulation in mice involves the administration of equine chorionic gonadotropin (eCG to promote follicle growth and then that of human chorionic gonadotropin (hCG to induce ovulation. Previously, some published studies reported that inhibin antiserum (IAS increased the number of ovulated oocytes in ddY and wild-derived strains of mice. However, the effect of IAS on the C57BL/6 strain, which is the most widely used inbred strain for the production of genetically engineered mice, has not been investigated. In addition, the combined effect of IAS and eCG (IASe on the number of ovulated oocytes in superovulation treatment has not been examined. In this study, we examined the effect of IAS and eCG on the number of ovulated oocytes in immature female mice of the C57BL/6 strain in superovulation treatment. Furthermore, we evaluated the quality of obtained oocytes produced by superovulation using IASe by in vitro fertilization (IVF with sperm from C57BL/6 or genetically engineered mice. The developmental ability of fresh or cryopreserved embryos was examined by embryo transfer. The administration of IAS or eCG had a similar effect on the number of ovulated oocytes in C57BL/6 female mice. The number of ovulated oocytes increased to about 3-fold by the administration of IASe than by the administration of IAS or eCG alone. Oocytes derived from superovulation using IASe normally developed into 2-cell embryos by IVF using sperm from C57BL/6 mice. Fresh or cryopreserved 2-cell embryos produced by IVF between oocytes of C57BL/6 mice and sperm from genetically engineered mice normally developed into live pups following embryo transfer. In summary, a novel technique of superovulation using IASe is extremely useful for producing a great number of oocytes and offspring from genetically engineered mice.

  16. 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)

    Tiberghien, P.; Longo, D.L.; Wine, J.W.; Alvord, W.G.; Reynolds, C.W.

    1990-01-01

    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

  17. 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

    Energy Technology Data Exchange (ETDEWEB)

    Tiberghien, P.; Longo, D.L.; Wine, J.W.; Alvord, W.G.; Reynolds, C.W. (Program Resources, Inc., Frederick, MD (USA))

    1990-10-01

    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.

  18. Streptococcus sinensis may react with Lancefield group F antiserum.

    Science.gov (United States)

    Woo, Patrick C Y; Teng, Jade L L; Leung, Kit-wah; Lau, Susanna K P; Tse, Herman; Wong, Beatrice H L; Yuen, Kwok-yung

    2004-11-01

    Lancefield group F streptococci have been found almost exclusively as members of the 'Streptococcus milleri' group, although they have been reported very occasionally in some other streptococcal species. Among 302 patients with bacteraemia caused by viridans streptococci over a 6-year period, three cases were caused by Streptococcus sinensis (type strain HKU4T, HKU5 and HKU6). All three patients had infective endocarditis complicating their underlying chronic rheumatic heart diseases. Gene sequencing showed no base differences between the 16S rRNA gene sequences of HKU5 and HKU6 and that of HKU4T. All three strains were Gram-positive, non-spore-forming cocci arranged in chains. All grew on sheep blood agar as alpha-haemolytic, grey colonies of 0.5-1 mm in diameter after 24 h incubation at 37 degrees C in ambient air. Lancefield grouping revealed that HKU5 and HKU6 were Lancefield group F, but HKU4T was non-groupable with Lancefield groups A, B, C, D, F or G antisera. HKU4T was identified by the Vitek system (GPI), API system (20 STREP) and ATB system (ID32 STREP) as 99 % Streptococcus intermedius, 51.3 % S. intermedius and 99.9 % Streptococcus anginosus, respectively. Using the same tests, HKU5 was identified as 87 % Streptococcus sanguinis/Streptococcus gordonii, 59 % Streptococcus salivarius and 99.6 % S. anginosus, respectively, and HKU6 as 87 % S. sanguinis/S. gordonii, 77 % Streptococcus pneumoniae and 98.3 % S. anginosus, respectively. The present data revealed that a proportion of Lancefield group F streptococci could be S. sinensis. Lancefield group F streptococci should not be automatically reported as 'S. milleri'.

  19. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Receptors for Theiler's murine encephalomyelitis virus: characterization by using rabbit antiviral antiserum

    International Nuclear Information System (INIS)

    Rubio, N.; Cuesta, A.

    1988-01-01

    An immunological assay was developed to characterize the binding of Theiler's murine encephalomyelitis virus to BHK-21 cell receptors. After absorption of the virus and formaldehyde fixation, rabbit antibodies and Staphylococcus aureus protein A labeled with 125 I formed a specific complex on the surfaces of the cells. The optimal multiplicity of infection in this system was 10 PFU per cell. The virus was internalized at 33 and 37 0 C, but internalization did not take place at 25 or 4 0 C. The binding was proportional to the number of cells and was significant within 30 s. Cell surface receptors were still active after fixation, and only intact viruses were bound, as demonstrated by the lack of binding of the purified, isolated virion proteins VP1, VP2, and VP3

  1. 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

    Protein A) as the affinity ligand, and differed from one another primarily with respect to the underlying base matrix. The remaining five matrices comprised various synthetic low molecular weight ligands immobilised on hydrophilic porous supports and these included: MEP HyperCel, MabSorbent AlP, Mab...

  2. Production of antiserum and development of radioimmunoassay for progesterone measurement in serum

    International Nuclear Information System (INIS)

    Abdalla, O.M; Abbas, S.H.

    2004-03-01

    The anti progesterone antibody was obtained by injection of local sudanese sheep (bevies arias) intramuscularly and subcutaneously using 11-a-hydroxy progesterone conjugated with bovine serum albumin and emulsified in friend's adjuvant. The blood samples collected one week after the 3 rd injection was tested for anti-progesterone antibodies. Different dilutions of antisera in Pbs containing 0.0.1 % BSA and 0.1 % Na N 3 (Ph 7.4) were made. Using these dilutions, polystyrene balls of 6.4 mm diameter were coated with a coating volume of 500 μ1/bead.The antibody dilution of 1/200000 was selected and used for assembling a kit to measure progesterone level in serum. Optimization of the assay condition resulted in the following recipes: Fifty μ1 of standard or Qc (Low, normal and high) from Bio Rad for 2 hours. The reaction medium aspirated, washed twice and read for 100 seconds in gamma counter. Assay validation tests including Linearity, recovery, reproducibility and comparability were done. In regards to linearity, the measured concentrations of progesterone in serum samples were plotted against the expected values. The correlation coefficient was 0.997 and a linear regression equation was Y=1.1019+0.9895 X. The mean recovery was 96.86% of the sample replicates ranged from 0.2% to 6%. between assay reproducibility was assessed fro the results obtained from 3 samples which were used for quality control in different assays. The results obtained were in the reference range and were close to each other. Comparison between local and American Dpc reagents for progesterone determination in serum showed high correlation high correlation where r 0.9492.(Author)

  3. Fimch antiserum inhibits the adherence of Escherichia coli to cells collected by voided urine specimens of diabetic women

    NARCIS (Netherlands)

    Meiland, Ruby; Geerlings, Suzanne E.; Langermann, Solomon; Brouwer, Ellen C.; Coenjaerts, Frank E. J.; Hoepelman, Andy I. M.

    2004-01-01

    PURPOSE: With the increasing problem of resistance in pathogenic microorganisms the development of nonantimicrobial therapies is important. Diabetes mellitus (DM) is associated with an increased incidence of urinary tract infections. The majority of Escherichia coli strains, which is the most

  4. 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)

    Clarke, A.H.; Ing, R.M.Y.; Jones, W.R.; Llewellyn-Jones, D.; Shutt, D.A.

    1974-01-01

    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.)

  5. 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.

  6. Serum sickness

    Science.gov (United States)

    ... the problem should be stopped. Avoid using that medicine or antiserum in the future. ... Call your provider if you received medicine or antiserum in the last 4 weeks and have symptoms of serum sickness.

  7. THE IMMUNOLOGICAL RESPONSE OF SOUTH AFRICAN ...

    African Journals Online (AJOL)

    Foot muscle antigens of the diploid Bulinus nala/ensis from Lake Sibayi showed complete immunological correspondence with antiserum prepared from foot muscle tissues of the diploid B. Iropicus. On the other hand, strong "non-identity" reactions occurred when B. nala/ensis antigens were tested with the antiserum ...

  8. The immunological response of South African Bulinus natalensis ...

    African Journals Online (AJOL)

    Foot muscle antigens of the diploid Bulinus natalensis from Lake Sibayi showed complete immunological correspondence with antiserum prepared from foot muscle tissues of the diploid B. tropicus. On the other hand, strong “non-identity” reactions occurred when B. natalensis antigens were tested with the antiserum ...

  9. TERATOGENIC EFFECTS OF TRANSPLACENTAL TRANSFUSION OF HETEROLOGOUS ANTISERA SIMULATED IN AN EXPERIMENTAL-MODEL USING INVITRO WHOLE RAT EMBRYO CULTURE

    NARCIS (Netherlands)

    VANDERZEE, DC; POELMANN, RE; ZWIERSTRA, RP; MENTINK, MMT; VERMEIJKEERS, C

    1991-01-01

    The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion

  10. Immunochemical analysis of apamin and its derivatives

    International Nuclear Information System (INIS)

    Vasilenko, S.V.; Kolesnikova, I.N.; Miroshnikov, A.I.; Komissarenko, S.V.

    1981-01-01

    The radioimmunologic analysis of apamin (neurotoxin of the venom of Apis mellifica honey bee) is carried out using its derivatives which have modifications of various aminoacidic remnants and antibodies against apamin. The curves of 125 J apamin binding with antiserum against apamin and the curves of inhibition with various nonlabelled binding effectors of 125 J-apamin with antiserum are obtained

  11. The ultrastructural localization of keratin proteins and carcinoembryonic antigen in malignant mesotheliomas.

    Science.gov (United States)

    Warhol, M J

    1984-09-01

    The immunoultrastructural localization of keratin proteins and carcinoembryonic antigen (CEA) in mesothelioma cells was accomplished with a low-temperature embedding colloidal gold technique. The keratin antiserum labeled only intermediate filaments. These filaments surrounded the cytoplasmic organelles and were inserted into desmosomes. The CEA antiserum labeled cytoplasmic vesicles and droplets. No definite labeling of microvilli was observed.

  12. Characterization of a rabbit polyclonal antibody against threonine-AMPylation

    Science.gov (United States)

    Hao, Yi-Heng; Chuang, Trinette; Ball, Haydn L.; Luong, Phi; Li, Yan; Flores-Saaib, Ruben D.; Orth, Kim

    2014-01-01

    An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins. PMID:21185336

  13. Partial passive protection with two monoclonal antibodies and frequency of feeding of hyperimmune anti-transmissible gastroenteritis virus (TGEV) serum for protection of three-day-old piglets from a TGEV challenge infection.

    Science.gov (United States)

    Wesley, R D; Woods, R D

    2001-07-01

    Passive protection experiments were conducted to determine the frequency and amounts of hyperimmune antiserum needed to block a transmissible gastroenteritis virus (TGEV) challenge infection and to identify monoclonal antibodies that are partially protective against TGEV. Hyperimmune antiserum or monoclonal antibodies were added to milk at each feeding or at selected feedings when the amount of antiserum was reduced. Three-day-old piglets were challenged with virulent virus that had been preincubated with antiserum or monoclonal antibodies. The results indicated that supplementing antiserum every other day was not efficacious for protection. Supplementing even small quantities of hyperimmune antiserum (0.5 ml) at least once a day in most cases was sufficient for piglet survival but did not prevent morbidity. Increasing the amount (>2 ml) and providing antiserum 3 times/day completely blocked the TGEV challenge infection. Two monoclonal antibodies were discovered that also provided passive protection for baby pigs. One monoclonal antibody, 5G1, had a high neutralizing titer, and the other, 6C4, was more effective in neutralizing and binding to virulent TGEV than to attenuated TGEVs. Both of these monoclonal antibodies were partially effective as supplements in milk for passive protection. Furthermore, these monoclonal antibodies were useful for boosting the efficacy of TGEV-neutralizing colostrum, which by itself was ineffective. These results show that other antigenic sites, different from the 4-well characterized epitopes on the S glycoprotein of TGEV, also are important for passive protection.

  14. Polyclonal VDAC3 antibody decreases human sperm motility: a novel approach to male contraception

    Directory of Open Access Journals (Sweden)

    Asmarinah Asmarinah

    2011-02-01

    Full Text Available Background: Voltage dependent anion channels (VDAC mediate transport of anions, cations and ATP which play an important role in sperm motility. This study was aimed to examine the effect of polyclonal VDAC3 antiserum to human sperm motility.Methods: Polyclonal VDAC3 antiserum used in this study was produced in rabbits by immunization of VDAC3-specific synthetic peptides.  Preimmunserum was collected before immunization and used for control experiment. Recognition of VDAC3 antiserum to antigen in human sperm was performed by western blot. Thirty sperm samples obtained from fertile men which had high quality of sperm motility were washed and collected by Percoll gradient. Sperm motility was assessed by means of evaluation of sperm velocity (seconds per 0.1 mm distance and the number of unmoved sperm (million per ml which were observed 0 minute, 30 minutes and 60 minutes after addition of VDAC3 antiserum and preimmunserum as a control. Both data were analyzed by SPSS 13.0 software.Results: VDAC3 antiserum recognized VDAC3 protein in human sperm. Statistical analysis demonstrated that there were increasing numbers of unmoved spermatozoa after addition of anti-VDAC3 antiserum in vitro for 60 minutes observation compared with preimmunserum (control. We found also that sperm velocity decreased signifi cantly after giving anti-VDAC3 antiserum in vitro for 0 minute, 30 minutes, and 60 minutes compared with pre-immunee serum (control.Conclusion: VDAC3 antiserum can decrease motility of human sperm. and may provide a novel principle of male contraception in the future. (Med J Indones 2011; 20:5-10Keywords: VDAC3 antiserum, sperm, motility, contraception

  15. Radioimmunoassay of buprenorphine

    Energy Technology Data Exchange (ETDEWEB)

    Bartlett, A.J.; Lloyd-Jones, J.G.; Rance, M.J.; Flockhart, I.R.; Dockray, G.J.; Bennett, M.R.D.

    1980-01-01

    Antisera to buprenorphine were obtained in rabbits immunised with 3-0-carboxymethylbuprenorphine and N-hemisuccinyl-norbuprenorphine conjugated to bovine serum albumin. Using the latter antiserum and tritium labelled buprenorphine a radioimmunoassay having good accuracy and precision was developed for concentrations as low as 50 picograms in 1 ml of plasma. The N-hemisuccinyl antiserum crossreacted with norbuprenorphine, and the 3-0-glucuronide conjugate with the 3-0-carboxy-methyl antiserum. Cross-reactivity of both antisera to other pharmacologically related compounds was negligible. The assay was employed to determine plasma buprenorphine concentration following its parenteral administration to dog and man.

  16. 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 american...

  17. Immunochemical cross-reactivity between albumin and solid-phase adsorbed histamine

    DEFF Research Database (Denmark)

    Poulsen, L K; Nolte, H; Søndergaard, I

    1990-01-01

    For production of an antibody against histamine, this was coupled to human serum albumin (HSA) and used for immunization of rabbits. To test the antiserum, an immunoradiometric assay was developed comprising solid-phase bound histamine, antisera and radiolabelled protein A. Titration and inhibition...... experiments revealed that histamine adsorbed onto a solid-phase could bind the antiserum. However, neither free histamine nor histamine coupled to unrelated carriers could inhibit the binding of antiserum to the solid-phase histamine. Cross-reactivity was demonstrated between HSA and solid-phase bound...... histamine, as the immunoradiometric assay was inhibited by HSA. This unexpected cross-reactivity was established, as a commercially available antiserum with specificity to HSA without histamine also bound to the solid-phase bound histamine. It is suggested that preparations of antibodies against histamine...

  18. Preparation of a potent anti-scorpion-venom-serum against the venom of red scorpion (Buthus tamalus.

    Directory of Open Access Journals (Sweden)

    Kankonkar R

    1998-10-01

    Full Text Available A number of children and adults, especially pregnant women succumb to the sting by red Scorpion (Buthus tamalus in Konkan region--particularly on the coastal line. No specific antiserum or any other antidote is available to treat a victim of scorpion bite and hence the need to prepare a potent antiserum. Red Scorpion (B. tamalus venom is a mixture of a number of protein moieties and neurotoxins of low molecular weight. Therefore, the venom is poor in antigenic composition and it is difficult to get antibodies specific to neutralise lethal factor/factors. Using Bentonite as an adjuvant and extending the period of immunization a potent antiserum has been prepared capable of neutralising the lethal factor/factors. In vivo testing carried out in albino mice, guinea pigs, dogs and langurs confirms this finding and shows that the antiserum is quite effective in neutralising the scorpion venom to save the life of envenomated animals.

  19. Immunochemical analyses of soluble lens proteins in some marine fishes

    Digital Repository Service at National Institute of Oceanography (India)

    Menezes, M.R.

    Soluble eye lens proteins of 10 fishes, belonging to the families Clupeidae, Hemirhamphidae, Lactaridae, Scombridae, Stromatidae, Psettodidae, Bothidae and Soleidae were studied by immunoelectrophoresis using the lens antiserum of Sardinella...

  20. Detection of the thraustochytrid protist Ulkenia visurgensis in a hydroid, using immunofluorescence

    Digital Repository Service at National Institute of Oceanography (India)

    Raghukumar, S.

    to November 1986 By treating the samples with antiserum prepared against this organism and conjugated with FITC stain, the protist was regularly found to occur in association with a hydroid Several cells of the organism were observed in the coelenteron...

  1. 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 ...... indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs....

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

    International Nuclear Information System (INIS)

    Schuettler, J.; White, P.F.

    1984-01-01

    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

  3. 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.

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

    International Nuclear Information System (INIS)

    Sundstrom, P.R.

    1986-01-01

    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 125 I, or metabolically with [ 35 S] methionine or [ 3 H] 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

  5. 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......+, ST-, LT- hemolytic E. coli (O139 or O-rough) was isolated were diagnosed as dead due to ED. Deaths due to ED in the control groups were 8.1% and 12.0% in herds A and C, respectively, compared with 0% and 0.7% in the corresponding serum groups. The difference between treatment and control groups...... was statistically significant (P immunization by intramuscular injection of a VT2e-specific antiserum can be used for protecting...

  6. Detection of pantothenic acid-immunoreactive neurons in the rat lateral septal nucleus by a newly developed antibody.

    Science.gov (United States)

    Mangas, Arturo; Yajeya, Javier; Gonzalez, Noelia; Husson, Marianne; Geffard, Michel; Coveñas, Rafael

    2016-01-01

    The available immunohistochemical techniques have documented restricted distribution of vitamins in the mammalian brain. The aim of the study was to develop a highly specific antiserum directed against pantothenic acid to explore the presence of this vitamin in the mammalian brain. According to ELISA tests, the anti-pantothenic acid antiserum used showed a good affinity (10-8 M) and specificity. The antiserum was raised in rabbits. Using an indirect immunoperoxidase technique, the mapping of pantothenic acid-immunoreactive structures was carried out in the rat brain. Pantothenic acid-immunoreactive perikarya were exclusively found in the intermediate part of the lateral septal nucleus. These cells were generally small, round, fusiform or pyramidal and showed 2-3 long (50-100 μm) immunoreactive dendrites. Any immunoreactive axons containing pantothenic acid were detected. The very restricted anatomical distribution of the pantothenic acid suggests that this vitamin could be involved in some specific neurophysiological mechanisms.

  7. Counterimmunoelectrophoresis in the diagnosis of bacterial meningitis

    DEFF Research Database (Denmark)

    Colding, H; Lind, I

    1977-01-01

    was performed with antisera to Neisseria meningitidis (groups A, B and C), Streptococcus pneumoniae (omni-serum and pools A to 1), and Haemophilus influenzae type b. Antigen was detected in 57% (72/126) of specimens in which cultures revealed these three kinds of microorganisms in CSF and in 12% (17....... influenzae type b. Specific diagnosis was achieved in 60% (170/283) of the specimens studied and could be extablished within 1 h in 85% (145/170) by the combined results of microscopy and CIE. Ten specimens, nine of which showed a reaction with antiserum to N. meningitidis group A, were positive by CIE only......./139) of the culture-negative specimens. CSF specimens from 21 patients with bacterial meningitis caused by other species were all negative in CIE, except four, three of which contained Escherichia coli antigen reacting with antiserum to N. meningitidis group B and one E. coli antigen reacting with antiserum to H...

  8. 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 same patients exhibited little immunoreactivity. Both the peptide antiserum and the polyclonal antiserum against the native protein immunoblotted a molecular weight 63,000 protein in nuclear extracts of tumor tissue, but not significantly in extracts of normal tissue. At the molecular level......, 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...

  9. ACTH, corticosterone, and beta-endorphin in rat blood plasma after prolonged immobilization stress

    Energy Technology Data Exchange (ETDEWEB)

    Kiyatkin, E.A.; Amiragova, M.G.; Kushlinskii, N.E.; Polyntsev, Yu. V.

    1986-01-01

    To assess functional relations between changes in ACTH, beta-endorphin (BE), and corticosterone (CS) levels, plasma concentrations of these hormones were studied in rats during the development of prolonged immobilization stress. Plasma hormone concentrations were determined by radioimmunoassay. The results were analyzed by standard statistical methods on a microcomputer. A particular feature about the kit used to determine BE was the presence of 50% cross-reactivity of the antiserum against beta-lipotrophin. To determine CS a highly specific antiserum produced by a laboratory was used.

  10. Translations on USSR Science and Technology, Biomedical and Behavioral Sciences, Number 42

    Science.gov (United States)

    1978-08-08

    an.tisera to A/FM/1/47 (HlNl) to the homologous titer, with antiserum to the strain A/ whale /Pacific Ocean/19/76 to one-eighth titer, with antiserum to A...with a virus isolated from whales 1.5 years prior to this event, indicates the possi- bility for importation of a virus into the human population...from rorquals caught " ^f^97^*£tion of South Pacific. This article presents the.results of ^ ^f epidemic strains and the influenza virus isolated

  11. 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.

  12. 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)

    Yanaihara, N.; Nishino, T.; Kodaira, T.; Imagawa, K.; Nishida, T.; Mihara, S.; Yanaihara, C.

    1979-01-01

    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.)

  13. Misidentification of Vibrio cholerae O155 isolated from imported shrimp as O serogroup O139 due to cross-agglutination with commercial O139 antisera

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Mazur, J.; Dalsgaard, Inger

    2002-01-01

    Fish and shellfish products imported into Denmark are routinely analyzed for pathogenic Vibrio spp., particularly Vibrio cholerae, if products originate from subtropical or tropical areas. A V. cholerae strain that agglutinated commercial O139 antiserum but not the O1, Inaba, or Ogawa antisera....... The strain contained two plasmids, in contrast to other O139 strains, which normally do not contain plasmids. The characteristics of the strain led to further agglutination testing with other antisera that are not commercially available, and the strain was found to agglutinate O155 antiserum in repeated...

  14. 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)

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

    1983-01-01

    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

  15. 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.

  16. 9 CFR 113.332 - Tenosynovitis Vaccine.

    Science.gov (United States)

    2010-01-01

    ... antigen. A known tenosynovitis antiserum and a known tenosynovitis antigen shall be used in the test. A... to each of 25 4-week-old or older tenosynovitis susceptible chickens. (iii) The vaccinates shall be... immunogenicity. The selected virus dose shall be established as follows: (1) Tenosynovitis susceptible chickens...

  17. A sandwich ELISA for porcine alpha-1acid glycoprotein (pAGP, ORM-1) and further demonstration of its use to evaluate growth potential in newborn pigs

    Science.gov (United States)

    A simple, reproducible sandwich ELISA was developed to measure porcine alpha-1 acid glycoprotein (pAGP, ORM-1) in pig plasma. Pig AGP isolated from serum was purchased and a polyclonal antisera was prepared in rabbits using the whole pAGP molecule as immunogen. The antiserum was affinity-purified...

  18. Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

    NARCIS (Netherlands)

    Wolvetang, E. J.; Wanders, R. J.; Schutgens, R. B.; Berden, J. A.; Tager, J. M.

    1990-01-01

    Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum

  19. Electron Microscopy of Intracellular Protozoa.

    Science.gov (United States)

    1980-08-01

    concentrations of antiserum were utilized, the addition of complement profoundly amplified the as socia - tion of trypanosomes and macrophages. Electron...of media for 120 mi. Please contrast with Fig. 7 (15 min incubation with immune serum) and Fig 9 (120 min with immune serum). Arrow indicates

  20. Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin

    NARCIS (Netherlands)

    Tytgat, K. M.; Büller, H. A.; Opdam, F. J.; Kim, Y. S.; Einerhand, A. W.; Dekker, J.

    1994-01-01

    Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic

  1. Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Collins, M T; Høiby, N

    1989-01-01

    with a monospecific rabbit antiserum raised against L. micdadei "common antigen" (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment...

  2. 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

    1994-01-01

    -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...

  3. Gastrointestinal expression and partial cDNA cloning of murine Muc2

    NARCIS (Netherlands)

    van Klinken, B. J.; Einerhand, A. W.; Duits, L. A.; Makkink, M. K.; Tytgat, K. M.; Renes, I. B.; Verburg, M.; Büller, H. A.; Dekker, J.

    1999-01-01

    To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM

  4. The Immunological Relationship between Typanosoma evansi and ...

    African Journals Online (AJOL)

    Trypanosoma evansi and Trypanosoma vivax show very high cross-reactivity in serology. This suggests that there is a close immunological relationship between the two parasite species and the possibility that antiserum against one species contains lytic antibodies against the other species was investigated. T. evansi and ...

  5. Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma

    DEFF Research Database (Denmark)

    Munkvad, S; Jespersen, J; Sidelmann, Johannes Jakobsen

    1990-01-01

    We present a new functional assay for the first complement component (C1) in plasma, based on its activation by inhibition of the C1-esterase inhibitor (C1-inh) when monospecific antiserum to C1-inh is added to the plasma. After maximal activation, we can determine the concentration of activated ...

  6. A Latex Agglutination Test for Capripoxvirus | Muinamia | Journal of ...

    African Journals Online (AJOL)

    disease, by a latex agglutination test (LAT) using the purified P32 antigen and guinea pig detector antiserum raised against the P32 antigen. The LAT and virus neutralization test (VNT) were used to screen one hundred livestock field sera for antibodies to Capripoxvirus, in comparison the LAT was simpler, rapid and 23% ...

  7. Medicinal plants used to treat Snake bite by Fulani Herdsmen in ...

    African Journals Online (AJOL)

    Their village settlement positions make it more difficult for them to assess antiserum, the only source of treatment available for snake bite, Therefore the only option left for them is the use of village surrounding medicinal plants for the treatment of the snake bite. Recent efforts on ethnopharmacology revealed several of these ...

  8. 21 CFR 660.33 - Testing of source material.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Testing of source material. 660.33 Section 660.33....33 Testing of source material. Except as provided in this section, a sample of each blood... tests shall be resolved by testing with at least one additional antiserum before concluding that the...

  9. 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...... are typically found during the chronic stage of the disease....

  10. Ontogeny and localization of γ-crystallin antigen in the developing pigeon (Columba livia) lens

    NARCIS (Netherlands)

    Brahma, S.K.; Rabaey, M.; Doorenmaalen, W.J. van

    Ontogeny and localization of the lens γ-crystallin antigen were investigated in the embryonic and post-embryonic pigeon lenses by the indirect immunofluorescence with antiserum from rabbit immunized with isolated pigeon lens γ-crystallin. The results show that γ-crystallin appears for the first time

  11. in Onchocerciasis

    African Journals Online (AJOL)

    The titre of the library was 108-10“ plaque forming units (pfu's)/ml. A kit from Clontech was employed for screening ac- cording to the instructions of the manufacturer, except that the blocked nitrocellulore filters were first reacted with the absorbed antiserum described above. Alter washing oil the unbound sera the filters were ...

  12. Immunochemical cross-reactivity between albumin and solid-phase adsorbed histamine

    DEFF Research Database (Denmark)

    Poulsen, L K; Nolte, H; Søndergaard, I

    1990-01-01

    For production of an antibody against histamine, this was coupled to human serum albumin (HSA) and used for immunization of rabbits. To test the antiserum, an immunoradiometric assay was developed comprising solid-phase bound histamine, antisera and radiolabelled protein A. Titration and inhibition...

  13. Studies on the antigenic properties of the Fd-fragment of a human G-myeloma protein (Daw)

    NARCIS (Netherlands)

    Zegers, Ben J.M.; Ballieux, R.E.

    The present investigation deals with an immunochemical approach in studies on the structure of Fd-fragments of human immunoglobulins. Rabbits were immunized with a preparation of Fd-fragment of a human G-myeloma protein (Daw) [1]. Detailed studies on the reactions of the rabbit antiserum with a

  14. 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 embry...

  15. Milk progesterone concentrations: an accurate early pregnancy ...

    African Journals Online (AJOL)

    by milk progesterone quantitation was determined as well as the diagnostic efficiency of the test for days 14- 24 ... Progesterone was quantitated utilizing a single antibody non- extraction iodine - 125radioimmunoassay employing ... with horseradish peroxidase as tracer. The antiserum against the bovine serum albumin ...

  16. Non-opiate [beta]-endorphin fragments and dopamine--IV [gamma]-type endorphins may control dopaminergic systems in the nucleus accumbens

    NARCIS (Netherlands)

    Ree, J.M. van; Wolterink, G.; Fekete, M.; Wied, D. de

    1982-01-01

    Chronic treatment with des-enkephalin-γ-endorphin (DEγE, β-endorphin 6–17) twice daily for 10 days into the nucleus accumbens of rats resulted in hypoactivity, while similar treatment with γ-endorphin antiserum led to a marked hyperactivity. This enhanced activity persisted for at least 3 days

  17. Amidated joining peptide in the human pituitary, gut, adrenal gland and bronchial carcinoids. Immunocytochemical and immunochemical evidence

    DEFF Research Database (Denmark)

    Bjartell, A; Fenger, M; Ekman, R

    1990-01-01

    .g., ACTH, beta-endorphin, Pro-tau-MSH, in the pituitary gland and adrenal medulla. The JP-N immunoreactive cells in the adrenal medulla were identified as a subpopulation of adrenaline-producing cells by means of an antiserum against phenylethanolamine N-methyltransferase. In the gut immunoreactive JP...

  18. Molecular Reassessment Of Relationships Within Vicieae Using ...

    African Journals Online (AJOL)

    Serologically, the legumins of V. faba and L. esculentum gave a total identity with P. sativum legumin antiserum under non-reducing conditions, whereas the ... et albumines des protéines des graines de petit pois (Pisum sativum), haricot (Vicia faba), lentille (Lens esculentum) et culin (Cicer arientinum) pour limiter les ...

  19. The effect of restricted suckling on LH and ovarian steroids after ...

    African Journals Online (AJOL)

    indicus cows. B.P. Louw, A.W. Lishman, A. van Niekerk, J. Grobbelaar and S.F. Lesch. Cedara College of Agriculture and Research Station, Pietermaritzburg. A total of 37 ... Keywords: Restricted suckling, beef cows, breed, LH, steroids. B.P. Louw*, A. .... changed from 1:100 000 to 1:160 000, and the incubation of antiserum ...

  20. Immunoreactive prolactin in the pituitary gland of cyprinodont fish at the time of hatching

    NARCIS (Netherlands)

    Schoots, A. F.; Ruijter, J. M.; van Kemenade, J. A.; Denucé, J. M.

    1983-01-01

    In the developing pituitary gland of embryos of the annual fish Cynolebias whitei and the medaka, Oryzias latipes, prolactin cells have been identified before hatching by means of a light-microscopic immunocytochemical method with antiserum against ovine prolactin. At the time of hatching, changes

  1. Annual Research Progress Report, Fiscal Year 1979,

    Science.gov (United States)

    1979-10-01

    exsanguinated by cardiac puncture and the serum collected. Diluting with phosphate buffered saline (PBS), pH 7.5, the optimum concentration of antiserum...and colleagues. The canals will be investigated for presence of pulpal tissue and debris as well as the elemental concentration of calcium and

  2. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  3. Isolation of cDNAs encoding subunit VIb of cytochrome c oxidase and steady-state levels of coxVIb mRNA in different tissues

    NARCIS (Netherlands)

    Taanman, J. W.; Schrage, C.; Ponne, N. J.; Das, A. T.; Bolhuis, P. A.; de Vries, H.; Agsteribbe, E.

    1990-01-01

    A full-length cDNA clone specifying the nuclear-encoded subunit VIb of human cytochrome c oxidase (COX) was isolated from a human skeletal muscle cDNA expression library. This was done with antiserum directed against the group of subunits VIa, b and c of bovine heart COX. A potential

  4. The role of sialoadenectomy and epıdermal growth factor (EGF) in ...

    African Journals Online (AJOL)

    USER

    2010-05-17

    May 17, 2010 ... result, epidermal growth factor was concluded to have an important role in skin development. Key words: Epidermal growth factor, ... sialoadenectomy on epiderm and the role of EGF and antiserum EGF in prevention of .... remarkable finding in skin healing of sialoadenectomy and normal rats. According to ...

  5. Increased muscle glucose uptake during contractions

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Galbo, Henrik; Richter, Erik

    1984-01-01

    in the presence of antiserum. 3-O-methylglucose uptake was increased markedly by contractions in fast-twitch red and white fibers that were severely glycogen depleted but not in slow-twitch red fibers that were not glycogen depleted. In hindquarters from ketoacidotic rats perfused exactly as by Berger et al., 3-O...

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

    NARCIS (Netherlands)

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

    2007-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

    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

  8. Evaluation of the protective efficacy of immunoglobulin Y (IgY ...

    African Journals Online (AJOL)

    user

    2012-09-06

    Sep 6, 2012 ... Saudi Arabia. Hens were used to produce IgY-antivenoms, Swiss outbreed mice were used to determine venom lethality, potency, the neutralizing potency of antivenom, in addition to other in vivo assay while rabbits were used to produce anti-IgY antiserum. Animal care was provided by expert personnel, ...

  9. Recombinant antivenoms based on mixtures of human antibodies against D. jamesoni toxins

    DEFF Research Database (Denmark)

    Pus, Urska; Harrison, Robert; Andersen, Mikael Rørdam

    Each year, more than 5 million people worldwide are affected by a snakebite, resulting in 150,000 deaths, and 400,000 amputations. The current medical treatment against envenoming is based on the administration of an animal-derived antiserum, containing antibodies against snake venom toxins. Due...

  10. Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera

    Digital Repository Service at National Institute of Oceanography (India)

    Vrinda, S.; Jasmin, C.; Sivakumar, K.C.; Jose, S.; Jose, B.; Philip, R.; BrightSingh, I.S.

    report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237 bp that codes for 79 amino...

  11. 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

    1982-01-01

    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...

  12. Novel development of a lateral flow immunoassay for rapid field detection of citrus tristeza virus

    Science.gov (United States)

    Maintenance of virus-free citrus in nurseries and orchards is essential to control spread of aphid-borne Citrus tristeza virus (CTV) in California. A lateral flow assay (LFA) test strip with a polyclonal antiserum made from virus particles produced in Nicotiana benthamiana plants inoculated with an ...

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

    International Nuclear Information System (INIS)

    Sekihara, H.; Ohsawa, N.

    1974-01-01

    A simplified method for evaluating serum dehydroepiandrosterone (DHEA) without chromatography was developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ 5 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- 3 H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α- 3 H. 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.)

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

    NARCIS (Netherlands)

    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.

    1990-01-01

    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,

  15. Keeping venomous snakes in the Netherlands: a harmless hobby or a public health threat?

    Science.gov (United States)

    van Genderen, P J J; Slobbe, L; Koene, H; Mastenbroek, R D L; Overbosch, D

    2013-10-01

    To describe the incidence of venomous snakebites and the hospital treatment thereof (if any) amongst private individuals who keep venomous snakes as a hobby. Descriptive study. Private keepers of venomous snakes were invited via the social media Facebook, Hyves, Twitter, Google Plus, Linked In and two large discussion forums to fill in an online questionnaire on a purely voluntary and anonymous basis. In the period from 1 September 2012 to 31 December 2012, 86 questionnaires were completed by individuals who keep venomous snakes as a hobby. One-third of the venomous snake keepers stated that they had at some point been bitten by a venomous snake. Out of those, two-thirds needed hospital treatment and one-third of those bitten required at least one, sometimes more, doses of antiserum. The chances of being bitten increased the more venomous snakes a person kept. An inventory of the collections of venomous snakes being kept further revealed that no antiserum exists for 16 of the species, including for the most commonly held venomous snake, the coral cobra. Keeping venomous snakes as a hobby is not without danger. Although in the majority of snakebite cases no antiserum had to be administered, there is nevertheless a significant risk of morbidity and sequelae. Preventing snakebites in the first place remains the most important safety measure since there are no antiserums available for a substantial number of venomous snakes.

  16. Role of glucagon in intestinal hyperemia associated with early experimental diabetes mellitus

    International Nuclear Information System (INIS)

    Yrle, L.F.; Smith, J.K.; Benoit, J.N.; Granger, D.N.; Korthuis, R.J.

    1988-01-01

    The role of glucagon as a blood-borne mediator of the intestinal hyperemia associated with experimental diabetes mellitus was assessed in anesthetized fasted (18-24 h) rats 4 wk after the administration of streptozotocin or its vehicle. Selective removal of pancreatic glucagon from the circulation was accomplished by the intravenous administration of a highly specific glucagon antiserum. Blood flow to the gastrointestinal tract and kidneys was measured with radioactive microspheres using the reference sample technique. Blood flows were increased by at least 60% in each segment of the gastrointestinal tract of diabetic animals compared with control rats. Glucagon antiserum had no effect on blood flows in the gastrointestinal tract of control animals. However, the antiserum produced a significant reduction in blood flow to the stomach (26%), duodenum (25%), jejunum (12%), and kidneys (16%) in diabetic rats. There was no change in blood flow to the ileum or colon of diabetic animals with antiserum administration. The results of this study support the hypothesis that glucagon mediates a portion of the hyperemia noted in the stomach, duodenum, and jejunum. However, glucagon does not appear to play a role in the genesis of the hyperemia noted in more distal segments of the gastrointestinal tract (ileum and colon). A possible role for glucagon in the maintenance of renal blood flow in diabetic rats is suggested

  17. Immuno disc assay for screening duck hepatitis B surface antigen in serum, liver tissue and cultured hepatocytes

    NARCIS (Netherlands)

    G.A. de Wilde (G.); R.A. Heijtink

    1993-01-01

    textabstractAn immuno disc assay (IDA) for semi-quantitative analysis of the surface antigen (DHBsAg) of duck hepatitis B virus (DHBV) is described. Unpurified antigen preparations were adsorbed onto punched-out nitrocellulose membrane discs. Rabbit antiserum raised against serum-derived

  18. Radioimmunoassay of norandrosterone

    International Nuclear Information System (INIS)

    Hampl, R.; Starka, L.; Putz, Z.; Protiva, J.

    1983-01-01

    A simple radioimmunoassay of the main nortestosterone metabolite, 3α-hydroxy-5α-estran-17-one (norandrosterone) is described and evaluated. The method employs 3 H-labelled ligand prepared from nortestosterone by three-step synthesis and rabbit antiserum against norandrosterone-17-carboxymethyloxime bovine serumalbumin. (author)

  19. Simple radioimmunoassay of cortisol in diluted samples of human plasma. [Tritium tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Connolly, T.M.; Vecsei, P.

    1978-09-01

    We describe a simple radioimmunoassay of plasma cortisol, which can be performed in 3 h and which requires no purification, heating, or refrigeration steps. The plasma proteins are inhibited through direct competition between them and the antiserum at room temperature, at which the antiserum's affinity exceed that of the binding proteins. Plasma, diluted with water, is incubated for 3 h at room temperature with (/sup 3/H) cortisol and the antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum was ..gamma.. = 0.954 (P < 0.001), and the slope was 0.661. With three other antisera it was ..gamma.. = 0.922 (P < 0.001) and slope 0.644. Plasmas with abnormal protein concentrations (i.e., from pregnant women, and after corticotropin adminsitration), tested to examine the validity of the method for routine use, and to define the role of the protein carriers, showed ..gamma.. = 0.859 (P < 0.001) and slope 0.726 for the four antisera used. Additional samples, assayed with diluted standards plus stripped plasma, showed a correlation with the usual extraction method of ..gamma.. = 0.945 (P < 0.001) and slope 1.026.

  20. Multiple Myeloma

    African Journals Online (AJOL)

    Three major criteria were used to establish the diagnosis: serum and urine protein chemistry, bone marrow morphology and the radiological appearances of bone. The fact that ... Serum and early morning urine samples preserved with sodium azide (1 ... chain typing were read between 2 and 4 hours after the antiserum had ...

  1. A sensitive radioimmunoassay for fentanyl

    International Nuclear Information System (INIS)

    Michiels, M.; Hendriks, R.; Heykants, J.

    1977-01-01

    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.) [de

  2. 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

    calves. An immunodiagnosis of H. somnus pneumonia was obtained in 15 of 17 lungs from which the bacterium had been isolated. Moreover, immunostained bacteria were also demonstrated in 20 lungs from which H. somnus had not been isolated. Thus, application of immunohistochemistry significantly enhanced......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...... for the determination of the sensitivity and specificity of antiserum that had been heterologously absorbed with antigens of cross-reacting bacteria, i.e. Pasteurella haemolytica and Pasteurella multocida. None of the antisera reacted with Actinomyces pyogenes. An antiserum raised against somatic antigens of the Danish...

  3. A simple robust assay for testosterone in male plasma using an 125I-radioligand and a solid-phase separation technique

    International Nuclear Information System (INIS)

    Dyas, J.; Read, G.F.; Riad-Fahmy, D.

    1979-01-01

    A radioimmunoassay for testosterone in male plasma utilising a gamma-emitting radioligand and a solid-phase antiserum is described. The radioligand was testosterone-3-(O-carboxymethyl)-oxime coupled to 125 I-iodohistamine, and the solid-phase antersum was prepared by coupling anti-testosterone-3-bovine serum albumin to cyanogen bromide activated cellulose. The new procedure retained much of the specificity associated with a published, specific radioimmunoassay using an antiserum raised against testosterone-11 α-BSA and a tritium radioligand and incorporating a dextran-coated charcoal separation procedure; values obtained by the two procedures were in excellent agreement (r = 0.98, n = 20). The combination of an 125 I-radioligand and a solid-phase separation technique greatly increased sample throughput and had the further advantage of reduced running costs and a greater potential for automation. The method gave satisfactory levels of sensitivity, precision, and accuracy. (author)

  4. Production of biological reagents for radioimmunoassay second antibody

    International Nuclear Information System (INIS)

    Borghi, V.C.; Silva, S.R. da; Bellini, M.H.; Lin, L.H.

    1992-02-01

    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)

  5. 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....../139) of the culture-negative specimens. CSF specimens from 21 patients with bacterial meningitis caused by other species were all negative in CIE, except four, three of which contained Escherichia coli antigen reacting with antiserum to N. meningitidis group B and one E. coli antigen reacting with antiserum to H...

  6. Tick paralysis: development of a vaccine.

    Science.gov (United States)

    Masina, S; Broady, K W

    1999-04-01

    The paralysis tick of Australia, Ixodes holocyclus, causes a severe toxicosis in domestic animals such as dogs and cats, livestock, and in some cases, humans. It is characterised by a rapidly ascending flaccid paralysis. The causative agent of the toxicosis is a neurotoxin(s) produced in the tick salivary glands. The current treatment for tick paralysis is in the form of a polyclonal dog antiserum. This antiserum treatment is expensive and effective only in the early stages of paralysis. The aim of current research is to develop a recombinant veterinary vaccine based on the tick neurotoxin peptide sequence. A successful vaccine would provide cost-effective, long-term protective immunity against tick-induced paralysis.

  7. Immunochemical and biological quantification of peanut extract

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Pedersen, Mona H; Platzer, Michael

    2003-01-01

    Biological standardization of allergen extracts is one of the steps in the characterization of an extract. The gold standard for determination of biological potency is the skin prick test, but histamine release (HR) has been used as a convenient ex vivo method for analyzing a large number...... of samples. We describe the use of rabbit basophils as a tool in biological standardization. Using peanut as a model allergen, it is described how rabbits immunized for production of antiserum may become sensitized and their basophils used for histamine release experiments. It is also possible to use rabbit...... antiserum to passively sensitize basophils derived from naive rabbits, but the sensitivity of this method is so far 100-1000 times lower than the direct histamine release. The rabbit histamine release results are compared to an ELISA developed by means of the same antisera and by passive sensitization...

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

    International Nuclear Information System (INIS)

    Gapp, D.A.; Ho, S.M.; Callard, I.P.

    1979-01-01

    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

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

    International Nuclear Information System (INIS)

    Ramli bin Abdullah

    1990-01-01

    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

  10. Radioimmunoassay of steroid hormones

    International Nuclear Information System (INIS)

    Hampl, R.; Starka, L.

    1976-01-01

    A list is given of steroids currently determined using the RIA method based on the reversible interaction of labelled and non-labelled steroids within the binding points of specific antibodies. Steroids do not have the properties of antigens, and antibodies are therefore obtained from conjugates of steroid derivatives with macromolecules where the steroid has the function of hapten. The most important synthetic methods of preparing the given derivatives are listed, and questions of the relation of the specificity of the antiserum and the structure of the respective derivative are discussed, the basic data characterizing the antiserum (titre, affinity and specificity) are described and methods are given used for measuring these variables. The technical aspects of RIA and the differences between the RIA determination of steroids and of other substances are given. The Amersham Radiochemical Centre antigens, kits and steroid hormones are recommended for use. (L.O.)

  11. Radioimmunoassay for chlorpromazine in plasma. [/sup 3/H tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Midha, K.K.; Loo, J.C.K.; Hubbard, J.W.; Rowe, M.L.; McGilveray, I.J.

    1979-01-01

    A radioimmunoassay for chlorpromazine in plasma is described. The antiserum was obtained by immunizing rabbits with a conjugate of bovine serum albumin and N-(2-carboxyethyl)desmethylchlorpromazine. It is specific for chlorpromazine and its minor active metabolite, N-desmethylchlorpromazine. Other known active or inactive chlorpromazine metabolites and other psychotropic drugs tested do not cross react with the antiserum. Less than 34 pg of the drug can be detected in 200 ..mu..L of plasma. As many as 100 samples can be processed in a day by one technician. Concentrations of chlorpromazine can be measured in 200-..mu..L samples of plasma collected as late as 48 h after a single oral 25-mg dose.

  12. Radioimmunoassay for chlorpromazine in plasma

    International Nuclear Information System (INIS)

    Midha, K.K.; Loo, J.C.K.; Hubbard, J.W.; Rowe, M.L.; McGilveray, I.J.

    1979-01-01

    A radioimmunoassay for chlorpromazine in plasma is described. The antiserum was obtained by immunizing rabbits with a conjugate of bovine serum albumin and N-(2-carboxyethyl)desmethylchlorpromazine. It is specific for chlorpromazine and its minor active metabolite, N-desmethylchlorpromazine. Other known active or inactive chlorpromazine metabolites and other psychotropic drugs tested do not cross react with the antiserum. Less than 34 pg of the drug can be detected in 200 μL of plasma. As many as 100 samples can be processed in a day by one technician. Concentrations of chlorpromazine can be measured in 200-μL samples of plasma collected as late as 48 h after a single oral 25-mg dose

  13. Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis.

    Science.gov (United States)

    Angelos, John A; Ball, Louise M; Hess, John F

    2007-11-15

    To determine if Moraxella bovoculi (M. bovoculi), a recently characterized coccoid Moraxella that was isolated from the eyes of calves affected with infectious bovine keratoconjunctivitis (IBK), and Moraxella ovis (M. ovis), originally isolated from sheep with conjunctivitis, possessed genes encoding RTX proteins, genomic DNA was amplified with oligonucleotide primers targeting RTX operon genes of Moraxella bovis (M. bovis). Complete classical RTX operons composed of RTXCABD genes closely linked to a putative secretion accessory protein encoding gene (tolC) were identified in M. bovoculi and M. ovis and were designated mbvCABDtolC and movCABDtolC, respectively. These genes were closely related to M. bovis mbxCABDtolC. Polyclonal rabbit antiserum against the carboxy terminus of M. bovoculi MbvA neutralized hemolytic activity of both M. bovoculi and M. ovis; this antiserum did not neutralize the hemolytic activity of M. bovis. M. bovoculi and M. ovis possess genes that encode proteins related to pathogenic factors of M. bovis.

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

    International Nuclear Information System (INIS)

    Maguire, K.P.; Burrows, G.D.; Norman, T.R.; Scoggins, B.A.

    1978-01-01

    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. 125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured

    International Nuclear Information System (INIS)

    Hasler, M.J.; Painter, K.; Niswender, G.D.

    1976-01-01

    We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding with a proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less

  16. Light-microscopic immunocytochemistry for Gentamicin and its use for studying uptake of the drug in kidney

    DEFF Research Database (Denmark)

    Fujiwara, Kunio; Shin, Masashi; Matsunaga, Hayato

    2009-01-01

    Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin......, kanamycin, or amikacin. The antiserum also detected glutaraldehyde-fixed GM, and this enabled us to develop an immunocytochemical method for detecting the uptake of GM in rat kidney. Twelve hours after a single intravenous administration of GM, immunocytochemistry revealed that GM accumulated in the S1, S2......, and S3 segments of the proximal tubules, as well as in the distal tubules and collecting ducts. By 12 h after injection, the drug was detected in cytoplasmic granules of the proximal tubule cells. However, early (1 h) after injection, drug accumulation was detected in the microvilli of these cells...

  17. 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)

    Breitenbuecher, R.

    1982-01-01

    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 21 0 C. 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) [de

  18. Methadone radioimmunoassay: two simple methods

    International Nuclear Information System (INIS)

    Robinson, K.; Smith, R.N.

    1983-01-01

    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- 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 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)

  19. Isolation of antibodies specific to sickle hemoglobin by affinity chromatography using a synthetic peptide

    International Nuclear Information System (INIS)

    Young, N.S.; Curd, J.G.; Eastlake, A.; Furie, B.; Schechter, A.N.

    1975-01-01

    Antibodies to hemoglobin have been studied with a radioimmunoassay which employs [ 14 C]carbamylated (= carbamoylated) hemoglobin S. An antiserum raised against hemoglobin S, which initially discriminated poorly between hemoglobins S and A, was fractionated by absorption to a column of Sepharose to which a synthetic peptide corresponding to the first 13 amino-acid residues of the β chain of sickle hemoglobin had been covalently bound. A subpopulation of the antiserum was eluted from this column with 4 M guanidine . HCl. These antibodies showed binding to hemoglobin S but not to hemoglobin A and this interaction could be inhibited by the synthetic peptide. These antibodies, of demonstrated fine structural specificity, may be useful in the detection of sickle hemoglobin and in the study of its structure in solution

  20. 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.

  1. Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods.

    Science.gov (United States)

    Farook, M A; Madan, N; Taju, G; Majeed, S Abdul; Nambi, K S N; Raj, N Sundar; Vimal, S; Hameed, A S Sahul

    2014-08-01

    White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR. © 2013 John Wiley & Sons Ltd.

  2. Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.

    OpenAIRE

    Djiane, J; Houdebine, L M; Kelly, P A

    1981-01-01

    Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and...

  3. 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

  4. Radioimmunoassay for the determination of low insulin levels

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Wajchenberg, B.L.; Okada, H.

    1988-08-01

    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) [pt

  5. 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...

  6. 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...... 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....

  7. A specific radio-immunoassay for Gonadotrophin-releasing hormone

    International Nuclear Information System (INIS)

    Hendricks, S.; Millar, R.; Pimstone, B.

    1975-01-01

    A specific antiserum has been made to synthetic gonadotrophin-releasing hormone (GnRH) conjugated to keyhole limpet haemocyanin and appears to be directed against amino acids 6 - 8 of this decapeptide. This has allowed the development of a radio-immunoassay for GnRH sensitive to 5 picograms per tube. Although it is easily measurable in hypothalamic extracts, we have failed to detect GnRH in plasma and urine from normal subjects and menopausal women

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

    International Nuclear Information System (INIS)

    Seren, E.; Bacci, M.L.

    1988-01-01

    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

  9. Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies.

    OpenAIRE

    Tuboly, T; Nagy, E; Derbyshire, J B

    1995-01-01

    Sera of pigs immunized with parts of the transmissible gastroenteritis virus (TGEV) spike (S) protein expressed by recombinant baculoviruses were tested, together with a TGEV hyperimmune antiserum, for their abilities to protect three-day-old piglets against TGEV infection. The piglets were infected with virulent TGEV and the sera were given orally 3 h before infection, together with the virus, and every 6 h postinfection during the 30 h of the experiment. Virus shedding was monitored by TGEV...

  10. Equine Infectious Anemia Virus from Infected Horse Serum

    Science.gov (United States)

    Nakajima, Hideo; Yoshino, Tomoo; Ushimi, Chuzo

    1974-01-01

    Equine infectious anemia virus was purified from infected horse serum samples. Electron microscope observation on negatively stained preparations of purified virus showed roughly spherical particles sized between 100 and 200 nm in diameter. In disrupted particles, an envelope was visible but no internal structure could be resolved. Since the purified virus fraction had a strong antigenic activity to antiserum in immunodiffusion reaction, these particles are thought to be the causative virus of equine infectious anemia. Images PMID:4372175

  11. Demonstration of Antigenic Identity Between Purified Equine Infectious Anemia Virus and an Antigen Extracted from Infected Horse Spleen

    Science.gov (United States)

    Nakajima, Hideo; Norcross, Neil L.; Coggins, Leroy

    1972-01-01

    Antigenic relationship between purified equine infectious anemia (EIA) virus and spleen-derived antigen from EIA-infected horses was examined by immunodiffusion. Identical antigenicity of these two antigens has been proven because precipitation lines formed between the two antigens and EIA antiserum connected with each other. The results indicate that the antigenic substance derived from infected spleen is a component of EIA virus. Images PMID:4629262

  12. Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers.

    OpenAIRE

    Herring, C. J.; West, C. M.; Wilks, D. P.; Davidson, S. E.; Hunter, R. D.; Berry, P.; Forster, G.; MacKinnon, J.; Rafferty, J. A.; Elder, R. H.; Hendry, J. H.; Margison, G. P.

    1998-01-01

    A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both ...

  13. Protein A gold identification of ureaplasmas on the bovine zona pellucida.

    Science.gov (United States)

    Britton, A P; Miller, R B; Ruhnke, H L; Johnson, W M

    1989-04-01

    The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Radioimmunological determination of parathormone in human serum

    International Nuclear Information System (INIS)

    Baret, A.; Morcellet, J.L.; Sauvan, R.; Piechowski, J.

    1975-01-01

    The radioimmunological determination of h PTH was studied, using reagents available on the market: purified bovine parathormone (b PTH) from the Wilson laboratories used for labelling and calibration, synthesized amino thermal peptide of b PTH (1-34 b PTH) from the Beckman laboratories, bovine antiparathormone guinea-pig antiserum from the Burrough Wellcome laboratories (AS 211/32). The results were analysed on standard curves, together with the seric PTH fractions for normal or renal-insufficient subjects [fr

  15. A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo

    OpenAIRE

    1989-01-01

    Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluore...

  16. Demonstration of carcinoembryonic antigen in human breast carcinomas by the immunoperoxidase technique.

    OpenAIRE

    Walker, R A

    1980-01-01

    Using an antiserum against carcinoembryonic antigen, which was free from non-specific cross-reacting antigen activity, carcinoembryonic antigen has been demonstrated in 45 out of 90 breast carcinomas by an indirect three-stage immunoperoxidase method. The presence of carcinoembryonic antigen appears to be related to good histological differentiation but not to histological type, lymph node metastasis, or recurrence within two years of primary diagnosis. It is suggested that the varying result...

  17. Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

    Science.gov (United States)

    Westin, M

    1975-06-01

    Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.

  18. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  19. Phytoferritin is synthesized in vitro as a high-molecular-weight precursor. Studies on the synthesis and the uptake in vitro of the precursors of ferritin and ferredoxin by intact chloroplasts.

    OpenAIRE

    van der Mark, F; van den Briel, W; Huisman, H G

    1983-01-01

    Evidence is presented that French-bean (Phaseolus vulgaris) seed ferritin is composed of one type of subunit with an apparent Mr of 26500. In normal and iron-loaded leaf tissues it is detected immunologically with an antiserum raised against purified bean seed ferritin and migrates in SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis with the same mobility as the bean seed ferritin subunit. The biosynthetic pathway of ferritin in normal and iron-loaded leaves was investigated. ...

  20. 9 CFR 130.14 - User fees for FADDL veterinary diagnostics.

    Science.gov (United States)

    2010-01-01

    ... diagnostics. 130.14 Section 130.14 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE....00 $155.00 $160.00 $165.00 Caprine antiserum, any agent 1 mL 184.00 189.00 195.00 202.00 Cell culture... slides or plates Slide 49.00 50.00 51.00 53.00 Reference animal tissues for immunohistochemistry Set 171...

  1. Diagnosis of Giardia duodenalis Infection using Dot Blot in Comparison with Microscopy.

    Science.gov (United States)

    Yazdani, Hajar; Sharafi, Seyedeh Maryam; Yousefi, Hoseynali; Hadipur, Mahbobeh; Sepahvand, Akram; Darani, Hossein Yousofi

    2016-01-01

    Giardia duodenalis is an intestinal flagellate parasite which spreads all over the world and is considered as a health problem in the most rural and low sanitation areas. Many diagnostic tests have been developed for the detection of Giardia parasite in stool samples but all of them have some disadvantages such as lack of sensitivity and specificity. In search for a simple and accurate test, diagnosis of Giardia infection using dot blot method has been investigated in this work. In this descriptive study, 30 stool samples which their infection with Giardia were confirmed by direct examination and formalin ether considered as case group. Thirty stool samples without Giardia infection according to formalin ether examination were also considered as a control group. Giardia cysts were isolated from the stool samples using sucrose method. In order to raise antiserum against Giardia cysts, the purified cysts were then sonicated and injected to a rabbit. A mono specific antiserum against the 66KDa band of Giardia cyst antigen was also prepared. The two antisera were used in the dot blot test. Finally, the sensitivity and specificity of the dot-blot method were estimated by considering formalin ether as the gold standard. When Poly specific antiserum was used, the sensitivity and specificity of the dot blot for detection of Giardia infection were 77% and 64% respectively. However the sensitivity and specificity of this assay were 97% and 64% respectively when monospecific antiserum was used. It seems that dot blot is an easy method for the diagnosis of Giardia especially in the rural areas. However more work is recommended for further development of this test.

  2. The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry.

    Science.gov (United States)

    Kerkhoven, R M; Croll, R P; Ramkema, M D; Van Minnen, J; Bogerd, J; Boer, H H

    1992-03-01

    VD1 and RPD2 are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD1/RPD2 gene. To this end, in situ hybridization with the cDNA probe of the VD1/RPD2 gene and immunocytochemistry with antisera specific to VD1 and RPD2 (the alpha 1-antiserum, Mab4H5 and ALMA 6) and to R15 (the alpha 1 and 16-mer antisera) were performed on alternate tissue sections. A VD1/RPD2 neuronal system comprising three classes of neurons (A1-A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD1 and RPD2) immunoreact with the alpha 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1-5 small and 1-5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the alpha 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3-4 medium-sized neurons and a cluster of 4-5 small neurons located in the pedal ganglion) immunoreact with the alpha 1-antiserum only. All neurons of the system are immunonegative to the R15 antisera. The observations suggest that the neurons of the VD1/RPD2 system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.

  3. Characterization of Antibody-Dependent Killing of Trypanosomes by Macrophages.

    Science.gov (United States)

    1985-01-01

    specific antibody, murine peritoneal macrophages and macrophage-like cell lines bind Trypanosoma rhodesiense parasites in vitro. Using a large bank of anti ...as T. cruzi are able to actively penetrate host cells. However, the mechanism of cellular uptake of T. rhodesiense is not known. We therefore...intracellular parasites. Am. J. Trop. Med. Hyg. 26:161. 18. Takayanagi, T. and Y. Nakatake. 1977. Trypanosoma fambiense: The binding activity of antiserum to

  4. 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.

  5. Radioimmunoassay of parathyroid hormone: past and future

    International Nuclear Information System (INIS)

    Yalow, R.S.

    1986-01-01

    In this report on radioimmunoassay of parathyroid hormone (iPTH) it was shown that the rate of disappearance of iPTH from plasma differed markedly in patients with primary hyperthyroidism or those with uremia and secondary hyperparathyroidism and that for each patient the rate of disappearance depended on the antiserum used for assay. The heterogeneity of iPTH in plasma was soon rapidly confirmed in many laboratories. (Auth.)

  6. 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

  7. 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...

  8. Comparison of capillary electrophoresis-based immunoassay with fluorescence polarization immunoassay for the immunodetermination of methamphetamine using various methamphetamine antibodies.

    Science.gov (United States)

    Choi, J; Kim, C; Choi, M J

    1998-11-01

    An accurate and simple immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was performed for the detection of methamphetamine (MA) in urine. The CE-LIF was conducted with an untreated fused-silica column using antiserum and a tracer of fluorescein isothiocyanate (FITC)-labeled MA. This CE-LIF system was compared with fluorescence polarization immunoassay (FPIA) in a TDx analyzer in the photo-check mode using the same FITC-labeled tracer and the same antiserum. Various antibodies, not only those prepared by our own immunogens but also those from commercial sources, were screened and characterized in both assay systems with regard to sensitivity, precision, and cross-reactivity. Both systems satisfied analytical precision and gave similar cross-reactivity patterns. However, the CE-LIF-based immunoassay was approximately one order superior to FPIA in sensitivity, requiring less volume of sample, antiserum, and tracer for the assay. Considering that the FPIA system is well known to be a useful tool for screening antibodies and detecting drugs, the CE-LIF-based immunoassay system, which is seemingly more advantageous than the FPIA system, appears to have great power for the characterization of antibodies and for the detection of MA in urine.

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

    International Nuclear Information System (INIS)

    Waller, H.

    1980-08-01

    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) [de

  10. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    International Nuclear Information System (INIS)

    Nigam, S.K.; Towers, T.

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

  11. Searching for a placental derived ES-62-like molecule to explain rheumatoid arthritis amelioration in pregnancy

    Directory of Open Access Journals (Sweden)

    Craig D. Scoville

    2014-11-01

    Full Text Available The majority of women with rheumatoid arthritis (RA experience disease amelioration during pregnancy for unclear reasons. One possible explanation pursued and described here is whether the placenta produces a protein similar to the immunomodulating protein, ES-62, excreted by filarial nematodes. This protein has also been shown to reduce disease activity in animal models of RA. Eleven human placentas were prepared and a polyclonal anti-ES-62 antiserum was used to identify if any ES-62-like molecule exists from human placental tissues. Any bands identified were then excised from the gel and sent for mass spectrometry and protein identification. The anti-serum showed consistent cross reactivity with the heavy chain from immunoglobulin G (IgG from the eleven human placentas by mass spectrometry. No primary sequence homology between the heavy chain of IgG and ES-62 was identified. The placenta does not produce an ES-62-like molecule. However the binding of the antiserum to the Fc region of IgG suggests that this may be a possible mechanism for rheumatoid factor production in some patients with chronic filarial infections.

  12. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.

    1979-01-01

    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 125 I 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 131 I 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 [pt

  13. Isolation of N-acetyl-beta-hexosaminidase from Acanthamoeba castellanii.

    Science.gov (United States)

    Baldwin, K M; Bowers, B

    1995-01-01

    The lysosomal enzyme N-acetyl-beta-hexosaminidase (beta hex) has been purified from Acanthamoeba castellanii growth medium by a three step procedure. The enzyme was precipitated with ammonium sulfate, partially purified on a DE52 column and purified to homogeneity on an affinity column. The purified beta hex appeared to be a monomer with a molecular mass of 58 kDa and a pI of approximately 5.8. The enzyme activity in growth medium at RT was stable for several months. The purified beta hex was enzymatically deglycosylated and injected into two rabbits to make polyclonal antibodies. One antiserum was specific for beta hex, but the other stained many bands on immunoblots of whole cell preparations. Using fluorescently labelled secondary antibodies we have determined that both antisera stain digestive vacuoles in the Acanthamoeba cytoplasm, and do not stain the contractile vacuole. The multi-specific antiserum had high avidity for beta hex, but also stained the carbohydrate portion of other molecules. These other molecules may be lysosomal enzymes as well, since the activity of several other lysosomal enzymes was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuolar structures in Acanthamoeba cytoplasm.

  14. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

    Directory of Open Access Journals (Sweden)

    Mengmeng ZHAO,Juanjuan QIAN,Jiexiong XIE,Tiantian CUI,Songling FENG,Guoqiang WANG,Ruining WANG,Guihong ZHANG

    2016-06-01

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.

  15. Specific immunologic recognition of the tumor-specific E2A-PBX1 fusion-point antigen in t(1;19)-positive pre-B cells.

    Science.gov (United States)

    Berendes, P; Hoogeveen, A; van Dijk, M; van Denderen, J; van Ewijk, W

    1995-08-01

    The t(1;19) translocation is the most commonly observed chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). Its presence among pre-B cell ALL cases, has been associated with a poor prognosis. Two genes, E2A and PBX1, are involved in this t(1;19) translocation. As a consequence, parts of the E2A and PBX1 genes are fused, resulting in a chimeric E2A-PBX1 gene, encoding chimeric E2A-PBX1 proteins. As such, the amino acid sequence at the fusion site represents a unique tumor-specific determinant. We report on the generation of a polyclonal antiserum, termed BP 1/19, raised against the tumor-specific E2A-PBX1 junction of E2A-PBX1 proteins. The specificity of antiserum BP 1/19 for the E2A-PBX1 fusion-point is demonstrated at the peptide and at the protein level. Furthermore, specific binding of antiserum BP 1/19 to t(1;19) positive cells was shown using immunofluorescence techniques. The study shows that: (1) the tumor-specific fusion-point epitope on E2A-PBX1 proteins is presented in an antigenic fashion, and (2) this particular fusion-point epitope can be used in immunological marker analysis using fluorescence microscopy.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 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

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

    International Nuclear Information System (INIS)

    Matsuhashi, Kazuo

    1974-01-01

    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 3 H-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.)

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

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) 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-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} 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 ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} 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-FABP{sub PM} and mGOT are closely related.

  19. Radioimmunoassay of extracted glucagon compared with three non-extraction assays

    International Nuclear Information System (INIS)

    Schenck, H. von; Nilsson, O.R.

    1981-01-01

    Radioimmunoassay of glucagon was performed with three different antisera, i.e. E7, 30K and 4305, all directed against the carboxyl-terminal region of glucagon and thus avoiding co-determination of glucagon-like polypeptides from the gut. Plasma samples from five healthy people subjected to various A-cell stimulation and suppression tests were used and immunoreactive glucagon assessed with the three antisera. Aliquots from all plasma samples were also extracted with acetone and glucagon re-assessed with antiserum E7. Even though all four baseline glucagon concentrations obtained were different, the glucagon profiles were comparable after superimposing the baselines. The differences in baseline concentrations of immunoreactive glucagon seem due to the interference of 'big plasma glucagon', a still unidentified factor in the E7 and 30K assays that can be precipitated by acetone. Since acetone extraction yielded the lowest baselines without altering the glucagon profiles, it is suggsted that the baseline glucagon concentrations of acetone-extracted plasma reflect the physiological level of the biologically active hormone. Using antiserum E7, our own antiserum, the normal range of glucagon values in acetone-extracted plasma samples from 22 healthy, fasting people of both sexes was 42+-16 ng/l (mean +- 2 S.D.). These values agree well with those obtained by other assay techniques. (Auth.)

  20. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Effect of gamma irradiation on toxicity and immunogenicity of Androctonus australis hector venom

    International Nuclear Information System (INIS)

    Abib, L.; Laraba-Djebari, F.

    2003-01-01

    An investigation was made of the radiosensitivity of the toxic and immunological properties of Androctonus australis hector venom. This venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60 Co source. The results showed that venom toxicity was abolished for the two radiation doses (1 and 2 kGy) with, respectively, 10 and 25 times its initial LD50 value. However, irradiated venoms were immunogenic, and the antibodies elicited by them were able to recognize the native venom by enzyme-linked immunosorbent assay. Antisera raised against these toxoids (1 and 2 kGy) had a higher neutralizing capacity and immunoreactivity against all components of native venom than did the antiserum produced against the native venom. The antiserum of rabbits immunized with 2-kGy-irradiated venom was more efficient than 1-kGy-irradiated toxoid antiserum. Indeed, in vivo protection assays showed that the mice immunized with 2-kGy-irradiated venom resisted lethal doses (i.p.) of A. australis hector venom. (author)

  2. Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo.

    Science.gov (United States)

    Hu, Xiao-Bo; Zhang, Yu-Jian; Zhang, Hui-Tang; Yang, Sheng-Li; Gong, Yi

    2003-11-01

    3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes. Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels. Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus. Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000. Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum. Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia. So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease. All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

  3. 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. © 2016 Wiley Periodicals, Inc.

  4. [Expression, purification and protective antigen analysis of cell wall protein MRP of Streptococcus suis type 2].

    Science.gov (United States)

    Wang, Ping-ping; Pian, Ya-ya; Yuan, Yuan; Zheng, Yu-ling; Jiang, Yong-qiang; Xiong, Zheng-ying

    2012-02-01

    To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. MRP represent an important protective antigen activity.

  5. Non-equilibrium method for the radioimmunoassay of clozapine in the presence of metabolites

    International Nuclear Information System (INIS)

    Rosenthaler, J.; Nimmerfall, F.; Sigrist, R.; Munzer, H.

    1977-01-01

    Cross-reactions with metabolites are an ever-recurring problem encountered in the use of radioimmunoassay techniques to determine active compounds in biological material. Metabolites may interfere with the assay of the parent drug to a variable extent. Taking 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine (clozapine) as an example, it was shown that the extent to which the antiserum produced interacts with the parent drug and the metabolites can be estimated by determining the equilibrium constants and the kinetics. In the present case, therefore, it was advantageous to carry out the radioimmunoassay in disequilibrium, i.e. in order to differentiate the metabolites from the parent drug, the sample was incubated with the antiserum for 10 min, after which the labelled antigen was added and the reaction mixture again incubated for a brief, exactly timed interval. It was shown that cross-reactions did not occur in mixtures of clozapine and its N-demethyl and N-oxide metabolites in the proportions 1 : 1 : 2 over a range of concentration of 1.5-48 ng clozapine per 100 μl human plasma. The equilibrium constants measured with the clozapine goat antiserum were as follows: clozapine 1.2 x 10 8 M -1 , the N-demethyl metabolite 4.6 x 10 7 M -1 and the N-oxide 3.7 x 10 7 M -1 (pH 7.5 and 20 0 C). (orig.) [de

  6. Initial characterization of a spontaneous interferon secreted during growth and differentiation of Friend erythroleukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Revel, M.F.E.M.; Kimchi, A.

    1982-12-01

    A gradual increase in the level of 2',5'-oligoadenylate synthetase takes place in Friend erythroleukemia cells after a shiftdown in the rate of cell growth. The increase is about 5-fold after entry of cells into the stationary phase of growth, but much higher (25-fold) when reduction in growth accompanies cell differentiation. In the latter case, the enzyme increase is similar to that which can be induced in these cells by exogeneous interferon (IFN). The increase in 2',5'-oligoadenylate synthetase was shown to be due to a spontaneous secretion of IFN by the cells themselves: it is completely abolished if antiserum to murine type I IFN is added to the culture medium. In attempts to isolate some of this spontaneously secreted IFN, we show that it is stable at pH 2, not neutralized by antiserum to type II IFN, and that it also differs from the known IFN species induced by Sendai virus in Friend cells. The major component of this spontaneously secreted IFN is 20,000 M/sub r/, and differs from the corresponding virus-induced 20,000-M/sub r/ IFN by its lower affinity for antiserum to type I IFN and its antigenic characterization as BETA-murine IFN. The major component of the spontaneous IFN also exhibits a higher ratio of antigrowth to antiviral activity than the Sendai-induced IFNs. The authors suggest that Friend cells produce this specific type of IFN for the regulation of their growth and differentiation.

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

    International Nuclear Information System (INIS)

    Troupel, Solange.

    1974-01-01

    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 [fr

  8. Insulin-like growth factor-binding protein-6 levels are elevated in serum of children with chronic renal failure: a report of the Southwest Pediatric Nephrology Study Group.

    Science.gov (United States)

    Powell, D R; Liu, F; Baker, B K; Hintz, R L; Durham, S K; Brewer, E D; Frane, J W; Tonshoff, B; Mehls, O; Wingen, A M; Watkins, S L; Hogg, R J; Lee, P D

    1997-09-01

    Previous studies suggest that growth retardation in children with chronic renal failure (CRF) results in part from inhibition of insulin-like growth factor (IGF) action by excess serum IGF-binding proteins (IGFBPs). Excess IGFBPs in CRF serum include IGFBP-1, -2, and -3 and a diffuse approximately 24- to 28-kDa IGFBP band identified by [125I]IGF ligand blot. The present studies characterized this diffuse approximately 24- to 28-kDa band. Initial studies identified this band as IGFBP-6, because it was immunoprecipitated by antiserum raised against a synthetic peptide of human IGFBP-6 (hIGFBP-6). Additional [125I]IGF ligand blots found that the immunoprecipitated band was 1) recognized by [125I]IGF-II but not [125I]IGF-1, 2) more abundant in CRF than in normal serum, and 3) more abundant in serum from dialyzed than nondialyzed prepubertal CRF children. Using the hIGFBP-6 antiserum in a specific and sensitive RIA, we found that serum IGFBP-6 levels were 4.7 +/- 1.7 nmol/L in 10 normal prepubertal children, 21.4 +/- 6.1 nmol/L in 44 nondialyzed prepubertal CRF children, 73.5 +/- 14.4 nmol/L in 7 dialyzed prepubertal CRF children, and 94.6 +/- 26.2 nmol/L in 14 dialyzed pubertal CRF children. IGFBP-6 levels were also elevated in 71 nondialyzed European children with CRF. In nondialyzed CRF children, serum IGFBP-6 levels 1) correlated inversely with the glomerular filtration rate, 2) did not correlate with height SD score, and 3) were not altered by 12 months of daily recombinant hGH treatment. In summary, a specific antiserum and RIA were used to demonstrate elevated levels of intact IGF-II-binding IGFBP-6 in serum of CRF children. We postulate that the excess IGFBP-6 may modulate the action of IGF-II on target tissues.

  9. 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.

  10. Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein.

    Science.gov (United States)

    Yeh, S D; Gonsalves, D

    1985-05-01

    The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.

  11. Parathyroid hormone in sodium-dependent hypertension

    International Nuclear Information System (INIS)

    Doris, P.A.; Harvey, S.; Pang, P.K.T.

    1987-01-01

    Plasma parathyroid hormone (pPTH) levels have been assessed in three separate radioimmunoassay systems in samples from Wistar-Kyoto rats. The animals were subjected to one of three dietary regimens throughout the study period: Group 1 animals consumed normal rat chow and drank tap water; Group 2 animals consumed normal rat chow and tap water was replaced with 0.05% saline solution; Group 3 animals consumed normal rat chow to which 2.5% CaCO 3 had been added and also drank 0.5% saline solution. Three assay systems were used to measure pPTH levels from trunk blood samples obtained by guillotine decapitation. One assay used an antiserum directed toward the vasoactive N terminal fragment 1-34 and produced pPTH measurements of 0.74 +/- 0.05 ng/ml in Gp 1 animals, 1.04 +/- 0.07 ng/ml in Gp 2 animals and 1.12 +/- 0.08 ng/ml in Gp 3 animals. This pattern was consistent with that obtained by another antiserum which had been raised against the intact 1-84 PTH molecule and produced values of 0.25 +/- 0.03 ng/ml in Gp 1 animals, 0.55 +/- 0.07 ng/ml in Gp 2 animals and 0.74 +/- 0.04 ng/ml in Gp 3 animals. Antiserum raised against the C-terminal did not show any difference in pPTH across groups. The authors conclude that saline consumption may increase some portions of circulating PTH. 26 references, 2 tables

  12. Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm.

    Science.gov (United States)

    Sakaue, Yuko; Bellier, Jean-Pierre; Kimura, Shin; D'Este, Loredana; Takeuchi, Yoshihiro; Kimura, Hiroshi

    2014-01-01

    Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.

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

    International Nuclear Information System (INIS)

    Geha, R.S.; Weinberg, R.P.

    1978-01-01

    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 125 I-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 125 I-TT but not of 125 I-DT to IgG derived from the donor of the immunizing F(ab') 2 anti-TT, but did not inhibit 125 I-TT binding to IgG derived from other donors. Finally, binding of 125 I-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

  14. The subcellular and organ distribution and natural form of histidyl-proline diketopiperazine in rat brain determined by a specific radioimmunoassay.

    Science.gov (United States)

    Yanagisawa, T; Prasad, C; Peterkofsky, A

    1980-11-10

    Histidyl-proline diketopiperazine is produced in brain as a product of the metabolism of thyrotropin-releasing hormone. A number of the previously observed central nervous system and pituitary activities resulting from an exposure to thyrotropin-releasing hormone appear to involve the conversion of the releasing factor to the cyclic dipeptide. In the present study, the development of a rabbit antiserum that is highly specific for histidyl-proline diketopiperazine is described; the antiserum has essentially no capability to bind thyrotropin-releasing hormone or a number of other related peptides. The antibody can also distinguish between the natural form of the cyclic dipeptide and a diastereomer containing D-proline. A procedure for extraction, with high yield, of histidyl-proline diketopiperazine from brain is described. With the aid of the specific antiserum it was found that the preponderance of the cyclic dipeptide in rat brain is bound to high molecular weight material, mainly in the range of Mr = 70,000; histidyl-proline diketopiperazine can be disassociated from this material by boiling in salt/methanol solution. The concentration of the dipeptide in rat brain is in the range of 275 to 565 pmol/brain, approximately 2.5 times the concentrations determined for thyrotropin-releasing hormone (113 to 210 pmol/brain). A study of the subcellular distribution of histidyl-proline diketopiperazine and thyrotropin-releasing hormone suggests that the releasing factor is concentrated in synaptosomal vesicles while the diketopiperazine is not. A determination of the regional distribution of thyrotropin-releasing hormone and histidyl-proline diketopiperazine indicated that both peptides are found in highest concentrations in pituitary and hypothalamus, but are detectable in other areas of brain as well.

  15. Hypersensitivity reactions to the Sabin vaccine in children with cow's milk allergy.

    Science.gov (United States)

    Parisi, C A S; Smaldini, P L; Gervasoni, M E; Maspero, J F; Docena, G H

    2013-02-01

    The Sabin vaccine is used world-wide, and most children with food allergies receive it without incident. However, in the 2009 vaccination campaign conducted in Argentina, four children experienced immediate-type hypersensitivity reactions following vaccination. We aimed to review the medical history of the affected children, study their allergic condition after the episodes and analyse the presence of allergenic vaccine components. Patients were selected based on their immediate allergic reactions following vaccination. They were assessed for allergies to cow's milk and hen's egg. The presence of cow's milk proteins in the vaccine was tested by various immunoassays involving cow's milk- or α-lactalbumin-specific polyclonal rabbit antiserum and patient sera. All of the patients had a history of milk allergy, and no history or current evidence of egg hypersensitivity was found. Levels of cow's milk- and Sabin vaccine-specific IgE were increased, and the result of a skin prick test with cow's milk proteins or the Sabin vaccine was positive in each patient. In addition, an ELISA using specific rabbit antiserum detected α-lactalbumin in the Sabin vaccine. When α-lactalbumin was employed as a soluble inhibitor in a competitive ELISA, binding to vaccine-coated plates by cow's milk- or α-lactalbumin-specific rabbit antiserum or by patient serum containing IgE was inhibited. We have demonstrated that these patients were allergic to cow's milk, and had circulating and mast cell-bound IgE antibodies specific to cow's milk proteins. We found that the Sabin vaccine contained α-lactalbumin, which may have been responsible for the reactions elicited following vaccination with the Sabin and dual viral vaccines in combination. © 2012 Blackwell Publishing Ltd.

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

    Science.gov (United States)

    Myerowitz, R; Proia, R L

    1984-09-01

    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 were obtained, 44 of which contained inserts in the plasmid DNA. Differential hybridization, with cDNA probes prepared from polysomal RNA enriched or depleted for beta-hexosaminidase messenger, was used to screen the recombinant plasmids for sequences encoding beta-hexosaminidase. One clone, p beta H alpha-1, containing a cDNA insert of approximately equal to 240 base pairs, was identified in this manner. The plasmid hybrid-selected a messenger from placental RNA that programed a translation system to synthesize the alpha-chain of beta-hexosaminidase. p beta H alpha-1 hybridized to an mRNA of approximately equal to 1.9 kilobases in preparations enriched separately in messenger for the alpha-chain or for both alpha- and beta-chains (by polysome immunoselection with antiserum against isolated alpha-chain or against beta-hexosaminidase A, respectively). It did not hybridize to an RNA preparation enriched for messenger of beta-chain by immunoselection with antiserum against beta-hexosaminidase B. The 1.9-kilobase mRNA was observed in poly(A)+ RNA preparations from control fibroblasts and from fibroblasts of a Tay-Sachs patient that synthesize an altered alpha-chain; however, it was not seen in similar preparations from fibroblasts of four Ashkenazi Tay-Sachs patients.

  17. Novel 125I radioimmunoassay for the analysis of Δ9-tetrahydrocannabinol and its metabolites in human body fluids

    International Nuclear Information System (INIS)

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

    1984-01-01

    A cannabinoid radioimmunoassay (RIA) that detects some of the major Δ 9 -THC metabolites is developed and evaluated for use in forensic science. It incorporates a novel 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 Δ 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

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

    International Nuclear Information System (INIS)

    McDonald, T.P.

    1978-01-01

    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

  19. FMRF-amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

    DEFF Research Database (Denmark)

    Jirikowski, G; Erhart, G; Grimmelikhuijzen, C J

    1984-01-01

    Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from...... the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material...

  20. Specific and sensitive radioimmunoassay for 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG)

    Energy Technology Data Exchange (ETDEWEB)

    Keeton, T.K. (Univ. of Texas Health Science Center, San Antonio); Krutzsch, H.; Lovenberg, W.

    1981-02-06

    Antibodies that specifically bind the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) were produced in rabbits after injection of a derivative of MOPEG conjugated with bovine thyroglobulin. A sensitive radioimmunoassay was devised with this antiserum, in which as little as 0.5 nanogram of MOPEG can be accurately measured with a final antibody dilution of 1:180. The antibody appears to be specific for MOPEG, since tritiated MOPEG was not displaced from the antibodies by norepinephrine, epinephrine, dopamine, serotonin, or their major metabolites, including MOPEG-sulfate (333 nanograms each).

  1. Development of a heterologous radioimmunoassay for the measurement of human prolactin in serum

    International Nuclear Information System (INIS)

    Vieira, J.G.H.; Russo, E.M.K.; Lengyel, A.M.J.; Maciel, R.M.B.

    1981-01-01

    The development of a heterologous radioimmunoassay method is described for the measurement of human prolactin in serum. Antisera were produced in rabbits using ovine prolactin as the immunogen; the best antiserum produced enebled the development of a simple and rapid method, with sufficient sensitivity (2ng/ml). The cross reactivity with human growth hormone and with placental lactogen was inferior to 0.03%. The labelling of human prolactin was done using the Lactoperoxidase method, followed by a column chromatography purification in a Sephadex G-100. Using 153 sera, the described method was compared with a homologous radioimmunoassay for human prolactin, and no significant difference was shown. (Author) [pt

  2. Design of Fab-based chimeric antibodies against Bothrops asper toxins

    DEFF Research Database (Denmark)

    M. Haack, Aleksander; B. Hallgren, Malte; U. W. Friis, Rasmus

    Snakebite is one of the world’s most neglected tropical diseases, with an estimated 5 million bites per year, resulting in about 125.000 deaths. The only current treatment for snakebite envenoming is antiserum derived from the blood of immunized mammals(typically horses). These antisera are expen...... are expensive to produce and carry a high risk of causing hyper-allergic reactions in human recipients due to their heterologous origin. Here we report the discovery of chimeric scFvs against Bothrops asper toxins....

  3. The radioimmunological determination of vasopressin in urine

    International Nuclear Information System (INIS)

    Horn, M.J. van der.

    1981-01-01

    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 125 I-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.)

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

    International Nuclear Information System (INIS)

    Bullock, S.; Hayward, C.; Manson, J.; Brock, D.J.H.; Raeburn, J.A.

    1982-01-01

    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)

  5. 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.

  6. Radioimmunoassay for arginine-vasotocin (AVT) in serum of Pekin ducks: AVT concentrations after adaptation to fresh water and salt water

    International Nuclear Information System (INIS)

    Moehring, J.; Schoun, J.; Simon-Oppermann, C.; Simon, E.

    1980-01-01

    A radioimmunoassay for arginine-vasotocin (AVT), the antidiuretic principle in birds, was developed using the high cross-reactivity of AVT with an AVP antiserum raised in rabbits. This assay is specific for the measurement of AVT in serum of birds. The sensitivity and precision is such that serum AVT concentrations above 0.5 fmol/ml can be measured quantitatively. A serum AVT concentration of 5.1 +- 1.4 fmol/ml was found in normally hydrated, fresh water adapted ducks with a serum osmolality of 293.7 +- 2.2 mosmol/kg. When the same animals were acutely hydrated, no or [de

  7. Radioimmunoassay method for the determination of cardiotonic glycosides

    International Nuclear Information System (INIS)

    1975-01-01

    A kit method for the in vitro determination of digoxin and digitoxin is described. The blood serum is mixed with the reagent which consists of an aqueous buffer solution containing a radiolabeled hapten for the glycoside. Antiserum with specific antibodies is added and the mixture is incubated. Thereafter, a thin strip of membrane mainly consisting of an ionexchanger is brought into contact with the mixture to separate the antibody bound hapten from the unbound hapten. The ratios of both are determined by counting the radioactive hapten

  8. Lymph node abscess due to Actinomyces viscosus in a cat.

    Science.gov (United States)

    Murakami, S; Yamanishi, M W; Azuma, R

    1997-11-01

    In a four-year-old male cat, a subcutaneous phyma about 3.5 cm in diameter was surgically removed from the left inframandibular region. Histopathologically, the phyma was found to be the swollen medial retropharyngeal lymph node containing an actinomycotic abscess. The filamentous organisms in the abscess stained positively by the Gram's, Grocott's and periodic acid-Schiff methods, and were negative by the Ziehl-Neelsen method. By the immunoperoxidase method, the organisms were specifically identified as Actinomyces viscosus serotype 2 by its antiserum absorbed with A. viscosus serotype 1 antigen.

  9. Norethindrone antisera: anomalous cross-reactivities with use of 3H-tetrahydro-reduced norethindrone as radioligand

    International Nuclear Information System (INIS)

    Watanabe, H.; Menzies, J.A.; Jordan, N.; Loo, J.C.

    1983-01-01

    Anomalous cross-reactions with the dihydro- and tetrahydro-reduced metabolites of norethindrone were observed utilizing antisera raised against norethindrone-3-bovine serum albumin. Whereas displacement of 3H-norethindrone from the antiserum by the metabolites was generally minimal, where one of the metabolites was used as radiotracer, displacement by the metabolites was equal to or greater than that achieved by norethindrone. This unexpected finding was examined for its usefulness in developing a radioimmunoassay system for norethindrone metabolites in plasma. The sensitivity of the resulting standard curve was such as to permit quantitation of pg amounts of the reduced metabolites

  10. Carcinoembryonic antigen (CEA)

    International Nuclear Information System (INIS)

    Ephraim, K.H.; Cox, P.H.; Hamer, C.J.A. v.d.; Berends, W.; Delhez, H.

    1977-01-01

    The carcinoembryonic antigen (CEA) is a complex of antigen determinants and also the carrier of these determinants. Chemically it is a glycoprotein. Its occurrence in blood serum or urine is correlated with malignant disease. Several radioimmunoassays (RIA) have been developed, one by Hoffmann-Laroche and one by the Rotterdam Radiotherapeutic Institute. Both methods and the Hoffmann assay kit are tested. Specifications are given for isolation of the antigen, preparation of the antiserum, and the execution of the RIA. Biochemical and clinical aspects are discussed

  11. Ultrastructural localization of RFamide-like peptides in neuronal dense-cored vesicles in the peduncle of Hydra

    DEFF Research Database (Denmark)

    Koizumi, O; Wilson, Douglas Clyde; Grimmelikhuijzen, C J

    1989-01-01

    -Araldite-embedded tissue labeled with 15-nm gold particles revealed a concentration of RFamide-like immunoreactivity over the granular cores of vesicles in epidermal ganglion cells. Gold-labeled, dense-cored vesicles were present in the perikaryon, long thin neurites, and axon terminals of these neurons. The aggregation...... of labeled dense-cored vesicles in an axon terminal on the myoneme of an epitheliomuscular cell suggests a possible function of RFamide-like peptides in neuromuscular transmission. Gold staining of dense-cored vesicles completely disappeared when the RFamide antiserum was preabsorbed with 10 micrograms...

  12. EXPRESSION OF BACTERIOOPSIN GENES IN ESCHERICHIA COLI

    OpenAIRE

    TSUJIUCHI, Yutaka; IWASA, Tatsuo; TOKUNAGA, Fumio

    1994-01-01

    An inducible expression vector pUBO was constructed with native codons in order to express the gene of Bacteriorhodopsin (BOP) in Escherichia coli (E. coli). Vector pUBO contains lac-promoter followed by the partial structural gene of lacZ and the structural gene of BOP. The expression of this fusion protein was detected by ELISA with anti-BOP antiserum. The fusion protein obtained from E. coli trnsformed with pUBO formed approximately 0.1% of the total protein of the E. coli membrane fraction.

  13. The black widow spider bite: differential diagnosis, clinical manifestations, and treatment options.

    Science.gov (United States)

    Shackleford, Rodney; Veillon, Diana; Maxwell, Nicole; LaChance, Lisa; Jusino, Tamara; Cotelingam, James; Carrington, Patrick

    2015-01-01

    Unrecognized and untreated black widow spider bites cause significant pain, impairment, and rarely death. The widow venom, a powerful neurotoxin known as a-latrotoxin, causes muscle pain, diaphoresis, tachycardia, flushing, and hypertension. Treatment is usually symptomatic with a combination of opioid analgesics and muscle relaxants. If symptom resolution fails, an equine IgG antiserum is available, but a high index of clinical suspicion coupled with a knowledgeable patient history often allows successful treatment, especially when the treating physician possesses awareness of this type of bite and its usual course and possible complications.

  14. 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.

  15. Radioimmunoassay of digoxigenin glycosides in Digitalis lanata

    International Nuclear Information System (INIS)

    Lehtola, T.; Hiltunen, R.; Schantz, M. v.; Huhtikangas, A.

    1981-01-01

    Radioimmunoassay (RIA) has been used in the analysis of digoxigenin glycoside contents of crude ethanolic extracts prepared from fresh leaf samples of Digitalis lanata EHRH. The commercial antidigoxin antiserum used in the assay was highly specific against the digoxigenin aglycone. Steroid derivatives with only slight structural differences from digoxigenin remained practically unassayed. The method is highly sensitive, and excellent values for accuracy, precision and correlation were obtained. Digoxigenin glycoside levels of second-year D. lanata leaves were found to be considerably higher than those of first-year leaves. (orig.)

  16. A radioimmunoassay for the detection of diethylstilboestrol and related stilbenes in biological fluids

    International Nuclear Information System (INIS)

    Hallahan, Cornelius; McGarry, Yvonne; Collins, J.D.

    1985-01-01

    A radioimmunoassay for the measurement of the synthetic anabolic agent diethylstilboestrol (DES) is described. It is based on a commercially available antiserum and a tritiated derivative of DES. The method can detect low concentrations of residues (less than 0.5 ng/ml) in small samples (0.05 to 0.2 ml) of biological fluids. DES was measured in plasma, bile and urine obtained from a calf slaughtered 22 days after subcutaneous implantation of 24 mg DES. The assay described is suitable as a rapid screening procedure for identifying animals treated with stilbene substances. (author)

  17. 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.

  18. Synthesis of nerve growth factor mRNA and precursor protein in the thyroid and parathyroid glands of the rat.

    OpenAIRE

    Dicou, E; Lee, J; Brachet, P

    1986-01-01

    Nerve growth factor (NGF) mRNAs were detected in the thyroid and parathyroid glands by hybridization to a preproNGF and a NGF-beta cDNA probe. The thyroid NGF transcript was identical in size to that found in the mouse submaxillary gland. Mature NGF-beta was not detected in the thyroid tissue either by immunoprecipitation or by immunohistochemical methods. In contrast, an antiserum directed against the predicted precursor protein sequence immunoprecipitated a protein of Mr 30,000-31,000 in th...

  19. Radioligand assays - method and application. 2

    International Nuclear Information System (INIS)

    Ziegler, M.; Keilacker, H.; Woltanski, K.P.; Besch, W.; Schubert, J.

    1980-01-01

    Glucagon antibodies were produced in rabbits using three immunogenic glucagon conjugates. Glucagon was coupled to bovine serum albumin by difluorodinitrobenzene (DFDNB), carbodiimide or glutardialdehyde. Rabbits immunized with glucagon conjugated to albumin using DFDNB produced sensitive antisera for radioimmunoassay specific for pancreatic glucagon. The affinity constant of the best antiserum was approximately 10 11 l/mol. Antisera raised against the two other glucagon conjugates were significantly less affine. All of these antisera showed inverse binding curves of 125 I-glucagon caused by positive cooperativity in dependence upon the antigen/antibody ratio. The race of rabbits used for immunization was without influence on the immune response. (author)

  20. Glucagon and glucagonlike immunoreactivity (GLI), ch. 18

    International Nuclear Information System (INIS)

    Faloona, G.R.

    1976-01-01

    The radioimmunoassay of glucagon and the problems concerning incubation damage of the labelled hormone and cross-reactivity with GLI are described. The commercially available 125 I-glucagon is purified by sephadex G-25 gel filtration. The requirements for the assay are described. The separation of bound and free-labelled glucagon is performed by adsorption using dextran-coated charcoal. A range of 50 to 240 pg per ml plasma was assayed. The assay of GLI, using a specific GLI-antiserum is identical to the procedure described for glucagon

  1. The use of radioassays in clinical and animal test trials on the physiology and pathophysiology of glucagon

    International Nuclear Information System (INIS)

    Boettger, I.

    1981-01-01

    Investigations in the field of glucagon physiology and pathophysiology were carried out employing sensitive, specific and precise radioimmunoassays for glucagon, gut glucagon-like immunoreactivity, GLI, and insulin which still needed to be validated and other radioassays. It could be shown in the area of methodology that the glucagon-RIA using the specific antiglucagon-antiserum 30 K UNGER as reference surpasses three commercially available glucagon RIA kits. Norm ranges for the basal glucagon concentration were worked out and glucagon levels were established in function tests using arginine stimulation to raise, and glucose suppression to lower levels. (orig./MG) [de

  2. Detection of human spermatozoal peptides after conjugation to 125I-labelled human serum albumin

    International Nuclear Information System (INIS)

    Metler, L.; Skrabei, H.; Czuppon, A.B.

    1981-01-01

    Human spermatozoal peptides, liberated during autolysis of the cells, were fractionated by gel-filtration chromatography and thin-layer chromatography. After conjugation to 125 I-labelled human serum albumin, all fractions were assayed with rabbit antihuman spermatozoa antiserum. In earlier publications, human sperm-immobilizing and sperm-agglutinating sera were used for the detection of solubilized spermatozoal antigen. The low sensitivity of these tests necessitated a more sensitive test. The purpose of this work is to describe a solid-phase radioimmunoassay for the detection of antigenic peptides

  3. Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Rinehart, K L; Jacob, E

    1990-01-01

    Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Asn-NH2, we have purified a peptide from acetic acid extracts of the sea anemone Anthopleura elegantissima. By classical amino acid analyses, mass spectrometry, and 1H NMR spectroscopy, the structure of this peptide was determined as 3......-phenyllactyl-Leu-Arg-Asn-NH2. By using reversed-phase HPLC and a chiral mobile phase, it was shown that the 3-phenyllactyl group had the L configuration. Immunocytochemical staining with antiserum against Arg-Asn-NH2 showed that L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide) was localized in neurons of sea...

  4. Radioimmunoassay for rhesus monkey gonadotropins

    International Nuclear Information System (INIS)

    Faiman, C.; Stearns, E.L.; Winter, J.S.D.; Reyes, F.I.; Hobson, W.C.

    1975-01-01

    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.)

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

    International Nuclear Information System (INIS)

    Dahlen, H.G.

    1986-01-01

    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.)

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

    International Nuclear Information System (INIS)

    Silva, S.R.; Borghi, V.C.; Bellini, M.H.; Wajchenberg, W.A.J.

    1992-01-01

    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.)

  7. Discovery of Human IgGs against α-Cobratoxin for Development of Recombinant Antibody-based Antivenom

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Engmark, Mikael; Redsted Rasmussen, Arne

    , in which large mammals (typically horses) are immunized with snake venom and antiserum is derived from the animals blood. The incompatibility with the human immune system of these animal derived antivenoms leads to a range of side effects,such as serum sickness, anaphylaxis, and sometimes even death......More than 5.5 million people are bitten by venomous snakes per year on a global basis. This leads to approx. 125,000 deaths and 3 times as many amputations. Particularly Sub-Saharan Africa is affected by the problem. Current antivenoms are still being produced by a method developed in the 1890’s...

  8. Preparation of a cell line persistently infected with maedi/visna virus and production of viral antigens.

    Science.gov (United States)

    Suzuki, Kazuya; Oguma, Keisuke; Sentsui, Hiroshi

    2017-01-20

    We attempted to prepare a cell line that produces maedi/visna virus (MVV) and is free of contamination by other viruses and mycoplasmas. Three cell lines, which originated from a sheep, goat and bat, were infected with MVV and passaged approximately every 5 days. The cultured cells were then subjected to polymerase chain reaction analysis for MVV provirus. As a result, a cell line persistently infected with MVV was established from ZZ-R cells, which originated from the fetal goat tongue. The 50-fold concentrated culture fluid formed a precipitation line against reference antiserum.

  9. Changes in RFamide-Related Peptide-1 (RFRP-1)-Immunoreactivity During Postnatal Development and the Estrous Cycle

    DEFF Research Database (Denmark)

    Jørgensen, Sara R; Andersen, Mille D; Overgaard, Agnete

    2014-01-01

    and their distinct roles in vivo. In this study, we raised an antiserum selective for RFRP-1 and defined the distribution of RFRP-1-immunoreactive (ir) neurons in the rat brain. Next, we analyzed the level of RFRP-1-ir during postnatal development in males and females and investigated changes in RFRP-1-ir during....... The number of RFRP-1-ir neurons and the density of cellular immunoreactivity were unchanged from juvenile to adulthood in male rats during the postnatal development. However, both parameters were significantly increased in female rats from peripuberty to adulthood, demonstrating prominent gender difference...

  10. Serological comparison of selected isolates of Aeromonas salmonicida ssp. Salmonicida

    Science.gov (United States)

    Hahnel, G.B.; Gould, R.W.; Boatman, E.S.

    1983-01-01

    Eight isolates of Acronionus salmonicida ssp. salmonicida were collected during furunculosis epizootics in North American Pacific coast states and provinces. Both virulent and avirulent forms of each isolate, confirmed by challenge and electron microscopy, were examined. Serological comparisons by cross-absorption agglutination tests revealed no serological differences between isolates. Using the double diffusion precipitin test, a single band was observed when antigen from a sonicated virulent strain was reacted with antiserum against a sonicated, virulent strain absorbed with homologous, avirulent strain. The presence of the single band was eliminated by excess sonication.

  11. Synthesis and turnover of purine nucleoside phosphorylase in human lymphocytes

    International Nuclear Information System (INIS)

    Snyder, F.F.; Kwan, E.; Mably, E.R.; Neote, K.

    1986-01-01

    The authors study the synthesis of purine nucleoside phosphorylase during phytohumagglutinin induced T cell transformation and have examined the turnover of purine nucleoside phosphorylase in thte human lymphoblast WI-L2. A polyclonal antibody to purine nucleoside phosphorylase in the synthetic and turnover studies is used. Fluorography of purine nucleoside phosphorylase immunoprecipitates from WI-L2 cells are shown. Lymphoblast proteins were labelled during 16hour cultrue with 4,5-tritium-leucine. Samples were 20 ug of total protein, immunoprecipitate of 200 ug protein with control serum and purine nucleoside phosphorylase antiserum

  12. 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

    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)....

  13. Produção e purificação de anticorpos policlonais para Salmonella Enteritidis (Enterobacteriaceae Production and purification of polyclonal antibodies for Salmonella Enteritidis (Enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    Mario Augusto Ono

    2002-04-01

    Full Text Available O objetivo deste trabalho foi produzir e purificar anticorpos policlonais específicos para Salmonella Enteritidis (Enterobacteriaceae. O anti-soro foi produzido em coelhos, empregando-se flagelina purificada. O título e a especificidade foram determinados através do ensaio imunoenzimático - ELISA e a purificação por cromatografia de afinidade com sepharose Proteína A. As suspensões bacterianas foram cultivadas em cinco diferentes meios de cultura (infusão de cérebro coração - BHI, caldo tripticase soja, caldo lactosado, caldo nutriente - CN e água peptonada. Observou-se que dependendo do meio o título do anti-soro pode variar e os melhores resultados foram obtidos com BHI e CN. O anti-soro foi específico para Salmonella Enteritidis, apresentando porcentagens de reações cruzadas com Salmonella Typhimurium, Salmonella Infantis e Salmonella Newport de 16,0, 11,9 e 6,4%, respectivamente. Menores porcentagens foram obtidas com outras enterobactérias testadas. Esses resultados indicam a possibilidade da utilização desses anticorpos na padronização de ensaios imunológicos para a detecção de Salmonella EnteritidisThe purpose of this study was to produce and to purify specific polyclonal antibodies for Salmonella Enteritidis (Enterobacteriaceae. The anti-serum was raised in rabbits using purified flagelin. Anti-serum titer and specificity were determined by an immunoassay - ELISA and its purification was performed by sepharose protein A affinity chromatography. The bacteria suspensions were cultivated in five different media (brain heart infusion - BHI, tripticase soy broth, nutrient broth - NB, peptone water. Results have showed that anti-serum titers varied depending on which media type was used and BHI and NB media yielded the most significant results. The anti-serum produced was specific for Salmonella Enteritidis. Its cross-reactivity with Salmonella Thyphimurium, Salmonella Infantis and Salmonella Newport were 16.0, 11

  14. Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Norén, O; Sjöström, H

    1980-01-01

    Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl...... revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed...... protein....

  15. In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

    DEFF Research Database (Denmark)

    Beavan, L A; Davies, M; Couchman, J R

    1989-01-01

    The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently......-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane...

  16. Preparation and purification of 7-Iodoclonazepam for use in radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Goddard, C.P.; Law, B.; Mason, P.A.; Stead, A.H.

    1986-04-01

    A method is described for the preparation and purification of 7-(/sup 125/I)-Iodoclonazepam (5-(o-Chlorophenyl)-2,3-Dihydro-7-(/sup 125/I)-Iodo-1H-1,4-Benzodiazepin-2-one). The structure was confirmed by mass spectrometry using 7-(/sup 127/I)iodoclonazepam prepared by the same method. 7-(/sup 125/I)-Iodoclonazepam binds well to a benzodiazepine antiserum. Although readily displaced by all the benzodiazepines commercially available in the UK, it is not displaced by structurally related nonbenzodiazepines except at very high concentrations. 7-(/sup 125/I) Iodoclonazepam should therefore be useful for the development of a screening radioimmunoassay (RIA) for benzodiazepines.

  17. Specificity of immunoassays. Pt. 2

    International Nuclear Information System (INIS)

    Pratt, J.J.; Woldring, M.G.; Boonman, R.; Kittikool, J.

    1979-01-01

    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.) [de

  18. The molecular basis of the antigenic cross-reactivity between measles and cowpea mosaic viruses

    International Nuclear Information System (INIS)

    Olszewska, Wieslawa; Steward, Michael W.

    2003-01-01

    Two nonrelated viruses, cowpea mosaic virus (wtCPMV) and measles virus (MV), were found to induce cross-reactive antibodies. The nature of this cross-reactivity was studied and results are presented here demonstrating that antiserum raised against wtCPMV reacted with peptide from the fusion (F) protein of MV. Furthermore, the F protein of MV was shown to share an identical conformational B cell epitope with the small subunit of CPMV coat protein. Passive transfer of anti-wtCPMV antibodies into BALB/c mice conferred partial protection against measles virus induced encephalitis. The results are discussed in the context of cross-protection

  19. Human laminin isolated in a nearly intact, biologically active form from placenta by limited proteolysis

    DEFF Research Database (Denmark)

    Wewer, U; Albrechtsen, R; Manthorpe, M

    1983-01-01

    fragments cross-reacted with rat laminin in immunodiffusion and enzyme immunoassay, and a polyclonal antiserum against the fragments reacted with basement membranes in tissues in a manner identical with the 4E10 antibody. Electron microscopic images of the human peptic fragments showed structures similar...... to the cross-shaped images of murine laminins, although the short arms were truncated to various degrees or even absent. The isolated peptic fragments also displayed biological activity similar to that of murine laminins in that the outgrowth of neurites by neuronal cells was promoted on plates coated...... with the fragments....

  20. 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

    1983-01-01

    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...... of overlap and colinearity of actin and fibronectin filaments. The ability of keratinocytes to produce fibronectin suggests that these cells can contribute to the formation of the basement membrane in skin. The localisation of fibronectin and its close association with actin also suggests that it is involved...

  1. 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

    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...... 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...

  2. Intestinal Bacterial Infection Diagnosed by Histological Examination of Endoscopic Biopsy Specimens

    Directory of Open Access Journals (Sweden)

    Michiko Yuki

    2016-10-01

    Full Text Available Intestinal spirochetosis (IS in humans is characterized by spirochetal microorganisms attached to the luminal surface of the colonic epithelium. In the present case, attached organisms appeared as 3- to 4 μm-thick (average thickness, 3.4 μm basophilic fringes or haze in HE-stained endoscopic biopsy specimens. The basophilic fringes were clearly labeled by Treponema pallidum antiserum. Because IS is relatively rare in developed countries, thin basophilic fringes characteristic of IS are readily overlooked. Thus, the recognition of histological characteristics of this disease is important for its diagnosis.

  3. Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

    OpenAIRE

    Lobato, M F; Ros, M; Moreno, F J; García-Ruíz, J P

    1986-01-01

    Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was consta...

  4. The isolation of the γ subunit of fetal hemoglobin (HbF) and its use in a radioimmunoassay for HbF

    International Nuclear Information System (INIS)

    James, R.F.L.; Shuster, J.; Freedman, S.O.; Gold, P.

    1980-01-01

    A method is described for the purification, from fetal hemoglobin (HbF), of the fetal specific globin chain (γ chain) in its native state. In the absence of α chain (the globin chain common to all adult human hemoglobins) γ chain, when used as an immunogen, is able to express its unique antigenicity. Here, a specific, high titer antiserum raised against γ chain has been used to establish a sensitive radioimmunoassay for HbF. This approach may be applicable to the measurement of other normal and abnormal hemoglobins. (Auth.)

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

    Science.gov (United States)

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

    1985-01-01

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

  6. Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

    Science.gov (United States)

    Gogolewski, R P; Mackintosh, J A; Wilson, S C; Chin, J C

    1992-10-01

    Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.

  7. Potential threat of a new pathotype of Papaya leaf distortion mosaic virus infecting transgenic papaya resistant to Papaya ringspot virus.

    Science.gov (United States)

    Bau, H-J; Kung, Y-J; Raja, J A J; Chan, S-J; Chen, K-C; Chen, Y-K; Wu, H-W; Yeh, S-D

    2008-07-01

    A virus identified as a new pathotype of Papaya leaf distortion mosaic virus (PLDMV, P-TW-WF) was isolated from diseased papaya in an isolated test-field in central Taiwan, where transgenic papaya lines resistant to Papaya ringspot virus (PRSV) were evaluated. The infected plants displayed severe mosaic, distortion and shoe-stringing on leaves; stunting in apex; and water-soaking on petioles and stems. This virus, which did not react in enzyme-linked immunosorbent assay with the antiserum to the PRSV coat protein, infected only papaya, but not the other 18 plant species tested. Virions studied under electron microscope exhibited morphology and dimensions of potyvirus particles. Reverse transcription-polymerase chain reaction conducted using potyvirus-specific primers generated a 1,927-nucleotide product corresponding to the 3' region of a potyvirus, showing high sequence identity to the CP gene and 3' noncoding region of PLDMV. Search for similar isolates with the antiserum against CP of P-TW-WF revealed scattered occurrence of PLDMV in Taiwan. Phylogenetic analysis of PLDMV isolates of Taiwan and Japan indicated that the Taiwan isolates belong to a separate genetic cluster. Since all the Taiwan isolates infected only papaya, unlike the cucurbit-infecting Japanese P type isolates, the Taiwan isolates are considered a new pathotype of PLDMV. Susceptibility of all our PRSV-resistant transgenic papaya lines to PLDMV indicates that the virus is an emerging threat for the application of PRSV-resistant transgenic papaya in Taiwan and elsewhere.

  8. Development of radioimmunoassay for prolactin binding protein

    International Nuclear Information System (INIS)

    Raikar, R.S.; Sheth, A.R.

    1982-01-01

    Using a homogenous prolactin binding protein (PBP) preparations from rat seminal vesicle secretion, a sensitive and specific radioimmunoassay (RIA) for PBP has been developed. The assay was highly specific and showed no cross-reaction with other protein hormones from various species. The antiserum had an affinity constant (Ka) of 2.66 x 10 10 M -1 . The assay sensitivity was in the range of 0.5-1.0 ng of pure PBP per assay tube and the intra- and inter-assay coefficients of variations were 6-8% and 12-14.5% respectively. The overall recovery of PBP to the rat seminal vesicle secretion was 96.8%. Using this RIA, PBP levels in various biological fluids and reproductive tissues were measured. Azoospermic human semen contained significantly higher levels of PBP than normospermic semen. The seminal vesicle of rat exhibited the highest concentration of PBP. Administration of antiserum to PBP to mature male rats resulted in a significant reduction in the weight of ventral prostrate and serum prolactin levels were significantly elevated in these animals suggesting that the antibody raised against the PBP was capable of blocking prolactin receptors. (author)

  9. Cooperative Serum Bactericidal Activity Between Human Antibodies to Meningococcal Factor H Binding Protein and Neisserial Heparin Binding Antigen

    Science.gov (United States)

    Vu, David M.; Wong, Tracy T.; Granoff, Dan M.

    2011-01-01

    A meningococcal group B vaccine containing multiple protein antigens including factor H binding protein (fHbp) and Neisserial heparin binding antigen (NHba) is in clinical development. The ability of antibodies against individual antigens to interact and augment protective immunity is unknown. We assayed human complement-mediated bactericidal activity (SBA) in stored sera from six immunized adults before and after depletion of antibodies to fHbp and/or NHba. All six subjects developed ≥4-fold increases in SBA titer against a test strain with fHbp in the variant 1 group with an amino acid sequence that matched the vaccine antigen (GMT 95 percent of the SBA was directed against fHbp. Four subjects developed ≥4-fold increases in SBA titer against a test strain with a heterologous fHbp variant 2 antigen and a homologous NHba amino acid sequence that matched the vaccine antigen (GMT bactericidal anti-fHbp variant 1 antiserum with a mouse anti-NHba antiserum also augmented the anti-NHba SBA titer against this test strain. For meningococcal vaccines that target relatively sparsely-exposed antigens such fHbp or NHba, non-bactericidal antibodies against individual antigens can cooperate and elicit SBA. PMID:21241734

  10. A radioimmunoassay for lignin in plant cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Dawley, R.M.

    1989-01-01

    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 {beta}-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 {eta}g/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. {sup 125}I 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 NaClO{sub 2} delignification. The RIA was found to be less sensitive than expected, so several avenues for improving the method are discussed.

  11. Validation of a direct radioimmunoassay of melatonin in the blue fox

    International Nuclear Information System (INIS)

    Forsberg, M.; Madej, A.

    1987-01-01

    A direct radioimmunoassay procedure for the determination of melatonin in the blood of blue fox has been validated and applied. The assay required 50 μl of sample and standard, 100 μl of antiserum and 100 μl of ( 3 H)melatonin. After overnight incubation at 4 deg. C the antibody bound melatonin was separated from the free hormone with dextran-coated charcoal. Following centrifugation the antibody bound ( 3 H)melatonin was determined in a beta scintillation counter. The antiserum bound 30-35 % of the ( 3 H)melatonin at a final dilution of 1:36000. The non specific binding represented less than 5 % of the total radioactivity in all assays. The lowest detectable amount of melatonin was 2.6 fmol/tube, corresponding to 52.5 pmol/l. The inter-assay coefficient of variation at 178 and 510 pmol/l was 15.6 and 8.8 %, respectively. The precision profile, calculated from a 10-replicate standard curve, showed that the coefficient of variation decreased from 43 % at 84 pmol/l to 15 % at 336 pmol/l, and remainded at or below 10 % for concentrations exceeding 670 pmol/l. Plasma was collected from 2 male blue foxes at about hourly intervals during a 24 h period in September and assayed for melatonin. Maximum (421 pmol/l) and minimum (97 pmol/l) concentrations of the hormone were inversely related to light intensity. (author)

  12. 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.

  13. Measurement of insulin in human sera using a new RIA kit. 1

    International Nuclear Information System (INIS)

    Besch, W.; Woltanski, K.P.; Keilacker, H.; Diaz-Alonso, J.M.; Schulz, B.; Kohnert, K.D.; Ziegler, M.; Amendt, P.

    1987-01-01

    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)

  14. [Antigenic relations of several strains of Naegleria].

    Science.gov (United States)

    Zubiaur, E; Alonso, P

    1987-02-01

    In previous papers different aspects of one strain of Naegleria lovaniensis (Aq/9/1/45D) and two strains of N. gruberi (1518/le and 1518/lf) have been studied. From the results obtained it can be concluded that each strain behaves differently; no more similarities have been found between both N. gruberi strains, than between each of these and N. lovaniensis. Such an event has prompted us to characterize their antigenic relationships by means of an immunoprecipitation assay (double diffusion in plate). Each antiserum was tested against the different antigenic extracts. For N. lovaniensis, besides the whole extract, two fractions (particulate and soluble) and their respective antisera were assayed separately. No reaction occurred between any of the anti-N. lovaniensis sera and either of the two N. gruberi extracts. The antiserum to N. gruberi 1518/lf reacted only with its homologue and with N. lovaniensis antigens. Both N. lovaniensis fractions share some antigenic components being more complex the antigenic structure of the soluble fraction. Therefore no more similarities occur between both N. gruberi strains than between each one and N. lovaniensis, rather N. gruberi 1518/le exhibits more antigenic relationships with N. lovaniensis than with 1518/lf strains. In view of such results the species N. gruberi should be taxonomically reconsidered, criterium shared by other authors.

  15. Production of specific antisera for radioimmunoassay of human luteinizing hormone (LH) in the presence of human chorionic gonadotropin (hCG)

    International Nuclear Information System (INIS)

    Thorell, J.I.; Jeppsson, S.; Holmstrom, B.

    1976-01-01

    A specific radioimmunoassay for LH, which measures plasma LH in the presence of human chorionic gonadotropin (hCG) is described. Rabbits were immunized with highly purified native LH. One of the antisera with a difference in its reactivity against LH and hCG was further purified by affinity chromatography on a column with hCG coupled to Sepharose 4B. The adsorbed antiserum and 125 I-LH was used in a double antibody assay. The LH standard (MRC/68/40) efficiently inhibited the binding of 125 I-LH, and the standard curve showed a sensitivity of 0.5 ng/ml in the sample. hCG up to 10,000 ng/ml did not inhibit the binding of 125 I-LH. The plasma level of LH in pregnant women in the first trimester was low (1.3 +- 0.1 ng/ml). When LH was measured in fertile or menopausal women with or without stimulation with LH/FSH releasing hormone (LH-RH)/sup x/ the results agreed to those found with our conventional LH-assay based on antiserum against hCG

  16. Evaluation of radioimmunoassays using mono- and polyclonal antibodies for the quantitation of human chorionic gonadotropin in early pregnancy

    International Nuclear Information System (INIS)

    Heip, J.; Devroey, P.; Naaktgeboren, N.; Van Steirteghem, A.C.

    1986-01-01

    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

  17. Identificação de predadores de Orphulella punctata (de Geer (Orthoptera, Acrididae através da serologia Serological technique for identifying Orphulella punctata (de Geer (Orthoptera, Acrididae predators

    Directory of Open Access Journals (Sweden)

    Marta Cassaro-Silva

    2001-03-01

    Full Text Available Females of Orphulella punctata (de Geer, 1794 were captured in the field and kept in cages with food and boxes filled with moist soil for oviposition. The eggs, macerated with 0,85% saline solution and centrifugated, were used as the immunizing antigen for obtention of the specific antiserum (AS-O. Rabbits were immunized by the linphonodule injection method with two injections of this antigen, with an interval of 15 days between the 1st and 2nd inoculations. Serological tests were performed using double diffusion in agar gel with homologous and heterologous antigens. Specific serological reactions were obtained 14 days after the 1st inoculation. The antiserum of the 30th day gave 5, 2-4 and 1-4 precipitation lines respectively with eggs, female and male antigen. Such differences can be attributed to the high specificity of AS-O. Positive reaction was observed with arachnids and mirmeleontids captured in the field, indicating the predation on O. punctata.

  18. Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein

    Science.gov (United States)

    Franke, J.; Batts, W.N.; Ahne, W.; Kurath, G.; Winton, J.R.

    2006-01-01

    Fourteen reptilian paramyxovirus isolates were chosen to represent the known extent of genetic diversity among this novel group of viruses. Selected regions of the fusion (F) gene were sequenced, analyzed and compared. The F gene of all isolates contained conserved motifs homologous to those described for other members of the family Paramyxoviridae including: signal peptide, transmembrane domain, furin cleavage site, fusion peptide, N-linked glycosylation sites, and two heptad repeats, the second of which (HRB-LZ) had the characteristics of a leucine zipper. Selected regions of the fusion gene of isolate Gono-GER85 were inserted into a prokaryotic expression system to generate three recombinant protein fragments of various sizes. The longest recombinant protein was cleaved by furin into two fragments of predicted length. Western blot analysis with virus-neutralizing rabbit-antiserum against this isolate demonstrated that only the longest construct reacted with the antiserum. This construct was unique in containing 30 additional C-terminal amino acids that included most of the HRB-LZ. These results indicate that the F genes of reptilian paramyxoviruses contain highly conserved motifs typical of other members of the family and suggest that the HRB-LZ domain of the reptilian paramyxovirus F protein contains a linear antigenic epitope. ?? Springer-Verlag 2005.

  19. Radioimmunoassay for the tachykinin peptide physalaemin: detection of a physalaemin-like substance in rabbit stomach

    Energy Technology Data Exchange (ETDEWEB)

    Lazarus, L.H.; DiAugustine, R.P.

    1980-09-15

    An antiserum against the amphibian tachykinin physalaemin was specific for the NH/sub 2/-terminal region of this peptide. The cross-reactivity of antiserum PS-1 with uperolein was 2.03%, phyllomedusin, 0.36%, and kassinin, 0.013%; negligible (< 0.0001%) recognition was shown for substance P as well as with numerous other polypeptide hormones. The assay readily detected 1 pg (0.79 fmol) physalaemin with 50% displacement at 6.79 +- 1.98 pg (5.29 +- 1.54 fmol). Rabbit stomach, extracted by boiling followed by homogenization in 1.0 N formic acid, contained physalaemin-like immunoreactivity. The concentration detected in the antrum was 43.0 +- 7.3 ng/g dry weight with less in the fundus, pylorus, corpus, and duodenum. Chromatography of the extracts on Bio-Gel P4 columns in 0.1 N formic acid gave a single, symmetrical peak of immunoreactivity which eluted with an apparent molecular weight of approximately 1700. These data are the first to demonstrate the presence of a peptide with physalaemin-like immunoreactivity in mammalian tissues.

  20. A radioimmunoassay for human parathyroid hormone utilizing a goat anto-bovine PTH serum

    International Nuclear Information System (INIS)

    Mallette, L.E.

    1981-01-01

    An antiserum (NG-1) against bovine PTH (hPTH) generated in the domestic goat was characterized for use in the radioimmunoassay of PTH in human serum. When a carboxyterminal fragment of bPTH is used as radioligand, this antiserum detects only an antigenic site in the central region of the hPTH molecule. The synthetic hormone fragment, hPTH-(44-68), will displace 93% of the tracer, after which the addition of intact hPTH causes no further displacement. The assay does not detect the synthetic aminoterminal 1-34 fragment of the bovine or human hormones, nor the carboxyterminal fragment of the human hormone, hPTH-(53-84). Standard curves with bPTH-(1-84) and partially purified hPTH are not parallel, so that hPTH is used as standard. Serum from subjects with uraemia or primary hyperparathyroidism gives dilution curves parallel to that with the hPTH standard. The assay with NG-1 has been applied to the diagnosis of primary and secondary hyperparathyroidism, used to monitor the disappearance of PTH after parathyroidectomy, and for measurement of PTH in selective venous samples. (author)

  1. 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)

    Soares, Carlos R.J.

    1995-01-01

    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

  2. A thymoma as a cause of true ectopic hyperparathyroidism.

    Science.gov (United States)

    Rizzoli, R; Pache, J C; Didierjean, L; Bürger, A; Bonjour, J P

    1994-09-01

    Ectopic tumoral secretion of authentic PTH is rare, as only four cases have been convincingly documented by demonstrating the presence of PTH messenger ribonucleic acid in tumor tissue. We report the case of a 25-yr-old male with biochemical alterations typical of primary hyperparathyroidism (elevated calcemia and renal tubular reabsorption of calcium, decreased phosphatemia and maximal tubular reabsorption of phosphate, and increased intact PTH serum levels). Extensive cervical exploration did not reveal any abnormally enlarged parathyroid tissue, but excision of a palpable superior retrosternal mass led to the correction of all abnormal biochemical values. Histological analysis showed a predominantly epithelial thymoma, without any detectable parathyroid gland on serial slices. Tumor extracts contained immunoreactive PTH material, with serial dilutions paralleling PTH standards in an immunoradiometric assay. By contrast, immunoreactive PTH-related protein was absent. Furthermore, on Northern blot analysis, there was a PTH messenger ribonucleic acid transcript with a size similar to that found in parathyroid adenoma or hyperplasia. The thymoma epithelial cells stained positively with antiserum against PTH-(1-34), but negatively with antichromogranin-A antiserum. These results support the ectopic production of authentic PTH by a thymoma and indicate a novel tumoral cause of primary hyperparathyroidism.

  3. Immunoregulatory changes induced by total lymphoid irradiation. II. Development of thymus-leukemia antigen-positive and -negative suppressor T cells that differ in their regulatory function

    International Nuclear Information System (INIS)

    King, D.P.; Strober, S.

    1981-01-01

    BALB/c mice treated with total lymphoid irradiation (TLI) develop non-antigen-specific suppressor cells of the adoptive secondary antibody response and of the mixed leukocyte reaction. Suppressors of the adoptive anti-DNP response were eliminated by incubation of spleen cells with anti-Thy-1.2 or anti-thymus-leukemia (TL) antiserum and complement before cell transfer. Thymectomy before TLI prevented the appearance of the latter suppressor cells. On the other hand, suppressors of the MLR were eliminated by incubation of spleen cells with anti-Thy-1.2 but not anti-TL antiserum and complement. Thymectomy before TLI did not prevent their subsequent development. Thus, two subpopulations of suppressor T cells that differ in the expression of the TL surface antigen, dependence on the presence of the thymus, and in regulatory functions develop after TLI. The TL+, thymus-dependent cell suppresses the adoptive antibody response, and the TL-, thymus-independent cell suppresses the MLR

  4. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    International Nuclear Information System (INIS)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H + translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10 5 , 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ ≅ 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F 0 F 1 ATPases

  5. Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes.

    Science.gov (United States)

    Ammermann, Silke; Hillebrand, Helmut; Rhiel, Erhard

    2014-06-01

    Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5-19kDa and two additional polypeptides of 23 and 26kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Measurement of 3-0-methyldopamine in urine and plasma by a rapid and specific radioimmunoassay

    International Nuclear Information System (INIS)

    Faraj, B.A.; Camp, V.M.; Pruitt, A.W.; Isaacs, J.W.; Ali, F.M.

    1977-01-01

    Antiserum against 3-0-methyldopamine (MD) was produced in rabbits immunized with MD hapten conjugated to hemocyanin. The antiserum was used to develop a radioimmunossay (RIA) for MD. As little as 0.5 ng of MD in 0.1 ml can be detected. The major catecholamines and the phenolic aromatic amines (dopamine, norepinephrine, epinephrine, octopamine, and tyramine) and their metabolites (normetanephrine, metanephrine, homovanillic acid and 4-hydroxy-3methxymandelic acid) did not bind significantly to the antibody. The RIA of MD was used to assay the endogenous level of MD in urine and plasma of hospitalized children. In children (7 mo to 13 yr), average concentration of MD in plasma was found to be 0.47 +- 0.11 ng/ml, and in urine 0.15 +- 0.05 μg/mg of creatinine (45.0 +- 16.3 μg/24 hr). In children with neuroblastoma, there was a 3- to 10-fold increase in urinary excretion and plasma level of 3-0-methyldopamine. In adults, the average urine and plasma levels were found to be 87.4 +- 3.4 μg/24 hr and 0.6 +- 0.2 ng/ml. The diagnostic applicability of the RIA of MD is discussed

  7. Characterization of a Syrian Chickpea chlorotic stunt virus strain and production of polyclonal antibodies for its detection

    Directory of Open Access Journals (Sweden)

    Yaseen ALNAASAN

    2013-05-01

    Full Text Available Reverse transcription-polymerase chain reaction analysis with two primer sets of luteoviruses was used to characterize an isolate of Chickpea chlorotic stunt virus (CpCSv, genus Polerovirus, family Luteoviridae (SC402-08 collected from Lattakia, Syria, during the 2007‒2008 chickpea growing season. Sequence analysis revealed that the coat protein gene of the isolate shared nucleotide sequence identities ranging from 97 to 98% with the CpCSv isolates from Egypt, morocco and Syria. The capsid protein was separated as a protein of approximately 20 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis, and was visually detected by its reaction with CpCSV monoclonal antibody in Western blot. SC402-08 isolate of CpCSV was purified from faba bean-infected plants, and yielded 112–182 μg of purified virions kg-1 of infected tissue. The purified preparation was injected into a white rabbit, and an antiserum was obtained and used to detect CpCSv in infected tissues by tissue-blot immunoassay. The antiserum obtained was able to detect CpCSv by the immunoassay up to a dilution of 1:1,024,000.

  8. Evidence that lactose binding to CBP35 disrupts its interaction with CBP70 in isolated HL60 cell nuclei.

    Science.gov (United States)

    Sève, A P; Hadj-Sahraoui, Y; Felin, M; Doyennette-Moyne, M A; Aubery, M; Hubert, J

    1994-07-01

    We have previously reported that two carbohydrate-binding proteins (CBP35 and CBP70) can, under appropriate conditions of affinity chromatography, be isolated from HL60 cell nuclear extracts as a complex. Moreover, we have demonstrated that, during affinity chromatography, the CBP70-CBP35 association can be modified by the binding of lactose to CBP35. To determine whether the CBP70-CBP35 association could be disrupted in the nucleus upon lactose binding to CBP35, the behaviors of CBP70 and CBP35 were analyzed in membrane-depleted nuclei of HL60 cells, incubated with or without lactose. This study was performed by using an antiserum that cross-reacts with CBP35 and CBP70, an antiserum that was specifically raised against CBP70, and a monoclonal antibody (Mac 2) reactive against CBP35. Taken together, the results of indirect immunofluorescent staining, immunoblotting experiments, and quantitative flow-cytofluorometric analysis show that (i) CBP70 and CBP35 are present and colocalized in the nuclei incubated without lactose, (ii) all the CBP70 molecules left the nuclei incubated in the presence of lactose, while CBP35 molecules, probably bound to RNA, remained inside the nuclei, and (iii) glucose failed to have the same effect as lactose. These results strongly suggest that, in membrane-depleted nuclei, CBP35 and CBP70 interactions can be altered by a conformational change of CBP35 induced by the binding of lactose to its carbohydrate-recognition domain.

  9. Irisin inhibition of growth hormone secretion in cultured tilapia pituitary cells.

    Science.gov (United States)

    Lian, Anji; Li, Xin; Jiang, Quan

    2017-01-05

    Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, is well-documented to be a regulator of energy metabolism. At present, not much is known about its biological function in non-mammalian species. In this study, a full-length tilapia FDNC5 was cloned and its tissue expression pattern has been confirmed. Based on the sequence obtained, we produced and purified recombinant irisin which could induce uncoupling protein 1 (UCP1) gene expression in tilapia hepatocytes. Further, the rabbit polyclonal irisin antiserum was produced and its specificity was confirmed by antiserum preabsorption. In tilapia pituitary cells, irisin inhibited growth hormone (GH) gene expression and secretion and triggered rapid phosphorylation of Akt, Erk1/2, and p38 MAPK. Furthermore, irisin-inhibited GH mRNA expression could be prevented by inhibiting PI3K/Akt, MEK1/2, and p38 MAPK, respectively. Apparently, fish irisin can act directly at the pituitary level to inhibit GH transcript expression via multiple signaling pathways. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.

    Science.gov (United States)

    Wessels, M R; Muñoz, A; Kasper, D L

    1987-12-01

    We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.

  11. Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease

    International Nuclear Information System (INIS)

    van Duinen, S.G.; Castano, E.M.; Prelli, F.; Bots, G.T.A.B.; Luyendijk, W.; Frangione, B.

    1987-01-01

    Hereditary cerebral hemorrhage with amyloidosis in Dutch patients is an autosomal dominant form of vascular amyloidosis restricted to the leptomeninges and cerebral cortex. Clinically the disease is characterized by cerebral hemorrhages leading to an early death. Immunohistochemical studies of five patients revealed that the vascular amyloid deposits reacted intensely with an antiserum raised against a synthetic peptide homologous to the Alzheimer disease-related β-protein. Silver stain-positive, senile plaque-like structures were also labeled by the antiserum, yet these lesions lacked the dense amyloid cores present in typical plaques of Alzheimer disease. No neurofibrillary tangles were present. Amyloid fibrils were purified from the leptomeningeal vessels of one patient who clinically had no signs of dementia. The protein had a molecular weight of ∼ 4000 and its partial amino acid sequence to position 21 showed homology to the β-protein of Alzheimer disease and Down syndrome. These results suggest that hereditary cerebral hemorrhage with amyloidosis of Dutch origin is pathogenetically related to Alzheimer disease and support the concept that the initial amyloid deposition in this disorder occurs in the vessel walls before damaging the brain parenchyma. Thus, deposition of β-protein in brain tissue seems to be related to a spectrum of diseases involving vascular syndromes, progressive dementia, or both

  12. PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells

    Directory of Open Access Journals (Sweden)

    Amr A.A. Gamil

    2016-06-01

    Full Text Available The double-stranded RNA-activated protein kinase R (PKR is a Type I interferon (IFN stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells.

  13. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    Science.gov (United States)

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Fundamental studies on the development of C-peptide radioimmunoassay kit

    International Nuclear Information System (INIS)

    Nakazawa, Nobuhiko; Maki, Kentaro; Ogawa, Hiroshi; Ikeda, Osamu

    1976-01-01

    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 4 0 C, 48 hrs. for 1st. incubation and 4 0 C, 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.)

  15. Effect of radiation on normal hematopoiesis and on viral induced cancers of the hematopoietic system. Technical progress report, August 1, 1974--May 1, 1975

    International Nuclear Information System (INIS)

    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

  16. Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings

    Science.gov (United States)

    Ward, M. R.; Tischner, R.; Huffaker, R. C.

    1988-01-01

    Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.

  17. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-07-01

    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 lambdagt11 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/..beta..-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 /sup 125/I-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.

  18. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    International Nuclear Information System (INIS)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-01-01

    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 125 I-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

  19. Specificity of direct radioimmunoassays of unconjugated estrone and estradiol-17β in plasma

    International Nuclear Information System (INIS)

    Grenier, J.; Strauss, N.; Scholler, R.

    1977-01-01

    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.) [de

  20. Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean, Ceratomyxa shasta

    Science.gov (United States)

    Bartholomew, J.L.; Rohovec, J.S.; Fryer, J.L.

    1989-01-01

    Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.

  1. Variations in Loxosceles spider venom composition and toxicity contribute to the severity of envenomation.

    Science.gov (United States)

    de Oliveira, Kátia C; Gonçalves de Andrade, Rute M; Piazza, Roxane M F; Ferreira, Jorge M C; van den Berg, C W; Tambourgi, Denise V

    2005-03-15

    Envenomation by Loxosceles spiders causes two main clinical manifestations: cutaneous and systemic loxoscelism. The factors contributing to the severity of loxoscelism are not fully understood. We have analysed biochemical and toxicity variations in venom of L. laeta and L. intermedia, with the aim to find a correlation with the seriousness of loxoscelism. Differences in expression of proteins, glycoproteins and sphingomyelinase activity were observed between venom from male and female spiders and between venom from the two species. These differences were reflected in the toxicity of the venoms including the capacity to induce complement-dependent haemolysis, dermonecrosis and lethality. Comparative analysis of gender and species, showed that these biological activities were more prominent in venom from female spiders, especially from L. laeta. Antiserum raised against venom from females L. laeta spiders had the highest efficacy in neutralizing venoms of males and females of both species. These results indicate that the severity of loxoscelism depends, at least partially, on the species and sex of the spider and suggest that for accidents involving L. laeta an specific serum therapy is necessary. Furthermore, it emphasizes the efficacy of the antiserum produced against L. laeta female venom in neutralizing Loxosceles venoms from different species and gender.

  2. [Cloning, expression and antigenic analysis of VP1-VP4 gene encoding the structural protein of Coxsackie virus A16].

    Science.gov (United States)

    Song, Yuanbin; He, Sijie; Yu, Nan; Chen, Xinxin; Wang, Bin; Che, Xiaoyan; Zeng, Qiyi

    2012-12-01

    To clone and express VP1-VP4 genes encoding the structural proteins of Coxsackie virus A16 and analyze the antigenicity of the expressed recombinant proteins. The VP1-VP4 cDNAs were amplified with RT-PCR from the extracted viral RNA and cloned into pMD19-T vectors. The VP1-VP4 genes were inserted to the multi-cloning sites of the plasmid pQE30a, and the protein expressions in E. coli M15 were induced by IPTG. After purification by washing with 8 mol/L urea under denaturing condition, the recombinant proteins were identified by Western blotting and ELISA for their immunogenicity against rabbit antisera of Coxsackie virus A16 and enterovirus 71, respectively. The recombinant VP1-VP4 proteins were highly expressed in E. coli M15. The purified proteins could be recognized by rabbit antiserum of Coxsackie virus A16 and showed cross reactivity with the rabbit antiserum of Enterovirus 71. The recombinant Coxsackie virus A16 VP1-VP4 proteins obtained possess good antigenicity.

  3. Serodiversity of opsonic antibodies against Enterococcus faecalis--glycans of the cell wall revisited.

    Directory of Open Access Journals (Sweden)

    Christian Theilacker

    Full Text Available In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D. The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of →6-β-Galf-(1→3- β-D-Glcp-(1→ with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain and type 5 (CPS-D. Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4-3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen.

  4. Antigenic characteristics as taxonomic criterion of differentiation of Alternaria spp., pathogenic for carrot and parsley

    Directory of Open Access Journals (Sweden)

    Bulajić Aleksandra R.

    2007-01-01

    Full Text Available Identification of Alternaria genus species is a very complicated process which demands broadly designed investigations and studying of great number of properties which together can be considered as satisfying taxonomic criteria. The main objective of these investigations was examining the possibilities of applying the antigenic characteristics of Alternaria spp. phytopathogenic fungi as a taxonomic criterion, as well as introducing the serological methods for their identification. Conducting the examination of Alternaria spp., pathogenic for Apiaceae plants in Serbia, several isolates were obtained and identified as Alternaria radicina, A. petroselini, A. dauci and A. alternata, based on the conventional mycological methods and host range, as well as on molecular detection and partial characterization. The investigation included 12 isolates from plant leaves, seeds and soil which were pathogenic mainly to carrot and parsley and were identified as A. radicina, A. petroselini, A. dauci and A. alternate. Investigated isolates were compared with each other, as well as with standard isolates for the mentioned species (a total of 5 isolates, originating from USA and EU. During the investigation of serological characteristics of Alternaria spp. firstly a polyclonal antiserum was prepared against one isolate from Serbia identified as A. dauci. This antiserum was specific to Alternaria genus while there was no reaction with antigens from other phytopathogenic fungi genera (Fusarium, Rhizoctonia and Agaricus. Antiserum titer, determined by slide agglutination test, was 1/32. Antigenic characteristics of Alternaria genus fungi were examined by Electro-Blot-Immunoassay serological method (EBIA, Western blot, i.e. their protein profiles were compared. Investigated Alternaria spp. isolates showed different protein band profiles in gel and on nitrocellulose paper, and the observed differences were in complete correlation with the results of the previous

  5. 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

  6. Solid phase radioimmunoassay for plasma testosterone using a plastic microtiter tray

    International Nuclear Information System (INIS)

    Hosogi, Hidemi

    1975-01-01

    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.)

  7. The radioimmunoassay of plasma oxytocin in pregnancy and at parturition

    International Nuclear Information System (INIS)

    Kogure, Satohisa

    1976-01-01

    The titer of the antiserum obtained by inoculating oxytocin-bovine serum albumin antigen into mature male rabbits was 1:64,000 in the final dilution, and the rate of conjugation with labeled-oxytocin was 34%. The cross reaction rate of the antiserum was 0.01% or below, both for lysine vasopressin and arginine vasopressin. The sensitivity of the radioimmunoassay using this antiserum was 5μIU. When a known quantity of oxytocin was added to plasma for measurement, the mean recovery rate was about 93%. The blood oxytocin concentration was 6.1+-2.5μIU/ml (ml omitted hereafter) in the early stage of pregnancy, 12.5+-6.0μIU in the middle stage, and 27.0+-7.5μIU in the terminal stage. The oxytocin concentration in the maternal blood was 34.1+-4.9μIU in the second stage, the concentration in the umbilical arterial blood immediately after delivery 3.5+-5.3μIU, and that in the umbilical venous blood 30.0+-4.2μIU. In cases of caesarean section not in labor, the oxytocin concentration in the maternal blood was 27.1+-6.6μIU, that in the umbilical arterial blood 25.1+-5.4μIU, and that in the umbilical venous blood 25.4+-5.4μIU. In cases of caesarean section in labor, the oxytocin concentration in the maternal blood was 37.1+-7.1μIU, that in the umbilical arterial blood 31.4+-6.7μIU, and that in the umbilical venous blood 27.0+-7.8μIU. The half-life of the oxytocin in the peripheral blood in cases injected with oxytocin was 5-10 minutes. When prostaglandin F2α was administered intravenously, the oxytocin concentration in the peripheral blood was increased in cases in labor. (Chiba, N.)

  8. Functional cell mediated lympholysis I. Description of the assay

    International Nuclear Information System (INIS)

    Goeken, N.E.; Thompson, J.S.

    1981-01-01

    The anamnestic response by human bi-directional (BD) mixed lymphocyte cultures (MLC) to restimulation by cells of the original stimulating type is generally strikingly reduced as compared to that of standard one-way cultures. This difference was shown not to be related to a change in kinetics nor was it due to exhaustion of the media or soluble factors since fresh media did not ameliorate the effect nor were supernatants from BD cultures found to be suppressive. The relative inhibition was also not reversed by removal of the allogeneic cells by phenotype specific antiserum. Cytotoxic tests with donor and responder specific antisera revealed that the cells bearing that phenotype were dramatically reduced in BD as compared to one-way cultures. Thus, the diminished secondary response appears to be due to cytotoxic elimination of the responder cells. This allogeneic cytotoxicity is dependent on non-T, phagocytic, adherent cells. The phenomenon is called Functional Cell Mediated Lympholysis (F-CML). (author)

  9. Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

    International Nuclear Information System (INIS)

    Azarkh, Eugene; Robinson, Erin; Hirunkanokpun, Supanee; Afanasiev, Boris; Kittayapong, Pattamaporn; Carlson, Jonathan; Corsini, Joe

    2008-01-01

    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

  10. A radioimmunoassay for wheat gliadin to assess the suitability of gluten free foods for patients with coeliac disease

    International Nuclear Information System (INIS)

    Ciclitira, P.J.; Ellis, H.J.; Evans, D.J.; Lennox, E.S.

    1985-01-01

    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 1.9x10 -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. (author)

  11. Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column.

    Science.gov (United States)

    Wu, Zhihua; Li, Kun; Zhan, Shaode; Tong, Ping; Li, Xin; Yang, Anshu; Chen, Hongbing

    2017-09-14

    Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits' antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.

  12. Gamma radiation effects on crotoxin (toxin of crotalus durissus terrificus venom)

    International Nuclear Information System (INIS)

    Souza Filho, J.N.; Rogero, J.R.

    1988-01-01

    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 10 3 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) [pt

  13. Beta-endorphin radioimmunoassay: specificity studies

    Energy Technology Data Exchange (ETDEWEB)

    Colas-Linhart, N.; Perdrisot, R.; Petiet, A.; Bok, B.

    1986-01-01

    This note describes the technical details of a cerebrospinal fluid (CSF) B-Endorphin (B-End) radioimmunoassay (RIA). We used an antiserum raised in rabbits against human B-End which cross-reacs 100% with B-Lipotrophin (B-Lph). Thus, filtration chromatography is used to separate both peptides. The assay is sensitive (limit detection=14 pmoles/l), reproducible (the intra and inter assay coefficients of variation are 5 and 6% respectively). Specificity studies are performed to verify the cross-reactions with other opioid peptides and the non specific reactions with the biological fluid (CSF). In order to evaluate the effects of iodine-containing contrast media on the RIA, additional standard curves were analyzed in the presence of varying concentrations of two contrast materials.

  14. Radioimmunoassay of Pro-. gamma. -melanotropin, the amino-terminal fragment of proopiolipomelanocortin. [Swine

    Energy Technology Data Exchange (ETDEWEB)

    Ekman, R.; Hakanson, R.; Larsson, I.; Sundler, F.; Thorell, J.I.

    1982-08-01

    A RIA has been developed for natural porcine pro-..gamma..-MSH, the 103-amino acid peptide that represents the amino-terminal part of proopiolipomelanocortin. Rabbits were immunized with the purified peptide polymerized with glutaraldehyde. The antiserum is directed against the amino-terminial end of the antigen and does not cross-react with corticotropin, ..beta..-lipotropin, ..beta..-endorphin, ..gamma../sub 3/MSH, or ..gamma../sub 2/MSH. The minimum detectable concentration is 0.15 ng/ml standard pro-..gamma..MSH (15 pg/tube). Pro-..gamma..MSH-like immunoreactivity was detected in plasma and extracts of the hypothalamus and pituitary of pigs. Gel chromatography of these extracts revealed at least three immunoreactive peaks in the anterior and neurointermediate lobes of the pituitary, wheras two immunoreactive peaks were found in extracts of the hypothalalmus. (Endocrinology 111:578,1982)

  15. Development and evaluation of a radioimmunoassay for the detection of amphetamine and related compounds in biological fluids

    International Nuclear Information System (INIS)

    Mason, P.A.; Bal, T.S.; Law, B.; Moffat, A.C.

    1983-01-01

    A radioimmunoassay has been developed for the detection of amphetamine and its analogues in blood and urine without any pretreatment of the samples. It is based on a commercially available antiserum and a [ 125 I] iodinated derivative of amphetamine. The assay can detect low levels of amphetamine (less than 10 ng ml - 1 ) in very small samples (50 μl) of blood and urine. It is cheap (3 pence per test), rapid, simple to perform and is specific for compounds closely related to amphetamine. A high, positive correlation was obtained (r = 0.93) when results of the analyses of urine samples from volunteers who had ingested amphetamine were compared with those produced by gas chromatography - mass spectrometry. The assay has proved very useful for the detection of amphetamine and closely related compounds in biological fluids. (author)

  16. Radioimmunoassay of bovine growth hormone

    International Nuclear Information System (INIS)

    Dvorak, P.; Becka, S.; Krejci, P.; Chrpova, M.

    1978-01-01

    A radioimmunological method for quantitative determination of bovine growth hormone in blood plasma or serum for routine use was worked out. The antibody is also suitable for radioimmunoassay of bovine growth hormone. The chloramin T and lactoperoxidase methods were used. Standard b-STH isolated by one of the authors was labelled with I-125(Na 125 I, carrier free, Amersham). The I-125-hormone was separated on Sephadex G-50 or G-100 and stored in 0.05M phosphate pH 7.5 with 1% of human serum albumine at 25 deg C. The method was checked on 2500 samples. Rabbit antiserum to b-STH was prepared in sufficient amount to serve also for other laboratories interested in the method. (author)

  17. Homologous radioimmunoassay of human prolactin

    International Nuclear Information System (INIS)

    Felixberger, F.X.

    1980-01-01

    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.) [de

  18. 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...... was compared in naturally and experimentally (intraperitoneal injections) infected fry by use of immunohistochemistry. This study showed that F. psychrophilus could be detected in paraffin-wax-embedded tissue of rainbow trout fry by immunohistochemistry. The principal immunohistochemical findings in naturally...... and experimentally infected fry showed that there was a localization of bacteria in the monocyte-macrophage system, in skin lesions, and in the retina and the choroid gland of the eye. The dermal changes included superficial or deep ulcers extending to the subcutaneous tissue or the musculature accompanied...

  19. A novel pestivirus associated with deaths in Pyrenean chamois (Rupicapra pyrenaica pyrenaica).

    Science.gov (United States)

    Arnal, MaríaCruz; Fernández-de-Luco, Daniel; Riba, Landry; Maley, Maddy; Gilray, Janice; Willoughby, Kim; Vilcek, Stefan; Nettleton, Peter F

    2004-12-01

    During investigations into recent population decreases in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) 21 animals found dead or dying were necropsied. Immunohistochemistry revealed the presence of a pestivirus in organs from two of the 21 chamois. From one of these animals a pestivirus was isolated from the spleen, skin and serum. The virus had better growth in ovine than in bovine cells and was neutralized most effectively by an anti-border disease virus (BDV) reference antiserum. Using panpestivirus and genotype-specific primers selected from 5'-untranslated region (UTR) of the pestivirus genome, BDV RNA was demonstrated by RT-PCR. Comparison of the chamois sequences from 5'-UTR, entire N(pro) and E2 gene coding regions with those of other pestivirus genotypes revealed that this virus did not fall into any of the pestivirus genotypes identified so far. Results of phylogenetic analysis suggested that the chamois pestivirus was closely related to BDV and it was typed as BDV-4 genotype.

  20. 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.

  1. Radioimmunoassay of erythropoietin

    International Nuclear Information System (INIS)

    Goldwasser, E.; Sherwood, J.B.

    1981-01-01

    A brief review of the historical development of the radioimmunoassay (RIA) for serum erythropoietin is given. It has been shown that there is reasonable agreement between the results obtained by RIA and those obtained by the previously used bioassay. By RIA, a mean normal serum titre of 18 mu/ml erythropoietin has been determined in a study of 445 individuals. Serum erythropoietin results for patients with polycythaemia vera have not been shown to be significantly different from normal values but in patients with secondary polycythaemia, serum titres averaging 94 mu/ml have been found. The predicted physiological changes in erythropoietin titre have also been demonstrated in humans using the RIA; increasing after bleeding and decreasing after red cell administration. Studies of erythropoietin levels in experimental animals have shown that, with the particular antiserum used, the sensitivity of the RIA is markedly reduced. (U.K.)

  2. Homologous radioimmunoassay for human prolactin

    International Nuclear Information System (INIS)

    Reuter, A.M.; Kennes, F.; Gevaert, Y.; Franchimont, P.

    1976-01-01

    Although thee are descriptions of a range of radioimmunoassays for human prolactin in various biological fluids, only one of these is an homologous assay using human prolactin as the reference standard and tracer as well and an anti-human prolactin antiserum (Sinha, Y.N., Selby, F.W.; Lewis, U.; and Vanderlaan, W.P., 1973, J. Clin. Endocr., Vol. 36, 509). A homologous radioimmunoassay using human putuitary prolactin has been developed. The separation method is based on the double antibody solid phase system. Cross reactivity with human growth hormone (GH), placental lactogen (HPL), the pituitary protein hormones and prolactins of various species were studied as were values found in normal subjects in basal conditions and after a TRH injection. (author)

  3. Equine herpes virus 1 (EHV-1) in liver, spleen, and lung as demonstrated by immunohistology and electron microscopy.

    Science.gov (United States)

    Jönsson, L; Beck-Friis, J; Renström, L H; Nikkilä, T; Thebo, P; Sundquist, B

    1989-01-01

    Ten aborted foals, diagnosed as infected with Equine Herpes Virus 1 (EHV-1) on histopathological criteria, were examined for the presence of EHV-1 using immunohistology as the investigative instrument. The primary reagent was an antiserum specific for viral envelope glycoproteins. Immunohistology localised EHV-1 to areas of liver necrosis and to the cytoplasm of infected Kupffer cells and hepatocytes. Cytoplasmic immunolabelling was also prominent in reticular cells of the red pulp of the spleen and in intact and degenerated bronchiolar epithelium. Cytoplasmic immunolabelling was seen in morphologically unchanged cells and in cells containing intranuclear inclusion bodies. Three aborted foetuses with no histological signs of EHV-1 infection were negative when immunostained for EHV-1. Detection by electron microscopy of EHV-1 virions confirmed the EHV-1 specificity of the immunolabelling procedure.

  4. Serologic test-systems development: immunoassays for antibiotics. Progress report, May 16, 1980-September 30, 1981

    Energy Technology Data Exchange (ETDEWEB)

    Brake, R.; Hollstein, U.; Hindman, K.

    1983-04-01

    Progress on development of immunoassays for the antibiotics gentamicin, tetracycline, and tylosin is discussed. The development of the gentamicin assay was completed and the assay was transferred to the Beltsville Laboratories of the US Department of Agriculture (USDA) Food Safety and Quality Service (FSQS). The sensitivity (1 ppB) is 50-fold greater than we have previously reported, and is satisfactory for the current needs. At year's end, work was still in progress on both the tetracycline and tylosin assays. Tetracycline presents a more difficult problem of chemistry than we had anticipated. Immunoassay reagents synthesized by diazonium coupling were only weakly immunoreactive; analysis suggests that this coupling procedure damages the tetracycline structure. Several tetracycline derivatives that offer promising alternative coupling procedures were synthesized. Tylosin was successfully coupled to peroxidase. With this conjugate and antiserum, obtained from R. Mageau of the USDA, some immune-specific binding was demonstrated. Several problems with the tylosin assay remain to be resolved.

  5. 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...

  6. Cow's milk with active immunoglobulins against Campylobacter jejuni: effects of temperature on immunoglobulin activity.

    Science.gov (United States)

    Riera, Francisco; Alvarez, Alejandro; Espi, Alberto; Prieto, Miguel; de la Roza, Begoña; Vicente, Fernando

    2014-04-01

    Adult Holstein cows were injected with an antiserum against Campylobacter jejuni and immunoglobulin activities in vitro were determined in blood and milk several weeks after injection. The immunoactivity of immunoglobulins in milk was measured by an ELISA after different temperature-time treatments (60-91°C and 4-3600 s) at laboratory and pilot-plant scales. Kinetic and thermodynamic parameters were determined. An increase in immunoglobulin activity in milk was detected several days after injection. Optical densities increased by three- to seven-fold in this period. The activity started to decay 4-5 weeks after injection. Immunoglobulins maintained most of their in vitro activity under pasteurisation conditions (72°C and 15 s) and were denatured following first-order kinetics. The injection protocol applied allows milk with specific immunoglobulins against Campylobacter jejuni to be obtained. Traditional pasteurisation did not reduce this activity. © 2013 Society of Chemical Industry.

  7. Mapping of antigenic sites in human neuron-specific enolase by expression subcloning.

    Science.gov (United States)

    Quinn, G B; Reeves, I G; Day, I N

    1994-05-01

    Human serum neuron-specific enolase (NSE) is a marker of neurons and of small-cell carcinoma of the lung; improved immunoassays of NSE remain an important goal. Here, we used overlapping complementary DNA (cDNA) clones for reconstruction to express full-length recombinant NSE, and also to express a set of cloned subfragments through the prokaryotic expression vectors pUEX and pUBEX. Subfragments expressed as fusion proteins were used to characterize immunogenic and antigenic regions and epitopes and, expressed as affinity matrices, to derive purified, fractionated polyclonal antibodies. NSE epitope data can be visualized with yeast enolase-1 crystal structure coordinates: The two protein sequences align almost perfectly and are 61% identical. This approach demonstrates the complementarity of cDNA expression with techniques of polyclonal antiserum and monoclonal antibody production and with chemical peptide synthesis in the refinement of immunodiagnostic reagents.

  8. Immunodiffusion Studies of Purified Equine Infectious Anemia Virus

    Science.gov (United States)

    Nakajima, Hideo; Ushimi, Chuzo

    1971-01-01

    Antigenicity of purified equine infectious anemia (EIA) virus was examined by immunodiffusion against sera obtained from horses experimentally infected with EIA virus. The purified virus reacted with the infected horse serum, and virus-specific precipitating antibody was demonstrated. Furthermore, it was found that purified EIA virus reacted against the serum of horses infected with all strains of EIA virus which were antigenically different from one another. From the result, group-specific components of the virus rather than strain-specific ones were considered to be involved in the reaction. Serological reactivity was lost by adding antiserum from the infected horse to the antigen. The precipitating antibody usually appeared in the serum 1 to 2 weeks after the first febrile attack of EIA and remained for a longer period. Some characteristics of the purified antigen and specificity of the reaction for EIA are described. Images PMID:16557982

  9. Effects of gamma radiation on crotoxin (toxin of Crotalus durissus terrificus venom)

    International Nuclear Information System (INIS)

    Souza Filho, J.N.; Rogero, J.R.

    1988-07-01

    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 60 Co GAMMACELL in the concentration of 2 mg/ml 0.85% NaCl with dose rate of 1.19 x 10 3 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) [pt

  10. 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...... 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...... 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...

  11. Easy and reliable radioimmunoassay of serum androstenedione: age-related normal values in 252 females aged 2 to 70 years

    Energy Technology Data Exchange (ETDEWEB)

    Hummer, L.; Nielsen, M.D.; Christiansen, C. (Glostrup Hospital (Denmark))

    1983-06-01

    A simple, non-chromatographic radioimmunoassay for the measurement of androstenedione (A-dione) in serum and based on a commercially available antiserum is described. The antibody-bound fraction was separated from the free fraction by a gel-centrifugation procedure. The method was used to establish reference values for normal females, based on determination in 252 normal females from 2 to 70 years of age. The data support the statement that the decline with age after puberty in the overall production of A-dione is mainly caused by a reduction in the adreno-derived A-dione, since the mean concentration of A-dione in age-matched pre- and postmenopausal women was very similar.

  12. Small-scale extraction and radioiodination of human hormones for the substitution of imported radioimmunoassay reagents

    International Nuclear Information System (INIS)

    Gimbo, E.K.; Ribela, M.T.C.P.; Borghi, V.C.; Schwarz, I.; Morganti, L.; Araujo, E.A.; Bartolini, P.

    1988-01-01

    The methods for national production of radioimmunoassay reagents to substitute imported kits of: highly purified unlabelled hormones for radioiodination; 125 I-labelled hormones; and specific high titre antisera are presented. The extraction and purification of human growth hormone (hGH) and human luteinizing hormone (hGH) were done from human pituitaries. The 125 I-labelled hormones are obtained by stoichiometric methods. The 125 I-hGH, 125 I-hLH, I-hTSH and 125 I- h calcitonin were prepared and tested in internal and external quality control, in comparison with imported products. The parameters such as: maximum binding to specific antiserum (Bo), nonspecific binding (NSB), mean effective dose (ED 50), sensitivity and accuracy were evaluated. (M.C.K.) [pt

  13. Zea mI, the maize homolog of the allergen-encoding Lol pI gene of rye grass.

    Science.gov (United States)

    Broadwater, A H; Rubinstein, A L; Chay, C H; Klapper, D G; Bedinger, P A

    1993-09-15

    Sequence analysis of a pollen-specific cDNA from maize has identified a homolog (Zea mI) of the gene (Lol pI) encoding the major allergen of rye-grass pollen. The protein encoded by the partial cDNA sequence is 59.3% identical and 72.7% similar to the comparable region of the reported amino acid sequence of Lol pIA. Southern analysis indicates that this cDNA represents a member of a small multigene family in maize. Northern analysis shows expression only in pollen, not in vegetative or female floral tissues. The timing of expression is developmentally regulated, occurring at a low level prior to the first pollen mitosis and at a high level after this postmeiotic division. Western analysis detects a protein in maize pollen lysates using polyclonal antiserum and monoclonal antibodies directed against purified Lolium perenne allergen.

  14. Studies on a radioimmunoassay for human erythopoietin.

    Science.gov (United States)

    Lertora, J J; Dargon, P A; Rege, A B; Fisher, J W

    1975-07-01

    A highly purified erythropoietin (ESF) preparation (12,000 units per milligram of protein) was labeled with Na'125I using the Chloramine-T method. Undamaged immunoreative labeled ESF was separated from the damaged, nonimmunologically receiveESF by Sephadex G-150 fractionation. This undamaged immunreactive ESF was usedin radioimmunoassay for human erythropoietin. Separation of bound from free antigen was acheived using the double-antibody technique. Approximately 55 per cent binding wasobserved at an antiserum dilution of 1:1500. This assay appears to be sensitive enough to detect as little as 0.025 milliunits of the International Reference Preparation erythropoietin. The estimated levels of thid hormone in normal and anemic uremic human subjects suggests that immunoreactive serum erythropoietin levels are elevated above normal in anemia of uremia.

  15. 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

    Science.gov (United States)

    Afonso, Margarete Martins dos Santos; Duarte, Rosemere; Miranda, José Carlos; Caranha, Lindenbergh; Rangel, Elizabeth Ferreira

    2012-01-01

    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. PMID:22315621

  16. Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. 1

    Science.gov (United States)

    Sánchez, Federico; Campos, Francisco; Padilla, Jaime; Bonneville, Jean-Marc; Enríquez, Consuelo; Caput, Daniel

    1987-01-01

    Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665575

  17. Hypothalamic CART is a new anorectic peptide regulated by leptin.

    Science.gov (United States)

    Kristensen, P; Judge, M E; Thim, L; Ribel, U; Christjansen, K N; Wulff, B S; Clausen, J T; Jensen, P B; Madsen, O D; Vrang, N; Larsen, P J; Hastrup, S

    1998-05-07

    The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

  18. Plasma levels of triamcinolone acetonide as determined by radioimmunoassay after topical application

    International Nuclear Information System (INIS)

    In this paper a method for the radioimmunological determination of triamcinolone acetonide (TAAc) in plasma is described. The antiserum used in the radioimmunological determinations showed high specifity for TAAc; the minimal detectable amount was 0.2 ng. The course of the plasma concentrationtime curve of TAAc after intravenous injection into rats was analysed. The concentration of TAAc in the blood of the patients after topical application of the drug is very low. No detectable amount of TAAc was found in the blood of out-patients. Hospitalized patients treated with TAAc under plastic occlusion showed plasma levels ranging from 0.8 to 3.4 ng/ml. This level remained constant throughout the treatment. (orig.) 891 AJ [de

  19. A four-step sandwich radioimmunoassay for direct selection of monoclonal antibodies to allergen molecules

    International Nuclear Information System (INIS)

    Ley, V.; Corbi, A.L.; Sanchez-Madrid, F.; Carreira, J.C.

    1985-01-01

    A 4-step radioimmunoassay has been devised for direct identification of monoclonal antibodies (MAb) directed to IgE-binding molecules. Polyvinyl chloride wells coated with purified anti-mouse kappa chain MAb (187-1) were successively incubated with: (1) MAb-containing hybridoma supernatants, (2) allergen extract, (3) allergic patients' serum pool, and (4) 125 I-labeled anti-human IgE antiserum, to detect MAb-allergen-IgE complexes. MAb to allergens from Parietaria judaica pollen and Dermatophagoides mites have been selected with this screening procedure. The affinity-purified allergen molecules competed the binding of IgE to allergen extracts coated to paper discs in a RAST inhibition assay, confirming the anti-allergen specificity of the selected MAb. This screening method is sensitive enough to allow detection of MAb directed to poorly represented allergens. (Auth.)

  20. Spontaneous squamous cell carcinoma of the tongue and multiple bronchioloalveolar carcinomas in a Virginia opossum (Didelphis virginiana).

    Science.gov (United States)

    Kim, D Y; Mitchell, M A; De las Heras, M; Taylor, H W; Cho, D-Y

    2002-01-01

    Two primary tumours, squamous cell carcinoma of the tongue and multiple bronchioloalveolar carcinomas, were diagnosed in a Virginia opossum (Didelphis virginiana). Two oral masses were located in the right ventrolateral surface of the tongue, near the frenulum, and the lungs contained multiple, widely distributed, nodular masses. Microscopically, the oral masses were composed of invasive cords of pleomorphic, polyhedral cells, typical of squamous cells. The multiple pulmonary masses consisted of non-ciliated, cuboidal, columnar, or occasionally polyhedral cells arranged in an alveolar pattern with multifocal areas of necrosis. This is the first report of spontaneous oropharyngeal squamous cell carcinoma in the Virginia opossum. However, multiple pulmonary adenomas have been reported previously in this species, the lesions being similar to those in sheep pulmonary adenomatosis (jaagsiekte). In the present study, immunohistochemical examination of the pulmonary tumours with a rabbit polyclonal antiserum to jaagsiekte retroviral capsid protein proved negative. Copyright Harcourt Publishers Ltd.

  1. Identification of Naegleria fowleri in fresh isolates of environmental amoebae using a staphylococcal coagglutination technique.

    Science.gov (United States)

    Kilvington, S; White, D G

    1986-01-01

    A coagglutination test using Staphylococcus aureus Cowan strain 1 sensitized with specific antiserum successfully identified Naegleria fowleri in axenic and monoxenic culture by detection of a heat stable antigen released into the culture medium during growth. Strong coagglutination, visible to the naked eye, occurred within 30 sec when the sensitized reagent was mixed on a slide with medium from N. fowleri cultures. N. lovaniensis gave a weak cross-reaction which developed over one min clearly distinguishable from that of N.fowleri.N.gruberi and N. australiensis gave no reaction. The technique is proposed as part of a rapid and economical scheme for detecting N. fowleri in environmental samples very soon after primary isolation, with distinct advantages over conventional methods. Further development of the technique to detect other Naegleria spp. and the early diagnosis of primary amoebic meningoencephalitis are suggested as future applications.

  2. Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

    International Nuclear Information System (INIS)

    Yatani, Ryuichi; Tohgo, Yukihiro; Kunishima, Nobuyoshi.

    1975-01-01

    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.)

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

    DEFF Research Database (Denmark)

    Couchman, J R

    1987-01-01

    A heparan sulfate proteoglycan (HSPG) synthesized by murine parietal yolk sac (PYS-2) cells has been characterized and purified from culture supernatants. A monospecific polyclonal antiserum was raised against it which showed activity against the HSPG core protein and basement membrane specificity...... for multiple HSPGs was derived from the finding that skeletal neuromuscular junction and liver epithelia also did not contain this type of HSPG, though previous reports have indicated the presence of HSPGs at these sites. The PYS-2 HSPG was shown to be antigenically related to the large, low 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 represents...

  4. A potential role for tetranectin in mineralization during osteogenesis

    DEFF Research Database (Denmark)

    Wewer, U M; Ibaraki, K; Schjørring, P

    1994-01-01

    to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis...... 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 cells contained...... approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no insert (Mann Whitney rank sum test, p

  5. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    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 G s 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 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  6. INFLUENCE OF COMPOSITION OF POLYUNSATURATED FATTY ACIDS ON MICROVISCOSITY OF THE MEMBRANE OF ERYTHROCYTES OF THE NAVEL OF THE BLOOD AT HERPES INFECTION CONTAMINATIONS

    Directory of Open Access Journals (Sweden)

    N. A. Isutina

    2013-01-01

    Full Text Available The method of gas-liquid chromatography investigates composition of polyunsaturated fatty acids of membrane of erythrocytes, discharged of navel bloods neonatal from mothers who have transferred in the season gestation exacerbation herpes of an infection contamination and his influence on microviscosity of membrane. Essential infringements of data exchange of bonds in navel bloods neonatal with an exacerbation  herpes  infection  contaminations  (antiserum  capacity  IgG  to  virus  of  simple  herpes  of  1  type 1 : 12 800 which show deficiency essential ω-3 acids at simultaneous augmentation of the precursor proinflammatory eicosanoid ω-6 arachidonic acids, promoting augmentation of relative microviscosity of membrane of erythrocytes that will be one of probable causes of development of hypoxia are found.

  7. A potential role for tetranectin in mineralization during osteogenesis

    DEFF Research Database (Denmark)

    Wewer, U M; Ibaraki, K; Schjørring, P

    1994-01-01

    approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no insert (Mann Whitney rank sum test, p role during...... to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis...... 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 cells contained...

  8. Radio-immunological method for the determination of the thyroid function by an in vitro experiment in blood serum

    International Nuclear Information System (INIS)

    1977-01-01

    The author describes a procedure for the determination of the thyroid function by an in vitro experiment in blood serum whereby one mixes a blood serum sample with a solution of radioactive thyroxine so that a combination of free thyroxine from thyroxine combined with globulin is produced; after the mixture has been buffered, an antiserum is applied which contains an antibody for the immuno-reaction with thyroxine, and that finally the non-bonded thyroxine separates and the antibody bonded thyroxine forms a relative quantity for the measurement of the radioactivity, characterised in that for the buffering one applies only a sample of the blood serum under investigation in a controlled quantity which is smaller than the initial serum sample. The separation may also be carried out by the aid of the adsorption charcoal, polyethylene glycol, an ion exchanger, a second antibody, ethanol or ammonium sulphate; or carried out over a Sephadex column. (G.C.)

  9. Radioimmunoassay methods for the determination of L-triiodo-thyronine and thyroxine

    International Nuclear Information System (INIS)

    1976-01-01

    An improved, simplified radioimmunoassay method for the in vitro determination of L-triiodo-thyronine in unextracted blood serum is described which involves the use of a combination reagent constituted by a buffered solution containing radioactive L-triiodothyronine and an inhibitor for inhibiting the binding of L-triiodothyronine to thyroxine-binding globulin. Optionally the reagent may also include an antiserum containing an antibody capable of immunoreactivity with L-triiodothyronine. Packaged test kits for use in conveniently carrying out the radioimmunoassay are also provided. Certain salts of 8-anilino-1-naphtalene sulfonic acid, which may be regarded as purified forms of the acid, are preferably employed as inhibitors for inhibiting binding of L-triiodothyronine to thyroxine-binding globulin

  10. Rapid identification of mycoplasmas by indirect immunoperoxidase test using small square filter paper.

    Science.gov (United States)

    Imada, Y; Uchida, I; Hashimoto, K

    1987-01-01

    The indirect immunoperoxidase test using small, square filter paper was used for rapid identification of mycoplasmas. Colonies of type strains of 22 mycoplasma species, 3 acholeplasma species, and three Ureaplasma diversum serogroups were stained by this test with high sensitivity and specificity. All of 49 isolates from bovine materials and cell cultures were easily identified by this test, and the results agreed with those obtained by growth inhibition test. Use of filter paper made it possible to add different kinds of antisera or conjugates to the same agar plate simultaneously and also to save antiserum and conjugate. This test proved to be a simple and useful technique for rapid identification of many mycoplasma species grown on agar medium.

  11. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    Science.gov (United States)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  12. Immunostimulatory mouse granuloma protein.

    Science.gov (United States)

    Fontan, E; Fauve, R M; Hevin, B; Jusforgues, H

    1983-10-01

    Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.

  13. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  14. Radioimmunological determination of parathyroid hormone

    International Nuclear Information System (INIS)

    Dudczak, R.; Waldhaeusl, E.; Wolner, E.; Czembirek, H.

    1979-01-01

    Angiographic or scintigraphic localization of parathyroid adenomas is successful in only a small number of patients with hypercalcaemic hyperparathyroidism. This report is concerned with the preoperative localization of parathyroid adenomas by regional catheterization of the thyroid veins in patients with hypercalcaemic hyperparathyroidism ( n = 7). PTH was determined radioimmunologically, using an antiserum specific for PTHsub(1-84) and the carboxyl-terminal fragment of the hormone. Determination of regional differences in the plasma concentration of PTH permitted the preoperative localization of PTH secreting adenomas. The preoperative localization of parathyroid adenomas was confirmed in each instance by surgery. Thus, the regional determination of plasma PTH represents a tool to improve the preoperative localization of parathyroid adenomas in patients with hypercalcaemic hyperparathyroidism and, hence, to reduce the risk of an unsuccessful operation. (author)

  15. Quantitative determination of urinary tetrahydroaldosterone by a radioimmunological method. Contribution to the study of its excretion

    International Nuclear Information System (INIS)

    Carayon, Alain.

    1978-06-01

    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 [fr

  16. Actinomyces israelii in osteoradionecrosis of the jaws. Histopathologic and immunocytochemical study of five cases

    International Nuclear Information System (INIS)

    Happonen, R.P.; Viander, M.; Pelliniemi, L.; Aitasalo, K.

    1983-01-01

    Five surgically treated patients with osteoradionecrosis of the jaws are presented. The clinical history of the disease varied from 3 to 17 years. In three cases the progression of the disease was enhanced by surgical procedures performed in the irradiated area causing exfoliation of the premaxillary area in one case and spontaneous mandibular fracture in two cases. Actinomyces israelii was demonstrated in tissue sections of all five cases by using FITC-labeled specific antiserum and additionally with peroxidase-antiperoxidase method in one case. Candida was found in histologic sections of three cases. Radiation damage in the oral soft tissues and jawbones makes the atmosphere favorable for anaerobic microorganisms. The present results indicate that the role of A. israelii in the pathogenesis of osteoradionecrosis of the jaws has not been fully appreciated

  17. Correlation of pancreatic secretion and plasma cholecystokinin concentrations after various stimuli in dogs

    International Nuclear Information System (INIS)

    Dale, W.E.

    1986-01-01

    The objectives of this study were to develop a sensitive, specific, and reliable radioimmunoassay for plasma cholecystokinin and to use the cholecystokinin radioimmunoassay (CCK RIA) to determine the role of CCK in stimulating exocrine pancreatic secretion. Three rabbits were immunized with synthetic sulfated CCK-10 conjugated to keyholed limpet hemocyanin. Using 125 I-CCK-39 (iodo-gen) as tracer, it was found that antiserum R016 (titer = 1:256,000) recognized short and long forms of CCK and had negligible cross-reactivity with gastrin and other peptides. Plasma CCK levels and pancreatic secretion in fasted dogs (N = 6) were measured in response to a meat meal, intestinal perfusion with saline, graded amounts of sodium oleate (+/-atropine), a mixture of tryptophan/phenylalanine (Trp/Phe), or HCl, and i.v. infusion of graded doses of CCK-8 or CCK-39

  18. Radioimmunoassay of anabolic steroids

    International Nuclear Information System (INIS)

    Hampl, R.; Stranska, I.; Starka, L.; Picha, J.; Chundela, B.

    1978-01-01

    Alternative antisera against 17 α-methyltestosterone and 19-nortestosterone were raised and used for radioimmunoassay of anabolic steroids. Tritiated compounds were used as radioligands. The RIA method suitable for doping control is proposed for 17 α-alkylated anabolic steroids in both plasma and urine, using qoat antiserum against methyltestosterone-3-carboxymethyloxime-BSA. Sensitivity of the method was expressed as least amount of nonradioactive methandienone which, when added to normal urine or plasma, caused statistically significant decrease of measured supernatant radioactivity at 99% level. The amounts from 50 to 500 pg were tested, each in eight parallel determinations. The amounts of 100 pg for plasma and 200 pg for urine met these criteria. The respective coefficients of variation did not depend on the amount of steroid added in this range. They averaged 4.60% for plasma and 4.95% for urine, respectively. (T.I.)

  19. 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.

  20. Measurement of plasma canine C peptide by radioimmunoassay

    International Nuclear Information System (INIS)

    Besch, W.; Woltanski, K.P.; Fischer, U.; Kohnert, K.D.; Ziegler, M.

    1985-01-01

    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. Production of Anti-triiodothyronine sulfate antibody for radioimmunoassay applications

    International Nuclear Information System (INIS)

    Elbanna, I.M.; Ragab, M.T.

    2000-01-01

    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. Isolation of the etiological agent of primary amoebic meningoencephalitis from artificially heated waters

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, A.R.; Tyndall, R.L.; Coutant, C.C.; Willaert, E.

    1977-12-01

    To determine whether artificial heating of water by power plant discharges facilitates proliferation of the pathogenic free-living amoebae that cause primary amoebic meningoencephalitis, water samples (250 ml) were taken from discharges within 3,000 feet (ca. 914.4 m) of power plants and were processed for amoeba culture. Pathogenic Naegleria fowleri grew out of water samples from two of five lakes and rivers in Florida and from one of eight man-made lakes in Texas. Pathogenic N. fowleri did not grow from water samples taken from cooling towers and control lakes, the latter of which had no associated power plants. The identification of N. fowleri was confirmed by pathogenicity in mice and by indirect immunofluorescence analyses, by using a specific antiserum.

  3. Analysis of an immunodominant epitope of topoisomerase I in patients with systemic sclerosis.

    Science.gov (United States)

    Meesters, T M; Hoet, M; van den Hoogen, F H; Verheijen, R; Habets, W J; van Venrooij, W J

    1992-05-01

    In this paper an immunodominant epitope of Topoisomerase I is described. An epitope expression sublibrary was constructed from Topoisomerase I cDNA. The subclones were screened with an antiserum from a patient with systemic sclerosis (SSc). The positive clones defined one immunodominant B cell epitope (epitope III), which was located at the carboxyterminal part of the protein. The epitope, 52 amino acids in length, neither contains the p30gag sequence nor the suggested active site Tyr-723, both presumed antibody recognition sites. More than 70% of our anti-TopoI sera recognize this epitope III, indicating that it is a major recognition site of the anti-TopoI autoantibodies in SSc sera. DNA relaxation experiments show that all sera that recognize epitope III and most sera with antibodies to other epitopes inhibit Topoisomerase I activity.

  4. The application of immunoradiometric assay for rapid identification of Xanthomonas campestris pv. oryzicola in diseased rice seeds

    International Nuclear Information System (INIS)

    Wang Gongjin; Zhu Xiandai; Chen Yuling; Wang Faming

    1990-01-01

    A rapid immunoradiometric assay (IRMA) has been established by the combination of 125 I-protein A with serum immunoglobulin IgG from rabbits. This method for the determination of infected samples of rice, compared with many other traditional methods, is very simple. The experiments show that the sensitivity of IRMA approximates to 1000 germ cells/ml. The coefficient variation of intrabatch and interbatch (CV) is about 15%. The antiserum only has slight cross-reaction (4.7%) against X. campestris pv. oryzae and other pathogenic bacteria of rice. Of 331 types of rice seeds chosen from infected fields of six provinces, 269 were positive, and the positive rate was as high as 81.3%, which indicated that most rice seeds in these areas were infected with the pathogenic bacteria. The identification of natural inoculated rice seeds by IRMA is effective and prove to be reliable by means of inoculation method

  5. 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.

  6. Re-examination of human calcitonin radioimmunoassay

    International Nuclear Information System (INIS)

    Argemi, B.; Hours, M.C.; Even, F.; Garguilo, G.; Hollard, J.M.

    1978-01-01

    Evaluations of human immunoreactive calcitonin (IRCT) assay have been extensively reviewed. Labelled hormone was re-purified on carboxy-methyl-cellulose in order to isolate a fraction containing mainly monoiodinated calcitonin, which was found to be very stable. Two antisera with different immunochemical characteristics were used for incubation studies, one of which was incubated with unextracted and extracted plasma samples. The sensitivity of the assays was 60 pg/ml plasma. However, marked differences were observed in the results obtained by the three methods depending on the importance of the inhibitory effect of plasma on the binding of the tracer to antibodies. However, the absolute plasma IRCT level could not be related to the presence of calcitonin M, except in one case. Further studies are needed in order to ascertain the origin and the significance of the immunoreactive material which was detected in normal plasma by one antiserum. (author)

  7. A rapid, sensitive and specific radioimmunoassay for human chorionic gonadotrophin

    International Nuclear Information System (INIS)

    Kardana, A.; Bagshawe, K.D.

    1976-01-01

    A specific, quantitative assay for human chorionic gonadotrophin (hCG) in plasma has been developed using an hCG-β antiserum and an assay procedure lasting four hours. It employs a double antibody method and in a procedure prior to assay the two antibodies are pre-incubated in bulk and then stored in ampoules in lyophilised form ready for use. The assay procedure is a dis-equilibrium (sequential saturation) system in which the sample or unlabelled antigen is first incubated with the pre-incubated antisera followed by the addition of the labelled antigen and a further incubation. The antigen-antibody complex is separated from free antigen by vacuum filtration on glass fibre discs. The assay sensitivity is 100-200 picograms hCG/ml (0.68 mIU/ml) and is uninfluenced by normal concentrations of luteinising hormone in the sample

  8. Fibronectin distribution in epithelial and associated tissues of the rat

    DEFF Research Database (Denmark)

    Couchman, J R; Gibson, W T; Thom, D

    1979-01-01

    Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated...... parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring...... in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues...

  9. Recombinant-derived chicken growth hormone used for radioimmunoassay

    International Nuclear Information System (INIS)

    Proudman, J.A.

    1984-01-01

    The use of recombinant-derived chicken growth hormone (rcGH) in an avian growth hormone (GH) radioimmunoassay (RIA) procedure is described. Antiserum to turkey GH bound 125 I-labeled rcGH, and unlabeled rcGH or turkey GH displaced binding in a dose-related manner. The dose-response curves of sera and pituitary extract from chickens and turkeys were parallel to the rcGH standard curve. Sera from hypophysectomized (hypox) chickens and turkeys produced no dose-response and did not inhibit binding of labeled rcGH. Recovery of rcGH added to hypox sera was quantitative. Modification of the homologous turkey GH RIA protocol of Proudman and Wentworth (1) to use rcGH made possible either an increase in assay sensitivity or a 3-day reduction in incubation time

  10. 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

    The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was 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...

  11. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, B; Pallesen, L; Jensen, LB

    1997-01-01

    The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was 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...

  12. THE ANTIGENIC RELATIONSHIP BETWEEN PROTEUS X-19 AND TYPHUS RICKETTSIA

    Science.gov (United States)

    Castaneda, M. Ruiz

    1934-01-01

    A soluble specific substance was isolated from Mexican typhus Rickettsia which gave, with Proteus X-19 antiserum and typhus human serum, the same precipitation reactions as the polysaccharides extracted from B. proteus OX-19. The soluble specific substance extracted from Rickettsia and Proteus OX-19 is likely to be of a polysaccharide nature owing to the strong Molisch reactions obtained with such extracts, the heat stability and the negative protein reactions (biuret). Since, however, it still contains 7 per cent nitrogen, this is not certain. In the antigenic composition of both Proteus X-19 and typhus Rickettsia there is a common soluble specific factor which is responsible for the Weil-Felix reaction. PMID:19870282

  13. Studies on radioallergosorbent test (RAST)

    International Nuclear Information System (INIS)

    Okuda, Minoru; Usami, Atsushi; Unno, Tokuji

    1975-01-01

    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 125 I-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.)

  14. High molecular somatostatin, an interfering factor in radioimmunoassay

    International Nuclear Information System (INIS)

    Diel, F.; Schneider, E.; Baumann, H.

    1977-01-01

    Cyclic Tyr 1 -somatostatin (Tyr 1 -SRIF) is radioiodinated by the lactoperoxidase method. Purification is achieved by Sephadex G-25 adsorption chromatography. Specific anti-SRIF serum (FA1) has been raised in rabbits. A dose response curve is obtained in the range of 5 - 5,000 pg per tube using an antiserum dilution of 1:2,000. There is little cross-reaction with linear somatostatin and none with ocytocin, (lys-, arg-) vasopressin, valinomycin, polymyxin, insulin, glucagon, human growth hormone (hGH), and thyrotropin-releasing hormone (TRH). For recovery tests, extraction procedures are necessary. Thin-layer chromatography (TLC) and polyacrylamide-disc-electrophoresis (Disc-PAGE) are performed to identify the presumed high molecular 125 I-Tyr 1 -SRIF associate. This high molecular associate may represent an interfering factor in the radioimmunoassay for cyclic SRIF. (orig./AJ) [de

  15. 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...... 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 oxytocin/vasopressin-like peptides. With radioimmunoassays that are specific for either oxytocin or vasopressin, only very low amounts of immunoreactivity were measured. In addition, the dilution curves in these assays were not parallel to the standards, indicating that the antigenic determinant of hydra...

  16. A beta-endorphin radioimmunoassay: specificity studies

    International Nuclear Information System (INIS)

    Colas-Linhart, N.; Perdrisot, R.; Petiet, A.; Bok, B.

    1986-01-01

    This note describes the technical details of a cerebrospinal fluid (CSF) B-Endorphin (B-End) radioimmunoassay (RIA). We used an antiserum raised in rabbits against human B-End which cross-reacs 100% with B-Lipotrophin (B-Lph). Thus, filtration chromatography is used to separate both peptides. The assay is sensitive (limit detection=14 pmoles/l), reproducible (the intra and inter assay coefficients of variation are 5 and 6% respectively). Specificity studies are performed to verify the cross-reactions with other opioid peptides and the non specific reactions with the biological fluid (CSF). In order to evaluate the effects of iodine-containing contrast media on the RIA, additional standard curves were analyzed in the presence of varying concentrations of two contrast materials [fr

  17. An iodine-125 radioimmunoassay for the direct detection of benzodiazepines in blood and urine

    International Nuclear Information System (INIS)

    Goddard, C.P.; Stead, A.H.; Mason, P.A.; Law, B.; Moffat, A.C.; McBrien, M.; Cosby, S.

    1986-01-01

    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-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)

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

    DEFF Research Database (Denmark)

    Pærregaard, A; Angen, Øystein; Lisby, M

    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 preformed neurotoxin could not be excluded. The food history revealed consumption of a commercially produced banana/peach puree which was suspected as a possible source, and based on a precautionary principle this product was recalled. The case description illustrates a risk-management dilemma between...

  19. 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.

  20. Characteristics of the molecular diversity of the outer membrane protein A gene of Haemophilus parasuis

    Science.gov (United States)

    Tang, Cheng; Zhang, Bin; Yue, Hua; Yang, Falong; Shao, Guoqing; Hai, Quan; Chen, Xiaofei; Guo, Dingqian

    2010-01-01

    The molecular diversity of the gene encoding the outer membrane protein A (OmpA) of Haemophilus parasuis has been unclear. In this study, the structural characteristics, sequence types, and genetic diversity of ompA were investigated in 15 H. parasuis reference strains of different serovars and 20 field isolates. Three nucleotide lengths of the complete open reading frame (ORF) of ompA were found: 1098 base pairs (bp), 1104 bp, and 1110 bp. The OmpA contained 4 hypervariable domains, mainly encoding the 4 putative surface-exposed loops, which makes it a potential molecular marker for genotyping. Western blot analysis showed that the recombinant OmpAs of serovars 4 and 5 could cross-react with antiserum to all 15 serovars. Hence, although ompA of H. parasuis exhibited high variation among serovars, this variation did not seem to affect the strong antigenic characteristics of OmpA. PMID:20885850

  1. Dog and human acid beta-D-galactosidases are structurally similar.

    Science.gov (United States)

    Hubert, J J; O'Brien, J S

    1983-01-01

    The purification of dog liver acid beta-galactosidase is described. The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000. Antiserum raised against purified human liver acid beta-galactosidase cross-reacted with beta-galactosidase from dog liver, but not with those from cat liver or Escherichia coli. Tryptic peptide maps of the dog and human acid beta-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping. We conclude that dog and human acid beta-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid beta-galactosidase deficiency) is an excellent model for the same disease in man. Images Fig. 1. Fig. 3. PMID:6412684

  2. Cytotoxin production in phytopathogenic and entomopathogenic Serratia marcescens.

    Science.gov (United States)

    Escobar, M M; Carbonell, G V; Beriam, L O; Siqueira, W J; Yano, T

    2001-01-01

    In this work, culture filtrates of entomopathogenic and phytopathogenic Serratia marcescens strains induced cytotoxic effects on CHO, Vero and HEp-2 cell lines. Morphological changes on sensitive cells were characterized by cell rounding and detachment as soon as 30 min of incubation, culminating in cell death after 24 h. The cytotoxic effect was completely neutralized by specific antiserum indicating that occur antigenic similarity among cytotoxins produced by these strains. The toxicity assays on plants showed that the culture supernatants did not provoke any visible morphological change and did not affect their growth. By contrast, the plants treated with bacterial suspension showed disease symptom, such as shriveling and decay of stores bulbus in onion and lettuce plantlets. In conclusion, this study show that phytopathogenic and entomopathogenic S. marcescens may produce a cytototoxin similar to that produced by clinical isolates and it is toxic to different mammalian cell lines. These results are especially important for studies involving this bacterium as biological control agent.

  3. The role of carbohydrates in the radioimmunoassay of human low-molecular-mass kininogen

    International Nuclear Information System (INIS)

    Turpeinen, U.; Kaerkkaeinen, T.

    1985-01-01

    The immunoreactivity of human low-molecular-mass kininogen from Cohn plasma fraction IV was investigated after deglycosylations and carbohydrate modifications by radioimmunoassay using the conformation-specific antiserum. Removal of all sialic acids, 44% of amino sugars and 63% of neutral sugars did not alter the immunoreactivity of the protein but the periodate-treated concanavalin A fractions showed strikingly diminished immunoreactivity. A conformational change could account for the observed effect of periodate on the decreased reactivity of the protein in radioimmunoassay. Externally added carbohydrates had no effect on immunoreactivity. The results suggest that the carbohydrate part of kininogen is not involved in the immunoreactivity although it accounts for the observed lectin-binding heterogeneity. (Auth.)

  4. 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.

  5. 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.

  6. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans

    1990-01-01

    Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did...... not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...... for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we...

  7. Isolation and characterization of a specific receptor for human albumin on a group L Streptococcus.

    Science.gov (United States)

    Lämmler, C

    1988-08-01

    Certain group L streptococci demonstrate surface receptors for human albumin. Binding of 125I-albumin to group L streptococci could be inhibited by unlabelled albumin preparations from humans, dogs, mice and bovines, but not by albumin from rabbits. The albumin-binding proteins (ABP) could be solubilized from the streptococcal surface by hot acid treatment of the bacteria and isolated by affinity chromatography on human-albumin sepharose. ABP and specific antisera produced against ABP inhibited 125I-albumin binding to group L streptococci. The molecular weight of ABP determined by SDS-PAGE and Western blotting, was approximately 48,000 Dalton. ABP preparations of group G streptococci isolated from bovines and humans demonstrated cross reactivity with antiserum produced against group L streptococcal ABP.

  8. Process for preparation of a solid-phase radioimmunoassay support and use thereof

    International Nuclear Information System (INIS)

    Meriadec, B.; Roubertie, P.

    1979-01-01

    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.)

  9. Development of the two-part pattern during regeneration of the head in hydra

    DEFF Research Database (Denmark)

    Bode, Matthias; Awad, T A; Koizumi, O

    1988-01-01

    The head of a hydra is composed of two parts, a domed hypostome with a mouth at the top and a ring of tentacles below. When animals are decapitated a new head regenerates. During the process of regeneration the apical tip passes through a transient stage in which it exhibits tentacle......-like characteristics before becoming a hypostome. This was determined from markers which appeared before morphogenesis took place. The first was a monoclonal antibody, TS-19, that specifically binds to the ectodermal epithelial cells of the tentacles. The second was an antiserum against the peptide Arg......-Phe-amide (RFamide), which in the head of hydra is specific to the sensory cells of the hypostomal apex and the ganglion cells of the lower hypostome and tentacles. The TS-19 expression and the ganglion cells with RFamide-like immunoreactivity (RLI) arose first at the apex and spread radially. Once the tentacles...

  10. Serial cultivation of human scalp hair follicle keratinocytes.

    Science.gov (United States)

    Weterings, P J; Roelofs, H M; Vermorken, A J; Bloemendal, H

    1983-01-01

    A method is described for the serial cultivation of adult human hair follicle keratinocytes. Plucked scalp hair follicles, placed on bovine eye lens capsules as a growth substrate, give rise to quickly expanding colonies within a few days. After trypsinization, the cells are replated with irradiated 3T3 cells as 'feeders'. Using this combination of techniques the keratinocytes can be subcultured up to four times. In this way about 10(7) keratinocytes can be generated from one single hair follicle. Moreover, the technique enables cryogenic storage of the cells, allowing for instance, convenient transportation. Subcultured hair follicle keratinocytes can be plated on glass coverslips. This allows immunofluorescence studies. The keratin cytoskeletons visualized using an antiserum against human keratin.

  11. The vectorial release of nascent immunoglobulin peptides.

    Science.gov (United States)

    Bevan, M J

    1971-03-01

    A microsomal preparation from a mouse plasmacytoma, MOPC 47A, that secretes immunoglobulin A was used to study the release of nascent immunoglobulin peptides in vitro. Nascent chains were released with puromycin and characterized with specific antiserum against the immunoglobulin product of the tumour. When the tissue had been prelabelled with [(3)H]leucine the experiments were complicated by the large background of completed radioactive polypeptides in the microsomal preparation. Up to one-third of the released radioactivity in the microsomal preparation could be recognized as immunoglobulin. With [(3)H]-puromycin as the radioactive label, however, the results are much easier to interpret, although the proportion of released radioactivity that can be identified as immunoglobulin is lower (up to one-tenth). Both types of experiment demonstrate that all of the recognizable nascent immunoglobulin chains remain in association with the microsomal vesicles after release from the ribosomes.

  12. Increased muscle glucose uptake during contractions

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Galbo, Henrik; Richter, Erik

    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 1.5 h......-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 muscle may only increase if glycogen...

  13. [An avian strain of Escherichia coli with antigens common to the genus Salmonella].

    Science.gov (United States)

    Terzolo, H R; Zoratti de Verona, A; d'Empaire, M; Furowicz, A J

    1977-01-01

    On a commercial poultry farm, a large percentage (9%) of clinically healthy fowls had positive reaction to the plate test, with commercial polyvalent pullorum antigens. We could not isolate Salmonella from the positive birds. An strain, of Escherichia coli Balcarce (E. coli B) was isolated from the feces of one of the birds. The isolate was identified biochemically and the antigenic study showed correlation with E. coli 044 and the somatic fraction 1, 2, 8, 14 and 23 of the Salmonella genus. The common antigens were studied by agglutination, absorption and crossed immunodiffusion tests, comparing the isolated strain and the different Salmonella serotypes. Four pullorum polyvalent commercial antigens reacted with sera containing somatic agglutinins 1, and with the E. coli B antiserum. These observations confirm the high antigenic correlation between the genus of the Enterobacteriaceae family. It is indicated that for the diagnosis of avian salmonelosis rather than using a single serological tests, the isolation and identification of the etiological agent is required.

  14. Radioimmunoassay for serum paraquat

    International Nuclear Information System (INIS)

    Fatori, D.; Hunter, W.M.

    1980-01-01

    Two variants of a radioimmunoassay for the bipyridylium herbicide Paraquat are described. Both employ antiserum raised to Paraquat-BSA which has been covalently linked to particulate solid-phase support media. The rapid assay for clinical use employs a [ 3 H] Paraquat tracer, requires no agitation and yields results in the range 10-2500 ng/ml serum in 20 min from receipt of sample. The more sensitive assay, designed for research purposes, employs a 125 iodinated tracer, requires 2 h continuous agitation but can detect Paraquat at 0.1 ng/ml in simple aqueous solution or 0.25 ng/ml serum. Results from rapid clinical assay agree well with the existing colorimetric method. (Auth.)

  15. 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.

  16. Radioimmunoanalysis of the immune response and tissue lipoperoxidation of rats for low doses contaminated with tritiated water

    International Nuclear Information System (INIS)

    Bejan, A.; Turcu, Gr.

    1996-01-01

    The paper presents the evaluation of the humoral immune response and liver and kidney lipoperoxidation on rats acutely and chronically contaminated with low doses of tritiated water (HTO). The contamination doses for both situations were 0.5 cSv, 5 cSv and 10 cSv. By humoral immune response analysis we mean the measurement, through a radioimmunoanalysis (RIA) method, of the two intrinsic parameters of an antiserum, that is, the mean antibodies concentration and the mean antibodies affinity constant. We followed the liver and kidney lipidic peroxidation through malondialdehyde (MDA) formation assayed by the thiobarbituric acid (TBA) reaction. The humoral immune response and lipoperoxidation have been compared with the response of an uncontaminated group by the Student t test. (author). 17 refs., 2 tabs

  17. Occurrence and significance of Mallory bodies in morbidly obese patients. An immunohistochemical study

    DEFF Research Database (Denmark)

    Gluud, C; Christoffersen, Pernille Yde; Andersen, T

    1984-01-01

    Liver biopsies from 61 consecutive patients with morbid obesity (less than 60% overweight) and from 48 patients with alcoholic liver disease were examined for the presence of Mallory bodies. For the detection both routine haematoxylin and eosin stained sections and sections exposed...... liver disease. Using the immunohistochemical technique, Mallory bodies were found in the liver of 2 obese patients (3%) and in 36 patients with alcoholic liver disease (75%). None of the Mallory body positive obese patients showed signs of diabetes mellitus, cholestasis or hypocholesterolemia, but both...... to an immunohistochemical technique were employed. The latter uses an antiserum which recognizes antigenic determinants in Mallory bodies. Using haematoxylin and eosin staining. Mallory bodies were not detected in any of the biopsies from the obese patients, but found to be present in 63% of the patients with alcoholic...

  18. Increase of catechol-O-methyltransferase activity in rat brain microglia after intrastriatal infusion of fluorocitrate, a glial toxin.

    Science.gov (United States)

    Reenilä, I; Tuomainen, P; Soinila, S; Männistö, P T

    1997-07-25

    Striatal catechol-O-methyltransferase (COMT), monoamine oxidase B (MAO-B; an astroglial enzyme), alkaline phosphodiesterase I (PDE; a microglia/macrophage marker) and tyrosine hydroxylase (TH; catecholaminergic neuron marker) activities were analyzed biochemically 1-3 days after infusion of fluorocitrate, an astrocyte damaging agent. Astrocytes, microglia and neurons were stained immunohistochemically with specific antibodies (against glial fibrillary acidic protein, OX-42 and TH, respectively) and with COMT antiserum. Three days after fluorocitrate infusion the activity of MAO-B was reduced, whereas COMT and PDE activities were increased. The elevation of COMT immunoreactivity co-localized to microglial cells, but not to astrocytes. In conclusion, this is the first report indicating that microglia contains COMT activity which may be increased in pathological conditions.

  19. Antigenic determinant of the Lancefield group H antigen of Streptococcus sanguis.

    Science.gov (United States)

    Rosan, B; Argenbright, L

    1982-01-01

    Previous studies indicated that the teichoic acid isolated from strains of Streptococcus sanguis was group specific and defined the Lancefield group H streptococci. To determine the specific antigenic determinants, the antigen was extracted from a group H streptococcus (ATCC 903) by the phenol-water method and purified by column chromatography. The isolated antigen had a glycerol/phosphate/glucose molar ratio of 1:0.9:0.3; the lipid concentration was 7.6% of its dry weight. No nucleic acids were detected, and amino acids constituted approximately 2% of the dry weight. The minimum concentration of antigen required to sensitize erythrocytes for hemagglutination with a 1:1,000 dilution of either group H antiserum or antiteichoic acid serum was 0.02 microgram/ml. Hemagglutination inhibition studies suggested that the major antigenic determinant consisted of an alpha-glucose linked to the glycerol phosphate backbone. Images PMID:6185428

  20. Clinical Application of Immunofluorescence I. Grouping β-Hemolytic Streptococci

    Science.gov (United States)

    Smith, Thomas B.

    1965-01-01

    Smith, Thomas B. (Armed Forces Institute of Pathology, Washington, D.C.). Clinical application of immunofluorescence. I. Grouping β-hemolytic streptococci. J. Bacteriol. 89:198–204. 1965.—Procedures are described for the production of antistreptococcal serum in rabbits and for the preparation of group-specific conjugates for Lancefield groups A, C, and G. A modification of the conventional technique of absorption and inhibition to prevent cross-reactions with common antigens was used with excellent results. In addition, a promising new approach to eliminating cross-reactions of group A conjugate with antigens of groups C and G by dilution with group A-variant antiserum was tested. A complete method is introduced that enables the clinical laboratory to report whether group A streptococci are present in a given throat culture well within 24 hr after the physician collects the sample. Images PMID:14255663

  1. CLINICAL APPLICATION OF IMMUNOFLUORESCENCE. I. GROUPING BETA-HEMOLYTIC STREPTOCOCCI.

    Science.gov (United States)

    SMITH, T B

    1965-01-01

    Smith, Thomas B. (Armed Forces Institute of Pathology, Washington, D.C.). Clinical application of immunofluorescence. I. Grouping beta-hemolytic streptococci. J. Bacteriol. 89:198-204. 1965.-Procedures are described for the production of antistreptococcal serum in rabbits and for the preparation of group-specific conjugates for Lancefield groups A, C, and G. A modification of the conventional technique of absorption and inhibition to prevent cross-reactions with common antigens was used with excellent results. In addition, a promising new approach to eliminating cross-reactions of group A conjugate with antigens of groups C and G by dilution with group A-variant antiserum was tested. A complete method is introduced that enables the clinical laboratory to report whether group A streptococci are present in a given throat culture well within 24 hr after the physician collects the sample.

  2. Radioimmunoassay of aldosterone and its level in plasma and urine

    International Nuclear Information System (INIS)

    Putz, Z.; Hampl, R.; Veleminsky, J.; Starka, L.

    1981-01-01

    A method of plasma and urine aldosterone radioimmunoassay is described and evaluated. Highly specific antisera were obtained through rabbit immunization using partly aldosterone-18,21-dihemisuccinate-BSA, partly aldosterone-3-carboxymethyl-oxime-BSA, free of the 18-derivative. The use of a more specific antiserum against the 3-derivative permitted the determination to be performed immediately from the biological specimen extract. Hydrolysis with sulphuric acid in the presence of dichloromethane proved the most appropriate of the different techniques of releasing aldosterone-glucuronoside for the determination of the total urine hormone. The normal values (average +- 2 S. D.) of plasma aldosterone were 0.19 +- 0.067 nmol/l, those of the free urine hormone 0.918 +- 0.458 nmol/day, and the total urine aldosterone 15.3 +- 5.8 nmol/day. (author)

  3. Radioimmunoassay of deoxynivalenol in wheat and corn

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y.C.; Zhang, G.S.; Chu, F.S.

    With the availability of antibody against deoxynivalenol triacetate (DON-triacetate), a radioimmunoassay (RIA) for DON in wheat was developed. DON is extracted from the sample with acetonitrile-water defatted with hexane, and then reacted with acetic anhydride to form DON-triacetate. The reaction mixture is loaded onto a C-18 cartridge to remove excess reagents and impurities. Acetylated DON is eluted from the cartridge with 50% methanol in water, and then analyzed by radioimmunoassay utilizing antiserum against DON-triacetate and tritiated DON-triacetate. Overall recovery for DON added to wheat between 50 and 5000 ppb was 86% with a standard deviation of 7% and coefficient of variation of 8%. The limit of detection for DON was about 20 ppb. Analysis of 12 naturally contaminated wheat, corn, and mixed feed samples for DON revealed that RIA results agreed well with thin layer chromatographic analyses performed by other laboratories.

  4. Obtenção e avaliação de antígenos de Aspergillus fumigatus Obtention and evaluation of Aspergillus fumigatus antigens extraction

    Directory of Open Access Journals (Sweden)

    Vanda de Sá Lirio

    1992-08-01

    Full Text Available Antígenos de três amostras de A. fumigatus (354, 356, JIG e antissoro contra a mistura destes antígenos foram produzidos e avaliados imunoquimicamente. Os antígenos de filtrado de cultura foram obtidos após concentração com acetona conforme adaptação da técnica descrita por Coleman & Kaufman. Em prova de ID obteve-se 100% de positividade com os soros de pacientes com aspergilose estudados. Com relação aos soros heterólogos encontramos reatividade com soro de um paciente com candidíase e com soro de um paciente com histoplasmose; foi encontrado padrão idêntico de resposta quando se utilizou o antígeno de referência. O antissoro foi titulado por ID, CIE e RFC MI contra o antígeno específico apresentando títulos respectivos de 1:32, 1:32 e 1:128, e utilizado para reagir contra o mesmo antígeno por IEF, demonstrando 8 linhas de precipitação, sendo 5 na região anódica e 3 na região catódica. O perfil de bandeamento do antígeno em eletroforese utilizando gel de poliacrilamida (SDS-PAGE a 12,5% apresentou-se complexo com 26 sub-unidades protéicas, cujos pesos moleculares variaram de 18 a > 100kDa. Quando estes componentes foram eletrotransferidos e reagidos com o antissoro específico ("immunoblotting", verificou-se imunogenicidade em todas as frações bandeadas.Antigens from three strains of Aspergillus fumigatus (354, 356, and JIG and an antiserum against the mixing of these antigens have been produced, and evaluated immunochemically. The antigens were obtained through a modified Coleman & Kaufman technique (culture filtrate concentrated by acetone. Analysis by the immunodiffusion test (ID against homologous serum has yielded 100% sensitivity (with the studied sera. Concerning heterologous sera we found reactivity with a serum of a patient of candidiasis and another with histoplasmosis. The same result was obtained with a reference antigen in immunodiffusion, showing similar standards of response. Titration of the

  5. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    International Nuclear Information System (INIS)

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C.

    1989-01-01

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

  6. Tuberal hypothalamic neurons secreting the satiety molecule Nesfatin-1 are critically involved in paradoxical (REM sleep homeostasis.

    Directory of Open Access Journals (Sweden)

    Sonia Jego

    Full Text Available The recently discovered Nesfatin-1 plays a role in appetite regulation as a satiety factor through hypothalamic leptin-independent mechanisms. Nesfatin-1 is co-expressed with Melanin-Concentrating Hormone (MCH in neurons from the tuberal hypothalamic area (THA which are recruited during sleep states, especially paradoxical sleep (PS. To help decipher the contribution of this contingent of THA neurons to sleep regulatory mechanisms, we thus investigated in rats whether the co-factor Nesfatin-1 is also endowed with sleep-modulating properties. Here, we found that the disruption of the brain Nesfatin-1 signaling achieved by icv administration of Nesfatin-1 antiserum or antisense against the nucleobindin2 (NUCB2 prohormone suppressed PS with little, if any alteration of slow wave sleep (SWS. Further, the infusion of Nesfatin-1 antiserum after a selective PS deprivation, designed for elevating PS needs, severely prevented the ensuing expected PS recovery. Strengthening these pharmacological data, we finally demonstrated by using c-Fos as an index of neuronal activation that the recruitment of Nesfatin-1-immunoreactive neurons within THA is positively correlated to PS but not to SWS amounts experienced by rats prior to sacrifice. In conclusion, this work supports a functional contribution of the Nesfatin-1 signaling, operated by THA neurons, to PS regulatory mechanisms. We propose that these neurons, likely releasing MCH as a synergistic factor, constitute an appropriate lever by which the hypothalamus may integrate endogenous signals to adapt the ultradian rhythm and maintenance of PS in a manner dictated by homeostatic needs. This could be done through the inhibition of downstream targets comprised primarily of the local hypothalamic wake-active orexin- and histamine-containing neurons.

  7. Labelling of. beta. -endorphin (. beta. -END) and. beta. -lipotropin (. beta. -LPH) by /sup 125/I

    Energy Technology Data Exchange (ETDEWEB)

    Deby-Dupont, G.; Joris, J.; Franchimont, P. (Universite de Liege (Belgique)); Reuter, A.M.; Vrindts-Gevaert, Y. (Institut des Radioelements, Fleurus (Belgique))

    1983-01-01

    5 ..mu..g of human ..beta..-endorphin were labelled with 2 mCi /sup 125/I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 ..mu..Ci/..mu..g.Kept at + 4/sup 0/C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a /sup 125/I ..beta..-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-..beta..-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 ..mu..g of human ..beta..-lipotropin were labelled with 0.5 mCi /sup 125/I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 ..mu..Ci/..mu..g was obtained. It remained utilizable for 30 days when kept at + 4/sup 0/C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled ..beta..-LPH was 32% for an anti-..beta..-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.

  8. Isolation and characterization of flagellar filament from zoospores of Dermatophilus congolensis.

    Science.gov (United States)

    Hiraizumi, Mieko; Tagawa, Yuichi

    2014-09-17

    Highly motile zoospores from Dermatophilus congolensis bovine isolates from clinical dermatophilosis in Japan were obtained by culturing at 27°C in an ambient atmosphere on heart infusion agar supplemented with 5% defibrinated sheep blood for 72h or in heart infusion broth for 48h with gentle shaking. After vigorous mechanical agitation of the zoospore suspension, the flagellar filaments detached from motile zoospores and were isolated in the clear gelatinous part of the final pellet by differential centrifugation. Typical morphology of a flagellar filament, with a width of approximately 15nm, was observed in the isolated flagellar filament by electron microscopy. A single major protein (flagellin) band with an apparent molecular mass of 35kDa was detected in the flagellar filament of D. congolensis strain AM-1 and that of 33kDa was detected in strain IT-2 by SDS-PAGE. In immunoblot analysis of whole-cell proteins from seven isolates of D. congolensis, antiserum to strain AM-1 zoospores reacted with the 35-kDa antigen band of strain AM-1, but not with any antigen band of other strains in a similar molecular mass range. In contrast, antiserum to strain IT-2 zoospores reacted with antigen bands at 33kDa from six strains, except strain AM-1. Similar strain-specific reactions of these anti-zoospore sera with isolated flagellar filaments from strains AM-1 and IT-2 were confirmed by immunoblot, indicating the presence of antigenic variations of flagellins of D. congolensis zoospores. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus).

    Science.gov (United States)

    De Sa, P B; Hiebert, E; Purcifull, D E

    2000-01-01

    A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3' terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3'UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3'UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3'UTR. Phylogenetic analyses of the coat protein region and the 3'UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus.

  10. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

    1990-01-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  11. Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera.

    Science.gov (United States)

    S, Vrinda; C, Jasmin; K C, Sivakumar; Jose, Seena; Jose, Blessy; Philip, Rosamma; I S, Bright Singh

    2017-01-01

    Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. IgE and IgG cross-reactivity among Lol p I and Lol p II/III. Identification of the C-termini of Lol p I, II, and III as cross-reactive structures.

    Science.gov (United States)

    van Ree, R; van Leeuwen, W A; van den Berg, M; Weller, H H; Aalberse, R C

    1994-04-01

    In this study, the homologous C-termini of Lol p I, Lol p II, and Lol p III were shown to contain cross-reactive B-cell epitopes. This was demonstrated by inhibition studies with purified Lol p I, II, and III and synthetic peptides of their C-termini. It was ruled out that the observed cross-reactivity was caused by cross-contamination of the purified allergens. Both human IgE and IgG bound to the C-terminus of Lol p I. These antibodies were cross-reactive with Lol p II and, more specifically, with its C-terminus. Within a small panel of allergic patients, no cross-reactivity with Lol p III was found. A hyperimmune polyclonal rabbit antiserum against Lol p I also recognized the Lol p I C-terminus. As for human antibodies, cross-reactivity with Lol p II and its C-terminus was demonstrated. Cross-reactivity with Lol p III was demonstrated with C-terminal peptides, but not with native Lol p III. A polyclonal rabbit antiserum against Lol p II bound to the C-terminal peptides of both Lol p II and III. This binding was inhibited with Lol p I, confirming that cross-reactive structures exist not only on the C-termini of Lol p II and Lol p I, but also of Lol p III and Lol p I. The existence of cross-reactivity between Lol p I and Lol p II and III possibly contributes to the frequently observed cosensitization for these allergens in grass-pollen-allergic patients.

  13. Molecular cloning and characterization of a glutathione S-transferase encoding gene from Opisthorchis viverrini.

    Science.gov (United States)

    Eursitthichai, Veerachai; Viyanant, Vithoon; Vichasri-Grams, Suksiri; Sobhon, Prasert; Tesana, Smarn; Upatham, Suchart Edward; Hofmann, Annemarie; Korge, Günter; Grams, Rudi

    2004-12-01

    An adult stage Opisthorchis viverrini cDNA library was constructed and screened for abundant transcripts. One of the isolated cDNAs was found by sequence comparison to encode a glutathione S-transferase (GST) and was further analyzed for RNA expression, encoded protein function, tissue distribution and cross-reactivity of the encoded protein with other trematode protein counterparts. The cDNA has a size of 893 bp and encodes a GST of 213 amino acids length (OV28GST). The most closely-related GST of OV28GST among those published for trematodes is a 28 kDa GST of Clonorchis sinensis as shown by multiple sequence alignment and phylogenetic analysis. Northern analysis of total RNA with a gene-specific probe revealed a 900 nucleotide OV28GST transcriptional product in the adult parasite. Through RNA in situ hybridization OV28GST RNA was detected in the parenchymal cells of adult parasites. This result was confirmed by immunolocalization of OV28GST with an antiserum generated in a mouse against bacterially-produced recombinant OV28GST. Both, purified recombinant and purified native OV28GST were resolved as 28 kDa proteins by SDS-PAGE. Using the anti-recOV28GST antiserum, no or only weak cross-reactivity was observed in an immunoblot of crude worm extracts against the GSTs of Schistosoma mansoni, S. japonicum, S. mekongi, Eurytrema spp. and Fasciola gigantica. The enzyme activity of the purified recombinant OV28GST was verified by a standard 1-chloro-2, 4-dinitrobenzene (CDNB) based activity assay. The present results of our molecular analysis of OV28GST should be helpful in the ongoing development of diagnostic applications for opisthorchiasis viverrini.

  14. Differential co-localization with choline acetyltransferase in nervus terminalis suggests functional differences for GnRH isoforms in bonnethead sharks (Sphyrna tiburo).

    Science.gov (United States)

    Moeller, John F; Meredith, Michael

    2010-12-17

    The nervus terminalis (NT) is a vertebrate cranial nerve whose function in adults is unknown. In bonnethead sharks, the nerve is anatomically independent of the olfactory system, with two major cell populations within one or more ganglia along its exposed length. Most cells are immunoreactive for either gonadotropin-releasing hormone (GnRH) or RF-amide-like peptides. To define further the cell populations and connectivity, we used double-label immunocytochemistry with antisera to different isoforms of GnRH and to choline acetyltransferase (ChAT). The labeling patterns of two GnRH antisera revealed different populations of GnRH-immunoreactive (ir) cell profiles in the NT ganglion. One antiserum labeled a large group of cells and fibers, which likely contain mammalian GnRH (GnRH-I) as described in previous studies and which were ChAT immunoreactive. The other antiserum labeled large club-like structures, which were anuclear, and a sparse number of fibers, but with no clear labeling of cell bodies in the ganglion. These club structures were choline acetyltrasferase (ChAT)-negative, and preabsorption control tests suggest they may contain chicken-GnRH-II (GnRH-II) or dogfish GnRH. The second major NT ganglion cell-type was immunoreactive for RF-amides, which regulate GnRH release in other vertebrates, and may provide an intraganglionic influence on GnRH release. The immunocytochemical and anatomical differences between the two GnRH-immunoreactive profile types indicate possible functional differences for these isoforms in the NT. The club-like structures may be sites of GnRH release into the general circulation since these structures were observed near blood vessels and resembled structures seen in the median eminence of rats. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Immunogenic peptide mimotopes from an epitope of Escherichia coli O157 LPS.

    Science.gov (United States)

    Navarro, Armando; Hernández-Chiñas, Ulises; Licona-Moreno, Delia; Zenteno, Edgar; Cravioto, Alejandro; Eslava-Campos, Carlos A

    2016-11-01

    Escherichia coli O157:H7 is a subtype of Shiga toxin-producing E. coli that is associated with haemorrhagic colitis and haemolytic uremic syndrome (HUS). Studies of populations in endemic areas have reported that the presence of specific antibodies against the O157 lipopolysaccharide (LPS) is associated with a lower incidence of diarrhoea and HUS. Phage display and IgG anti-O157 LPS antibodies were used in the present study to select peptide mimotopes of O157 LPS expressed in protein III of the M13 phage. Synthetic peptides (SP) were designed using the derived amino acid sequences obtained from DNA nucleotides of 63 selected phagotopes. The LxP/YP/SxL motif was identified in five of the phagotope amino acid sequences. Antibody responses against the phagotopes and their corresponding SPs were evaluated. SP12, one of the designed SP, induced the production of antibodies against the homologous peptide (1:800) and O157 LPS (1:200). The specificity of anti-SP12 antiserum was confirmed by analyzing its response to SP3, an SP with a different amino acid sequence than that of SP12, as well as against an E. coli LPS different from O157. Competitive studies with SP12 and O157 LPS showed a significant decrease in anti-SP12 and anti-LPS O157 antiserum responses against SP12 and O157 LPS, respectively. Eighteen (82%) of the 22 human serum samples with positive reactivity against E coli O157 LPS reacted with SP12 SP (cut-off >0.4). These results support the idea that SP12 is an immunogenic mimotope of O157 LPS. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  16. The E7 protein of human papillomavirus 8 is a nonphosphorylated protein of 17 kDa and can be generated by two different mechanisms.

    Science.gov (United States)

    Iftner, T; Sagner, G; Pfister, H; Wettstein, F O

    1990-11-01

    The E7 protein of human papillomavirus (HPV) 8 shows no in vitro transforming activity, in contrast to E7 of the genital HPVs, but seems to be involved in the control of viral DNA replication. To determine whether functional differences between the E7 proteins of HPV16 and HPV8 were reflected in differences in their biochemical properties, we characterized the E7 protein of HPV8 expressed from the late simian virus 40 promoter in COS 7 cells. An E7-specific antiserum was obtained by immunizing rabbits with a beta-gal-E7 fusion protein containing all of the E7 polypeptide. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS 7 cells a protein with a low mobility of 17 kDa in SDS-PAGE. The high sedimentation rate in nondenaturing glycerol gradients pointed to an interaction with other proteins or to the existence of E7 oligomers. An association with the retinoblastoma protein could, however, be excluded. The 5' ends of HPV8 transcripts derived from the E6-E7 region in G418 selected rodent cells, which were mapped by nuclease S1 digestion, are located upstream of ORFs E6 and E7, respectively. No evidence for an E6*I-like mRNA was found. In COS 7 cells transfected with a plasmid expressing the E6-E7 region of HPV8 under the control of the late SV40 promoter, however, the E7 protein was translated from a polycistronic mRNA potentially encoding E6 and E7. These data indicate that the E7 protein of HPV8 may be expressed in two ways: (i) through translation of an E7-specific mRNA, as with HPV6 and HPV11, or (ii) through internal initiation of translation at the ATG of E7.

  17. [Construction and expression of the fusion gene CCK39/UreB in recombinant Escherichia coli BL-21(DE3)].

    Science.gov (United States)

    Zhang, Weiyang; Li, Yan; Wu, Tongshan; Luo, Wenhua; Hu, Bin; Hu, Wenfeng

    2009-01-01

    The aims of this research were to construct prokaryotic expression vector containing fusion gene of Cholecystokinin 39 (CCK39) of pig and Urease subunit B (UreB) of coliform bacteria, and then to express the fusion protein in recombinant Escherichia coli BL21(DE3). The CCK39 gene was amplified by RT-PCR from the extracted total RNA of pig's duodenum, and the UreB gene was then amplified by PCR from the extracted plasmid DNA of bacillus of coliform bacteria from pig's intestinal content. Then the CCK39 and the UreB were inserted into the prokaryotic expression vector pET43a(+) to construct a recombinant fusion expression vector pET43a(+)/CCK39/UreB and then, the recombinant vector was identified by PCR, endonuclease digestion and sequence analysis. It was identified that the gene fragment of CCK39 at length of 117 bp and UreB at length of 324 bp were amplified and cloned into the vector pET43a(+) successfully. The recombinant vector was transformed into Escherichia coli BL21(DE3) and induced the expression of CCK39/UreB fusion protein with a molecular mass of approximately 80 kD by using isopropylthio-beta-D-galactoside (IPTG) as inducer. The fusion protein was mostly located in the cytoplasm and it was soluble. The soluble protein was collected and purified by Ni2+-NTA column chromatograph and then reached a purity of more than 95%. It was proved by western blotting that the fusion protein could react with rabbit anti-CCK8 antiserum and rabbit anti-UreB antiserum. Therefore, the expressed fusion protein has good antigenicity. This work established a good foundation for further study on the production of anti-CCK/Urease vaccines.

  18. Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

    Directory of Open Access Journals (Sweden)

    Asmarinah

    2017-11-01

    Full Text Available Objective: To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3 protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded. Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly. Conclusions: Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

  19. Effect of gamma irradiation on the behavioral properties of crotoxin

    Directory of Open Access Journals (Sweden)

    E.G. Moreira

    1997-02-01

    Full Text Available Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996 Brazilian Journal of Medical and Biological Research, 29: 629-632. Therefore, we decided 1 to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2 to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g were used. Crotoxin (100, 250, and 500 µg/kg was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source was administered at the doses of 100, 250, and 500 µg/kg for the hole-board test, and at the doses of 1000 and 2500 µg/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05. Crotoxin decreased the social interaction time (s at the doses of 100, 250 and 500 µg/kg (means ± SEM from 51.6 ± 4.4 to 32.6 ± 3.7, 28.0 ± 3.6 and 31.6 ± 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1 crotoxin may be an anxiogenic compound, and 2 in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum

  20. Radioimmunoassay for biopterin

    International Nuclear Information System (INIS)

    Nagatsu, Toshiharu; Yamaguchi, Tokio; Kato, Takeshi; Sugimoto, Takashi; Matsuura, Sadao.

    1979-01-01

    Specific antibodies against biopterin, neopterin, and 6,7-dimethylpterin were prepared and a new type of radioimmunoassay, to which these pterins were subjected was developed. This new type of radioimmunoassay was used to determine biopterin in human urine. Specific antiserum against biopterin did not crossreact significantly with tetrahydro-biopterin, dihydro-biopterin, neopterin, 6,7-dimethylpterin, pterin, or folic acid and showed 30% binding of biopterinyl-caproyl- ( 125 I ) tyramide at a dilution of 1:800. The antisera against neopterin and dimethylpterin also showed a high specificity and did not cross-react significantly with the other pterins. The sensitivity of the radioimmunoassay was increased about ten-fold by pre-incubating the antiserum with 6,7-dimethylpterinyl-caproyl to remove antibodies against the caproic acid moiety of the biopterin conjugate. The recovery of biopterin or tetrahydro-biopterin added human urine was nearly 100% according to this type of radioimmunoassay, and the biopterin concentrations in the urine that were obtained by means of this type of radioimmunoassay showed a fairly good agreement with the values obtained by means of bioassay. The biopterin contents per milliliter of human urine showed considerable variations depending on the subjects, but those per milligram of creatinine were found to be fairly constant in normal subjects. Therefore, biopterin concentrations per milligram of creatinine in human urine may be a good indicator of biopterin metabolism in clinical chemistry. This new type of radioimmunoassay is simple, highly specific, and reproducible. Therefore, it may be very useful for the screening of atypical phenylketonuria due to biopterin deficiency and also for the study of the pathogenesis of various biopterin metabolic diseases. (J.P.N.)

  1. Kinetics of two-site immunoradiometric ('sandwich') assays

    International Nuclear Information System (INIS)

    Rodbard, D.; Feldman, Y.; Jaffe, M.L.; Miles, L.E.M.

    1978-01-01

    The mathematical model of the ideal two-site IRMA developed in the accompanying report, which assumes that both the first (solid phase) and second (labeled) antisera are 'homogeneous', fails to provide an explanation for the frequent occurrence of a paradoxical fall in the dose-response curve in the high-dose region ('high-dose hook effect'). Accordingly, the model has been extended in an attempt to provide a theoretical basis for these phenomena. Two mechanisms were considered: (A) heterogeneity of the first antiserum (which is bound to the solid phase), i.e. presence of two or more classes of sites with moderately to widely differing affinity constants; and (B) incomplete washing of the solid phase after the first reaction. This leads to an expanded system of five differential equations, five equations for conservation of mass, and fourteen rate constants. In the presence of a heterogeneous first antiserum, a high dose 'hook effect' will occur. Rapid dissociation of the ligand from the low affinity class of sites during the second 'wash' procedure will accentuate this effect. Thus, heterogeneity of binding sites is a sufficient explanation for the high-dose hook effect and is a likely mechanism in practice, since the antisera which are coupled to the solid phase have usually not been fractionated. The present studies also show that, even if dealing with homogeneous first antibody, one may still observe a high-dose hook effect if washing is incomplete after the first incubation with the antigen. Using computer simulation studies, factors affecting the magnitude of the 'high-dose hook effect' have been explored. (author)

  2. The involvement of luteinizing hormone (LH) and Pregnancy-Associated Glycoprotein family (PAG) in pregnancy maintenance in the pig.

    Science.gov (United States)

    Panasiewicz, Grzegorz; Majewska, Marta; Szafrańska, Bozena

    2004-07-01

    The paper presents the effect of in vivo immuno-neutralization of porcine luteinizing hormone (pLH) by species-homologous porcine antiserum (anti-pLH) administrations on pregnancy maintenance and immunodetection of the PAG proteins in precipitated plasma proteins of pregnant gilts. Pregnant gilts were passively immunized with 100 ml of porcine anti-pLH (titer 1:10 000) by multiple intravenous infusions performed from 37(th) to 42(nd) day post coitum (dpc; 12-h intervals). Blood samples of pregnant gilts were taken 12 times daily from 35 until 50 dpc. Concentrations of progesterone (P(4)) and pLH were determined by radioimmunoassays in systemic blood plasma of treated gilts and control pregnant gilts. The immuno-neutralization of peripheral pLH with the use of homologous anti-pLH serum resulted in a significant reduction (pgilts only, but abortion did not occur. In the remaining four passively immunized pregnant gilts, plasma P(4) concentration was increased (pgilts. In addition, various anti-pPAG sera were purified by sequential adsorptions with endometrial proteins of cyclic gilts. Western blotting demonstrated the expression of the PAG proteins in precipitated plasma proteins of pregnant gilts. In conclusion, the passive immuno-neutralization of porcine LH by species-homologous antiserum (anti-pLH) did not affect the pregnancy maintenance. Thus, the maintenance of mid-pregnancy in gilts may depend also on other than LH luteotrophic factors. In addition, Western analysis of precipitated plasma proteins of pregnant pigs suggests a role of the PAG family during pregnancy in the pig.

  3. Exposure to environmental tobacco smoke measured by cotinine 125I-radioimmunoassay

    International Nuclear Information System (INIS)

    Knight, G.J.; Palomaki, G.E.; Lea, D.H.; Haddow, J.E.

    1989-01-01

    We describe a polyclonal-antiserum-based 125 I-radioimmunoassay for cotinine that is suitable for measuring nonsmokers' passive exposure to tobacco smoke in the environment. The standard curve ranged from 0.25 to 12.0 micrograms/L, with an estimated lower limit of sensitivity of 0.2 microgram/L (95% B/Bo = 0.2 microgram/L; 50% B/Bo = 4.0 micrograms/L). The median within-assay CVs for patients' samples with cotinine values from 0.4 to 1.3, 1.4 to 2.4, 2.5 to 4.6, and 4.7 to 15.6 micrograms/L were 13.9%, 7.2%, 5.1%, and 5.7%, respectively. Between-assay CVs for two quality-control sera with average values of 1.53 and 3.68 micrograms/L were 14.3% and 7.8%, respectively. Analytical recoveries of cotinine from smokers' sera diluted in zero calibrant ranged from 91% to 116%. Cotinine values determined on 79 paired sera and urines from nonsmokers showed significant correlation with self-reported exposure to environmental tobacco smoke (r = 0.49, P less than 0.001 for sera; r = 0.57, P less than 0.001 for urine). The log of the values for serum and urine cotinine were also significantly correlated (r = 0.85, P less than 0.001). Evidently, polyclonal antiserum can be used to develop a cotinine assay for measuring exposure to environmental tobacco smoke that compares well with that described for monoclonal-based assays

  4. Identification of Vibrio harveyi proteins involved in the specific immune response of Senegalese sole (Solea senegalensis, Kaup).

    Science.gov (United States)

    Medina, A; Mancera, J M; Martínez-Manzanares, E; Moriñigo, M A; Arijo, S

    2015-11-01

    Senegalese sole cultures are frequently affected by Vibrio harveyi disease outbreaks. Vaccines in aquaculture are one of the most successful methods of preventing fish pathologies; however, these vaccines are usually composed of inactivated whole cells containing a wide pool of antigens, and some do not induce any protection against pathogens. Thus, the aim of this study was to identify immunogenic proteins of V. harveyi involved in the specific antibody production by Senegalese sole. S. senegalensis specimens were immunized, by intraperitoneal injection, with V. harveyi bacterin supplemented with inactivated extracellular polymeric substances (ECP) and Freund incomplete adjuvant to obtain polyclonal antiserum. One month later, specimens were re-inoculated with the same antigens. Sera from immunized fish were collected two months post first immunization. Strong specific immune response to V. harveyi antigens was detected by ELISA using bacterin (limit dilutions of sera were 1:64000), ECP (1:4000) and outer membrane proteins (OMP) (1:4000) as antigens. Presence of immunogenic proteins in V. harveyi ECP and OMP were determined by 2D-PAGE. For Western Blot analysis some gels were transferred onto nitrocellulose membranes and incubated with sera from S. senegalensis specimens immunized against V. harveyi. 2D-PAGE and Western Blot showed at least five reactive proteins in the ECP and two in the OMP fraction. The spots that clearly reacted with the sole antiserum were excised from stained gel, and analyzed by mass spectrometry (MALDI/TOFTOF). A database search was then performed, using MASCOT as the search method. According to the results, the five ECP spots were identified as Maltoporine, protein homologous to Metal dependent phosphohydrolase, two porins isoforms of V. harveyi and a protein homologous to the cell division protein FtsH. Reactive proteins in the OMP fraction were identified as the protein 3-hydroxyisobutyrate dehydrogenase and a protein homologous to acid

  5. Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested.

    Science.gov (United States)

    Leoff, Christine; Saile, Elke; Rauvolfova, Jana; Quinn, Conrad P; Hoffmaster, Alex R; Zhong, Wei; Mehta, Alok S; Boons, Geert-Jan; Carlson, Russell W; Kannenberg, Elmar L

    2009-06-01

    The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.

  6. Toxic Shock Syndrome Toxin-1-Mediated Toxicity Inhibited by Neutralizing Antibodies Late in the Course of Continual in Vivo and in Vitro Exposure

    Directory of Open Access Journals (Sweden)

    Norbert Stich

    2014-05-01

    Full Text Available Toxic shock syndrome (TSS results from the host’s overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs. There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1 neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation, determination of IL-2 release in the cell supernatant (ELISA, and IL-2 gene activation (real-time PCR (RT-PCR. Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure.

  7. Characterization of Melon necrotic spot virus Occurring on Watermelon in Korea

    Directory of Open Access Journals (Sweden)

    Hae-Ryun Kwak

    2015-12-01

    Full Text Available Melon necrotic spot virus (MNSV was recently identified on watermelon (Citrullus vulgaris in Korea, displaying as large necrotic spots and vein necrosis on the leaves and stems. The average occurrence of MNSV on watermelon was found to be 30–65% in Hapcheon and Andong City, respectively. Four isolates of the virus (MNSV-HW, MNSV-AW, MNSV-YW, and MNSV-SW obtained from watermelon plants in different areas were non-pathogenic on ten general indicator plants, including Chenopodium quinoa, while they infected systemically six varieties of Cucurbitaceae. The virus particles purified by 10–40% sucrose density gradient centrifugation had a typical ultraviolet spectrum, with a minimum at 245 nm and a maximum at 260 nm. The morphology of the virus was spherical with a diameter of 28–30 nm. Virus particles were observed scattered throughout the cytoplasm of watermelon cells, but no crystals were detected. An ELISA was conducted using antiserum against MNSV-HW; the optimum concentrations of IgG and conjugated IgG for the assay were 1 μl/ml and a 1:8,000–1:10,000 dilutions, respectively. Antiserum against MNSV-HW could capture specifically both MNSV-MN from melon and MNSV-HW from watermelon by IC/RT-PCR, and they were effectively detected with the same specific primer to produce product of 1,172 bp. The dsRNA of MNSV-HW had the same profile (4.5, 1.8, and 1.6 kb as that of MNSV-MN from melon. The nucleotide sequence of the coat protein of MNSV-HW gave a different phylogenetic tree, having 17.2% difference in nucleotide sequence compared with MNSV isolates from melon.

  8. Type XII and XIV collagens mediate interactions between banded collagen fibers in vitro and may modulate extracellular matrix deformability.

    Science.gov (United States)

    Nishiyama, T; McDonough, A M; Bruns, R R; Burgeson, R E

    1994-11-11

    Type XII and XIV collagens are very large molecules containing three extended globular domains derived from the amino terminus of each alpha chain and an interrupted triple helix. Both collagens are genetically and immunologically unique and have distinct distributions in many tissues. These collagens localize near the surface of banded collagen fibrils. The function of the molecules is unknown. We have prepared a mixture of native type XII and XIV collagens that is free of contaminating proteins by electrophoretic criteria. In addition, we have purified the collagenase-resistant globular domains of type XII or XIV collagens (XII-NC-3 or XIV-NC-3). In this study, we have investigated the effect of intact type XII and XIV and XII-NC-3 or XIV-NC-3 on the interactions between fibroblasts and type I collagen fibrils. We find that both type XII and XIV collagens promote collagen gel contraction mediated by fibroblasts, even in the absence of serum. The activity is present in the NC-3 domains. The effect is dose-dependent and is inhibited by denaturation. The effect of type XII NC-3 is inhibited by the addition of anti-XII antiserum. To elucidate the mechanism underlying this phenomenon, we examined the effect of XII-NC-3 or XIV-NC-3 on deformability of collagen gels by centrifugal force. XII-NC-3 or XIV-NC-3 markedly promotes gel compression after centrifugation. The effect is also inhibited by denaturation, and the activity of type XII-NC3 is inhibited by the addition of anti-XII antiserum. The results indicate that the effect of XII-NC-3 or XIV-NC-3 on collagen gel contraction by fibroblasts is not due to activation of cellular events but rather results from the increase in mobility of hydrated collagen fibrils within the gel. These studies suggest that collagen types XII and XIV may modulate the biomechanical properties of tissues.

  9. Separation of cell-dependent antibody (CDA) and inhibitory antibody by protein-A affinity chromatography and the effect of fractions on antibody-dependent cellular cytotoxicity (ADCC).

    Science.gov (United States)

    Sato, N; Yabuki, Y; Toh, K; Ishii, Y; Kikuchi, K

    1979-01-01

    The nature of cell-dependent antibody (CDA) and the mechanism of inhibition of antibody-dependent cellular cytotoxicity (ADCC) were studied in the ADCC assay system in which culture cells of methylcholanthrene-induced rat fibrosarcoma (KMT-50) were used as target cells, xenogeneic antiserum (rabbit anti-KMT-50) as the CDA, and human peripheral blood leucocytes (PBL) as effector cells, respectively. By using protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-KMT-50 serum, CDA was shown to bind protein A. Complement dependent-cytotoxicity (CDC), however, was demonstrated in both the adsorbed fraction (eluate) and the non-adsorbed fraction (effluent) to protein A from the same affinity column chromatography. These data confirmed that CDA was IgG with an intact Fc portion. Inhibition of ADCC occurred by pretreatment of effector cells with rabbit anti-effector (human PBL) serum even with extremely small amounts of antiserum. Such inhibition was demonstrated with the eluate but not with the effluent from protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-effector serum. F(ab')2 fragments of the same eluate (IgG) did not inhibit the ADCC activity. These data showed that the inhibition of ADCC was induced by the blocking of Fc receptors of effector cells with the Fc portions of IgG in anti-effector serum. The data obtained indicate the usefulness of protein A in separation and analysis of CDA and in investigation of the inhibitory mechanisms of ADCC. Images Figure 2 PMID:437836

  10. Interplay of thymus and bone marrow regeneration in x-irradiated mice

    International Nuclear Information System (INIS)

    Hiesche, K.-D.

    1975-01-01

    aim of the prepresent investigation was to study the modifying effects of bone marrow cells on regeneration, after X-irradiation, of thymus and bone marrow cell populations. Data are presented which indicate that the cellular composition of the thymus and, in particular, the frequency of the stem cells in the organ at the time of radiation exposure determines thymic regeneration for about two weeks after irradiation. After this period, regeneration depends on new precursors from the bone marrow which have previously seeded the thymus. In contrast to the thymus, cellular restoration of the bone marrow is already initially dependent on the number of protected or transplanted marrow cells. Two phases in the recovery of thymic PHA-reactivity after irradiation were observed: one initial phase which is independent on the number of the available bone marrow cells, and a subsequent phase during which PHA-reactivity is slightly increased in mice irradiated with partly protected bone marrow in comparison to in total body irradiated animals. During the entire observation period, PHA-reactivity remains at a low level not exeeding 50 % of that in untreated mice. In contrast the thymus is fully repopulated with regard to the number of nonreactive cells. Alternative pathways of thymocyte development within the thymus are discussed. Bone marrow X cells were shown to be as sensitive to in vitro treatment with a specific H-2 antiserum as were lymphocytes from normal bone marrow. This finding was teken to indicate that the X cells represent a particular lymphoid cell type. A xenogeneic rabbit-anti-mouse embryo antiserum was more toxic to pre-irradiated bone marrow, with high proportion of X cells, than to bone marrow from untreated mice, using in vitro cytotoxicity test. A possible embryonic character of the X cells is discussed. (author)

  11. Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

    International Nuclear Information System (INIS)

    Patters, M.R.; Landsberg, R.L.; Johansson, L.-A.; Trummel, C.L.; Robertson, P.R.

    1982-01-01

    Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated 45 Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis. (author)

  12. Granulocyte-colony stimulating factor levels in bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis

    Science.gov (United States)

    Ashitani, J.; Mukae, H.; Taniguchi, H.; Ihi, T.; Kadota, J.; Kohno, S.; Matsukura, S.

    1999-01-01

    BACKGROUND—Granulocyte-colony stimulating factor (G-CSF) is known as a potent neutrophil chemotactic glycoprotein in vitro but its contribution to chemotactic activity in neutrophil mediated lung diseases is not yet known. The aims of this study were to determine whether G-CSF is present in high concentrations in bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis (IPF, also called cryptogenic fibrosing alveolitis), a neutrophil mediated lung disease, and to what extent G-CSF in BAL fluid contributes to neutrophil accumulation in the lung of patients with IPF.
METHODS—G-CSF concentrations in BAL fluid samples from 16 healthy volunteers, 24 patients with IPF, and 73 patients with non-IPF lung disease were measured by enzyme linked immunosorbent assay. The relationship between G-CSF concentrations and neutrophil count in BAL fluid was also examined. Neutrophil chemotactic activity (NCA) was measured in BAL fluid in healthy volunteers and patients with IPF. The contribution of G-CSF to overall NCA in lungs with IPF was assessed by repeating the measurement of NCA after a complete neutralisation of G-CSF bioactivity by anti-human G-CSF antiserum.
RESULTS—Detectable levels of G-CSF were found in BAL fluid of 83% of patients with IPF while the levels in all healthy volunteers were below the detection limit. In patients with IPF a significant correlation was observed between the BAL fluid neutrophil count and the concentration of G-CSF in the BAL fluid. The neutrophil count also correlated significantly with percentage forced vital capacity. In BAL fluid samples from patients with IPF the mean NCA value was reduced by 35% after neutralisation with an anti-human G-CSF antiserum.
CONCLUSIONS—G-CSF may be involved in enhancing neutrophil accumulation in the lungs of patients with IPF.

 PMID:10525561

  13. Small-scale extraction and radioiodination of human hormones for the substitution of imported radioimmunoassay reagents

    International Nuclear Information System (INIS)

    Gimbo, E.K.; Carvalho Pinto Ribela, M.T. de; Borghi, V.C.; Schwarz, I.; Dias, L.E.M.; Araujo, E.A. de; Bartolini, P.

    1988-08-01

    One of the main objectives of our laboratory is the national production of radioimmunoassay (RIA) reagents so to substitute the use of expensive imported commercial kits or in-bulk reagents such as the following: a) highly purified unlabelled hormones for radioiodination, useful also for preparing secondary standards and specific antisera; b) 125 I-labelled hormones; c) specific high titre antisera. The extraction and purification of human growth hormone (hGH) and human luteinizing hormone (hLH) has been already carried out. We have recently obtained quite purified, active and stable preparation starting from a small number of human pituitaries, the whole process being carried out in just one or two weeks for hGH an hLH respectively. For what concernes 125 I-labelling, we basically utilize two techniques: the Classical Chloramine T method described by Hunter and Roth's stoichiometric method, both followed by a quantitative and qualitative analysis of the reaction mixture, set up in our laboratory. The results of such comparison will be discussed. 125-I-hGH, 125-I-hLH, 125-I-hTSH and 125-I-h Calcitonin (unlabelled synthetic product donated by CIBA, Switzerland) have been prepared and tested in internal and external quality control, in comparison with good quality imported products. Satisfactory results have been obtained through the evaluation of these parameters: maximum binding to specific antiserum (Bo), nonspecific binding (NSB), mean effective dose (ED 50), sensitivity, precision and accuracy. Concerning antisera production, specific and high titre rabbit anti-hGH and anti-hLH antibodies have been obtained, while guinea-pig anti-h Calcitonin antiserum is being produced. Together with 125-I-Calcitonin it will be used in the setting up of the RIA of this hormone whic allows the early detection of Medullary Thyroid Carcinoma. (author) [pt

  14. Growth hormone inhibits activation of phosphatidylinositol phospholipase C in adipose plasma membranes: evidence for a growth hormone-induced change in G protein function.

    Science.gov (United States)

    Roupas, P; Chou, S Y; Towns, R J; Kostyo, J L

    1991-03-01

    Pituitary growth hormone (GH) functions physiologically to oppose the actions of insulin on carbohydrate and lipid metabolism by interfering with metabolic events that occur after insulin binds to its receptor. Which postreceptor effects are involved is presently unknown. Recently, we found that insulin rapidly stimulates a phosphatidylinositol phospholipase C (PI-PLC) in adipose tissue of obese (ob/ob) mice and that this effect of insulin is blocked by treatment of the animals with S-carboxymethylated human GH (RCM-hGH), a derivative having mainly anti-insulin activity. The activation of this PI-PLC by insulin is also inhibited by pertussis toxin. Thus, this study was performed to examine whether the inhibitory effect of GH on the activation of this PI-PLC is exerted at the level of signal transmission by guanine nucleotide binding proteins (G proteins). We found that the nonhydrolyzable GTP analogue, guanosine 5'-[gamma-thio]triphosphate, stimulated basal PI-PLC activity in plasma membranes of adipose tissue of saline-treated ob/ob mice, but it did not stimulate the enzyme in adipose membranes from RCM-hGH-treated mice. Also, RCM-hGH treatment markedly inhibited pertussis toxin-catalyzed ADP ribosylation of G protein alpha subunits in the membranes, suggesting some modification of the G proteins by GH. Immunoblot analysis of adipose membranes from saline- and RCM-hGH-treated mice using antiserum AS/7 (anti-Gi1 alpha and anti-Gi2 alpha) or antiserum EC/2 (anti-Gi3 alpha) showed no difference in the amount of Gi alpha-like protein between the groups. These findings suggest that GH interferes with the ability of a putative Gi-like protein to mediate the activation of PI-PLC in adipose membranes without altering the expression of the G protein.

  15. Passive immunization to outer membrane proteins MLP and PAL does not protect mice from sepsis.

    Science.gov (United States)

    Valentine, Catherine H; Hellman, Judith; Beasley-Topliffe, Laura K; Bagchi, Aranya; Warren, H Shaw

    2006-01-01

    Multiple older studies report that immunoglobulin directed to rough mutant bacteria, such as E. coli J5, provides broad protection against challenge with heterologous strains of Gram-negative bacteria. This protection was initially believed to occur through binding of immunoglobulin to bacterial lipopolysaccharide (LPS). However, hundreds of millions of dollars have been invested in attempting to develop clinically-effective anti-LPS monoclonal antibodies without success, and no study has shown that IgG from this antiserum binds LPS. Identification of the protective mechanism would facilitate development of broadly protective human monoclonal antibodies for treating sepsis. IgG from this antiserum binds 2 bacterial outer membrane proteins: murein lipoprotein (MLP) and peptidoglycan-associated lipoprotein (PAL). Both of these outer membrane proteins are highly conserved, have lipid domains that are anchored in the bacterial membrane, are shed from bacteria in blebs together with LPS, and activate cells through Toll-like receptor 2. Our goal in the current work was to determine if passive immunization directed to MLP and PAL protects mice from Gram-negative sepsis. Neither monoclonal nor polyclonal IgG directed to MLP or PAL conferred survival protection in 3 different models of sepsis: cecal ligation and puncture, an infected burn model, and an infected fibrin clot model mimicking peritonitis. Our results are not supportive of the hypothesis that either anti-MLP or anti-PAL IgG are the protective antibodies in the previously described anti-rough mutant bacterial antisera. These studies suggest that a different mechanism of protection is involved.

  16. Identification of mRNA transcripts and immunohistochemical localization of Na/H exchanger isoforms in gerbil inner ear.

    Science.gov (United States)

    Bond, B R; Ng, L L; Schulte, B A

    1998-09-01

    Recent physiological and pharmacological studies have implicated involvement of the Na/H exchanger (NHE) in regulating inner ear ion homeostasis, but the cellular distribution of this membrane transporter remains unknown. Here reverse transcription and the polymerase chain reaction (RT-PCR) were employed to screen adult gerbil inner ears for mRNA transcripts encoding the four best characterized isoforms of NHE. PCR products spanning selected segments of NHE mRNAs were cloned and sequenced. The putative housekeeping gene NHE-1 was found to be expressed and the 459 bp product shared 98.7% amino acid homology with rat sequence. NHE-2, NHE-3 and NHE-4 cDNA transcripts likewise were detected and the PCR products shared 100, 99.4 and 88.9% amino acid homology, respectively, with their rat counterparts. In addition, the cellular distribution of NHE isoforms 1 and 3 was mapped in the gerbil inner ear by immunostaining with polyclonal antisera against rat antigens. In the cochlea, the antiserum against NHE-1 reacted strongly at the basolateral membrane of strial marginal cells as well as with inner and outer hair cells and spiral ganglion neurons. Less intense staining for NHE-1 was present in subpopulations of fibrocytes in the spiral limbus and in inferior and superior areas of the spiral ligament. In the vestibular system dark and transitional cells expressed abundant NHE-1 as did hair cells and vestibular ganglia neurons. Immunostaining with the antiserum against NHE-3 was limited to the apical surface of marginal cells in the stria vascularis. Based on these data, NHE-1 likely functions primarily to maintain intracellular pH levels in cells where it is found in high abundance. NHE-3, on the other hand, possibly participates in the vectorial transcellular movement of Na+ by strial marginal cells thus helping to maintain the extremely low Na+ level in cochlear endolymph.

  17. Primary afferent and spinal cord expression of gastrin-releasing peptide: message, protein, and antibody concerns.

    Science.gov (United States)

    Solorzano, Carlos; Villafuerte, David; Meda, Karuna; Cevikbas, Ferda; Bráz, Joao; Sharif-Naeini, Reza; Juarez-Salinas, Dina; Llewellyn-Smith, Ida J; Guan, Zhonghui; Basbaum, Allan I

    2015-01-14

    There is continuing controversy relating to the primary afferent neurotransmitter that conveys itch signals to the spinal cord. Here, we investigated the DRG and spinal cord expression of the putative primary afferent-derived "itch" neurotransmitter, gastrin-releasing peptide (GRP). Using ISH, qPCR, and immunohistochemistry, we conclude that GRP is expressed abundantly in spinal cord, but not in DRG neurons. Titration of the most commonly used GRP antiserum in tissues from wild-type and GRP mutant mice indicates that the antiserum is only selective for GRP at high dilutions. Paralleling these observations, we found that a GRPeGFP transgenic reporter mouse has abundant expression in superficial dorsal horn neurons, but not in the DRG. In contrast to previous studies, neither dorsal rhizotomy nor an intrathecal injection of capsaicin, which completely eliminated spinal cord TRPV1-immunoreactive terminals, altered dorsal horn GRP immunoreactivity. Unexpectedly, however, peripheral nerve injury induced significant GRP expression in a heterogeneous population of DRG neurons. Finally, dual labeling and retrograde tracing studies showed that GRP-expressing neurons of the superficial dorsal horn are predominantly interneurons, that a small number coexpress protein kinase C gamma (PKCγ), but that none coexpress the GRP receptor (GRPR). Our studies support the view that pruritogens engage spinal cord "itch" circuits via excitatory superficial dorsal horn interneurons that express GRP and that likely target GRPR-expressing interneurons. The fact that peripheral nerve injury induced de novo GRP expression in DRG neurons points to a novel contribution of this peptide to pruritoceptive processing in neuropathic itch conditions. Copyright © 2015 Solorzano et al.

  18. Study on the relationship between enkephalin and neonatal asphyxia

    International Nuclear Information System (INIS)

    Hu Dian; Jin Zhijun; Xiong Ying; Cao Liping; Gu Hang; Hong Xinru; Wang Chenghai

    2003-01-01

    Objective: To evaluate the role of enkephalin in the neonatal asphyxia. Methods: Near-term pregnant rabbits were asphyxiated. The rabbit fetuses were delivered by cesarean section and were asphyxiated with the 1 min Apgar scores ranging at 2 to 7. All the neonatal rabbits delivered by asphyxiated mother rabbits were randomized into four groups: neonatal asphyxia untreated (asphyxia group), neonatal asphyxia treated with ICI 174864 ( ICI group), neonatal asphyxia treated with enkephalin anti-serum (anti-serum group), neonatal asphyxia treated with normal rabbit serum (serum group). A group of intact neonatal rabbits delivered by nonasphyxiated mother rabbits were used as control group. All the study neonatal rabbits were scored according to the respiration, heart beat, skin color, muscle tone, reflexes at 1, 5, 10, 15 and 30 min after cesarean delivery. The concentration of enkephalin was measured by radioimmunoassay. Results: 1) The levels of enkephalin in hypothalamus, pituitary and peripheral blood in asphyxia group were (635.2±57.6), (452.7±37.5) and (297.6±28.4) ng/L, respectively; in the control group, (185.7±29.6), (150.9±21.4) and (121.5±19.9) ng/L, respectively. The levels of enkephalin in asphyxia group were significantly higher than that in the control group (P 0.05). Conclusions: The neonatal asphyxia was associated with enkephalin. The changes of enkephalin levels may play an important role in the pathophysiological changes in neonatal asphyxia; delta-receptors may play certain role in conducting the enkephalin effect

  19. Dynamics of virus shedding and in situ confirmation of chelonid herpesvirus 5 in Hawaiian green turtles with Fibropapillomatosis

    Science.gov (United States)

    Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schettle, Nelli; Ackermann, Mathias

    2015-01-01

    Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

  20. Cholinergic urethral brush cells are widespread throughout placental mammals.

    Science.gov (United States)

    Deckmann, Klaus; Krasteva-Christ, Gabriela; Rafiq, Amir; Herden, Christine; Wichmann, Judy; Knauf, Sascha; Nassenstein, Christina; Grevelding, Christoph G; Dorresteijn, Adriaan; Chubanov, Vladimir; Gudermann, Thomas; Bschleipfer, Thomas; Kummer, Wolfgang

    2015-11-01

    We previously identified a population of cholinergic epithelial cells in murine, human and rat urethrae that exhibits a structural marker of brush cells (villin) and expresses components of the canonical taste transduction signaling cascade (α-gustducin, phospholipase Cβ2 (PLCβ2), transient receptor potential cation channel melanostatin 5 (TRPM5)). These cells serve as sentinels, monitoring the chemical composition of the luminal content for potentially hazardous compounds such as bacteria, and initiate protective reflexes counteracting further ingression. In order to elucidate cross-species conservation of the urethral chemosensory pathway we investigated the occurrence and molecular make-up of urethral brush cells in placental mammals. We screened 11 additional species, at least one in each of the five mammalian taxonomic units primates, carnivora, perissodactyla, artiodactyla and rodentia, for immunohistochemical labeling of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), villin, and taste cascade components (α-gustducin, PLCβ2, TRPM5). Corresponding to findings in previously investigated species, urethral epithelial cells with brush cell shape were immunolabeled in all 11 mammals. In 8 species, immunoreactivities against all marker proteins and ChAT were observed, and double-labeling immunofluorescence confirmed the cholinergic nature of villin-positive and chemosensory (TRPM5-positive) cells. In cat and horse, these cells were not labeled by the ChAT antiserum used in this study, and unspecific reactions of the secondary antiserum precluded conclusions about ChAT-expression in the bovine epithelium. These data indicate that urethral brush cells are widespread throughout the mammalian kingdom and evolved not later than about 64.5millionyears ago. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Multimerized HIV-gp41-derived peptides as fusion inhibitors and vaccines.

    Science.gov (United States)

    Nomura, Wataru; Mizuguchi, Takaaki; Tamamura, Hirokazu

    2016-11-04

    To date, several antigens based on the amino-terminal leucine/isoleucine heptad repeat (NHR) region of an HIV-1 envelope protein gp41 and fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of gp41 have been reported. We have developed a synthetic antigen targeting the membrane-fusion mechanism of HIV-1. This uses a template designed with C3-symmetric linkers and mimics the trimeric form of the NHR-derived peptide N36. The antiserum obtained by immunization of the N36 trimeric antigen binds preferentially to the N36 trimer and blocks HIV-1 infection effectively, compared with the antiserum obtained by immunization of the N36 monomer. Using another template designed with different C3-symmetric linkers, we have also developed a synthetic peptide mimicking the trimeric form of the CHR-derived peptide C34, with ∼100 times the inhibitory activity against the HIV-1 fusion mechanism than that of the monomer C34 peptide. A dimeric derivative of C34 has potent inhibitory activity at almost the same levels as this C34 trimer mimic, suggesting that presence of a dimeric form of C34 is structurally critical for fusion inhibitors. As examples of rising mid-size drugs, this review describes an effective strategy for the design of HIV vaccines and fusion inhibitors based on a relationship with the native structure of proteins involved in HIV fusion mechanisms. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 622-628, 2016. © 2015 Wiley Periodicals, Inc.

  2. Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface.

    Science.gov (United States)

    Handley, P S; Carter, P L; Fielding, J

    1984-01-01

    Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology. Images PMID:6197404

  3. Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

    Energy Technology Data Exchange (ETDEWEB)

    Patters, M.R.; Landsberg, R.L.; Johansson, L.A.; Trummel, C.L.; Robertson, P.R. (Department of Periodontology, University of Connecticut, School of Dental Medicine, Farmington, Connecticut, U.S.A.)

    1982-01-01

    Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated /sup 45/Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis.

  4. Comparison of pig, rabbit and mouse IgG response to Streptococcus suis serotype 2 proteins and active immunization of mice against the infection.

    Science.gov (United States)

    Quessy, S; Dubreuil, J D; Caya, M; Létourneau, R; Higgins, R

    1994-07-01

    The aim of this study was to compare the IgG response of different animal species to Streptococcus suis serotype 2 proteins and to evaluate the immunogenic potential of these proteins in the mouse experimental model of infection. The protein profiles of ten different S. suis capsular type 2 isolates were compared by Western blotting using antisera produced in mice, rabbits and pigs against the reference strain. Strains were grown overnight in Todd-Hewitt broth, harvested by centrifugation, processed in a French press cell and digested with lysozyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then performed and proteins transferred to nitrocellulose. The rabbit antiserum recognized seventeen common immunoreactive proteins, of which, proteins of 33, 44, 96, 122 kDa were present in all strains. Two, 128 and 136 kDa proteins were recognized by swine serum in many strains. An additional protein of 30 kDa was recognized by the mouse antiserum. These seven proteins, originating from the reference strain, were excised directly from polyacrylamide gels, mixed with incomplete Freund's adjuvant and given to groups of five mice on days 0 and 10. Immunoglobulin G response to each protein was monitored on day 20 using Western blots. Mice were then experimentally infected on day 21. Results indicated that vaccination with proteins of 33, 44, 128 and 136 kDa resulted in an IgG response and protection against the challenge with the reference strain, but gave only a partial protection against another virulent S. suis serotype 2 strain.

  5. Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus

    Directory of Open Access Journals (Sweden)

    Qian Biao

    2007-03-01

    Full Text Available Abstract Background Infectious salmon anaemia (ISA virus (ISAV, an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE protein encoded on segment 6 and fusion (F protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV. Results In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1 so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the

  6. Production of antisera and development of radioimmunoassay for serum T3, T4, and ferritin

    International Nuclear Information System (INIS)

    Elhag, Omer Mohamed Abdalla

    1998-05-01

    In this study twelve local rabbits and sixteen New-zealand rabbits were subjected to immunization against T3 and T4 immunogens. Two local sheep (ovis aris) were immunized against human liver ferritin. The T3 and T4 immunogens were prepared by conjugation of the haptens to carrier proteins (bovine serum albumin ''BSA'' and horse serum protein ''HSP''), using water soluble carboiimide as coupling agent. The local and New-zealand rabbits were immunized against these conjugates emulsified in freund's complete adjuvant (FCA) in the first and second injections, and emulsified in freund's incomplete adjuvant (FIA) in the following injections. The blood samples obtained from rabbits after each injection were tested for antibodies as well as for the effect of immunization on rabbits biochemical and haematological parameters. The blood samples obtained from sheep were tested for anti-ferritin antibodies using crude antiserum, then this antiserum was purified using ammonium sulphate. A part of it was adsorbed physically onto polystyrene beads while the other part was linked chemically to magnitisable particles inorder to develop to IRMAs. The purified antiferritin antibody was diluted 200,000 folds before being coated to polystyrene beads, and different dilutions were tried with coupling to magnetic solid phase. Optimization and validation procedures for the two IRMAs ferritin were performed. The results obtained showed poor response of rabbits to immunization against T3 and T4 immunogen conjugates, where the percent bound (B%) of tracer with the antibody ranged from (0.0-22%) for local rabbits using charcoal seperation technique, and (0.0-2.9%) using second antibody precipitation technique. The B% for the antiserum obtained from New-zealand rabbits ranged from (0.0-18.1) using second antibody precipitation technique. Serum T3, T4 and TSH of the immunized rabbits were measured and found to be not significantly different form the controls (p=0.2211, 0.098, 0.35 respectively

  7. Purification and production of monospecific antibody to the hemagglutinin from Subtype H5N1 influenza virus

    Directory of Open Access Journals (Sweden)

    Simson Tarigan

    2010-12-01

    Full Text Available The purpose of this study was to purify the hemagglutinin from H5N1 virus and to generate monospecific antibody appropriate for production of sensitive and specific immunoassay for H5N1 avian influenza. For this purpose, a local isolate H5N1 virus (A/Ck/West Java/Hamd/2006 was propagated in chicken embryos. The viral pellet was dissolved in a Triton-X-100 solution, undissolved viral particles were pelleted by ultracentrifuge, and the supernatant containing viral surface glycoproteins (Hemagglutinin and neuraminidase was collected. The neuraminidase in the supernatant was absorbed by passing the supernatant through an Oxamic-acid-superose column. After dialyzing extensively, the filtrate was further fractionated with an anion exchange chromatography (Q-sepharose column. Proteins adsorbed by the column were eluted stepwisely with 0.10, 0.25, 0.25 and 0.75 M NaCl in 20 mM Tris, ph 8. Hemagglutinin (H5 was found to be eluted from the column with the 0.5 M NaCl elution buffer. The purified H5 was free from other viral proteins based on immunoassays using commercial antibodies to H5N1 nucleoprotein and neuraminidase. When used as ELISA’s coating antigen, the purified H5 proved to be sensitive and specific for hemagglutinin H5. Cross reactions with other type-A-influenza virus, H6, H7 dan H9, were negligibly low. For the production of monospecific antiserum, the purified H5 was separated with SDS-PAGE, the band containing the H5 monomer was cut out , homogenised and injected into rabbits. The antiserum was capable of detecting the presence of inactivated H5N1 virus in a very dilute suspension, with a detection limit of 0.04 heagglutination (HA unit. The purified hemagglutinin and the serum raised against it should be useful for developing specific, sensitive and affordable immunoassay for H5N1 avian influenza.

  8. Mitis group streptococci express variable pilus islet 2 pili.

    Directory of Open Access Journals (Sweden)

    Dorothea Zähner

    Full Text Available Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2-encoded pili to facilitate adhesion to eukaryotic cells.PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains.This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to link strain-specific bacterial interactions and/or tissue tropisms with

  9. Botulism: a laboratory investigation on biological and food samples from cases and outbreaks in Brazil (1982-2001 Botulismo: investigação laboratorial de amostras biológicas e de alimentos de casos e surtos no Brasil (1982-2001

    Directory of Open Access Journals (Sweden)

    Dilma Scala GELLI

    2002-12-01

    Full Text Available Laboratory investigation of botulism from 1982 to 2001 confirmed the occurrence of eight positive outbreaks/cases of botulism in Brazil. From those, type A botulism was observed in seven of them. Biological material of one case (serum and feces was positive in the first step of the bioassay, but the amount of sample was not sufficient for typification. One of the outbreaks that occurred in 2001 was negative for botulinum toxin in samples of serum, gastric washing and feces, collected eight days before the onset of the symptoms in the affected person who was clinically diagnosed as presenting the disease. Other two cases presenting compatible clinical diagnoses presented negative results. However, in those cases, the collection of samples was (1 after antiserum administration or (2 later than eight days of the onset of symptoms. Investigation was performed by mouse bioassay, as described in the Compendium of Methods for the Microbiological Examination of Foods (compiled by American Public Health Association - APHA11, using specific antiserum from Centers for Disease Control (CDC, USA.A investigação laboratorial de botulismo durante 1982-2001, confirma a ocorrência de surtos/casos de botulismo no Brasil. Dentre estes, a toxina botulínica tipo A foi encontrada em 7. O material biológico de 1 caso (soro e fezes foi positivo para a primeira etapa do bioensaio, mas a quantidade do material não foi suficiente para a sua tipificação. Um surto, ocorrido em 2001, foi negativo para a presença de toxina botulínica em amostras de soro, lavado gástrico e fezes coletadas antes de 8 dias da instalação dos sintomas na pessoa afetada com diagnóstico clínico compatível com a doença. Outros 2 casos com diagnóstico clínico compatível foram negativos, porém nestes a coleta de amostras foi (1 depois da administração de anti-soro ao paciente e (2 em período superior a 8 dias do início dos sintomas. A investigação laboratorial foi realizada

  10. Expression and characterization of the UL31 protein from duck enteritis virus

    Directory of Open Access Journals (Sweden)

    Zhu Dekang

    2009-02-01

    Full Text Available Abstract Background Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product. Results The entire ORF of the UL31 was cloned into pET 32a (+ prokaryotic expression vector. Escherichia coli BL21(DE3 competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa was produced on the SDS-PAGE. Over expressed 6×His-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs. Conclusion In this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the

  11. Expression of Galpha14 in sweet-transducing taste cells of the posterior tongue

    Directory of Open Access Journals (Sweden)

    Kim Soochong

    2008-11-01

    Full Text Available Abstract Background "Type II"/Receptor cells express G protein-coupled receptors (GPCRs for sweet, umami (T1Rs and mGluRs or bitter (T2Rs, as well as the proteins for downstream signalling cascades. Transduction downstream of T1Rs and T2Rs relies on G-protein and PLCβ2-mediated release of stored Ca2+. Whereas Gαgus (gustducin couples to the T2R (bitter receptors, which Gα-subunit couples to the sweet (T1R2 + T1R3 receptor is presently not known. We utilized RT-PCR, immunocytochemistry and single-cell gene expression profiling to examine the expression of the Gαq family (q, 11, 14 in mouse taste buds. Results By RT-PCR, Gα14 is expressed strongly and in a taste selective manner in posterior (vallate and foliate, but not anterior (fungiform and palate taste fields. Gαq and Gα11, although detectable, are not expressed in a taste-selective fashion. Further, expression of Gα14 mRNA is limited to Type II/Receptor cells in taste buds. Immunocytochemistry on vallate papillae using a broad Gαq family antiserum reveals specific staining only in Type II taste cells (i.e. those expressing TrpM5 and PLCβ2. This staining persists in Gαq knockout mice and immunostaining with a Gα11-specific antiserum shows no immunoreactivity in taste buds. Taken together, these data show that Gα14 is the dominant Gαq family member detected. Immunoreactivity for Gα14 strongly correlates with expression of T1R3, the taste receptor subunit present in taste cells responsive to either umami or sweet. Single cell gene expression profiling confirms a tight correlation between the expression of Gα14 and both T1R2 and T1R3, the receptor combination that forms sweet taste receptors. Conclusion Gα14 is co-expressed with the sweet taste receptor in posterior tongue, although not in anterior tongue. Thus, sweet taste transduction may rely on different downstream transduction elements in posterior and anterior taste fields.

  12. Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

    Directory of Open Access Journals (Sweden)

    Robert A Eagle

    Full Text Available The activating immunoreceptor NKG2D is expressed on Natural Killer (NK cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised.We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy.We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique

  13. Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

    Science.gov (United States)

    Eagle, Robert A; Flack, Gillian; Warford, Anthony; Martínez-Borra, Jesús; Jafferji, Insiya; Traherne, James A; Ohashi, Maki; Boyle, Louise H; Barrow, Alexander D; Caillat-Zucman, Sophie; Young, Neil T; Trowsdale, John

    2009-01-01

    The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

  14. Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

    Directory of Open Access Journals (Sweden)

    Taverne Marcel AM

    2010-02-01

    Full Text Available Abstract Background The involvement of placental lactogen (PL in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG via a foetal catheter (in vivo study. The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions. Methods Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 μg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000. Result(s Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P Conclusion(s The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.

  15. Development of a melatonin RIA and observation on the plasma melatonin contents in rat models of chronic hyperirritable-depression

    International Nuclear Information System (INIS)

    Rong Yang; Sun Acheng; Ma Cong; Zhao Zhong; Gui Yuning; Li Jianjun; Wang Guangkai; Guo Xiazhen

    2005-01-01

    Objective: To establish a new melatonin assay and to investigate the changes of plasma melatonin content in rat models of chronic hyperirritable-depression. Methods: Quality melatonin antiserum was obtained from immunization of Newzealand white rabbit with melatonin immunogen derived from conjugation of melatonin to bovine thyroglobulin using formaldehyde. Radioiodinated melatonin was used as tracer and a melatonin assay was developed through non-equilibrium competition. Twenty rat models of chronic hyperirritable-depression were prepared with multiple randomly-combined stimuli as previously reported. Plasma and pineal body tissue contents of melatonin in the models were examined in midsummer (n=10) and mid-winter (n=10) with the newly developed melatonin RIA. Contents of melatonin were also determined in 20 control rats. Results: The antiserum possessed very low cross-reaction rate with several melatonin analogous tested (0.09%-2.3%). At the titer of 1:1800, the maximal combination rate was 41%. The affinity constant was 1.7 x 10 9 L/M. The specific radioactivity of the tracer 125 I-melatonin was 55 μCi/μg, with radio-chemical purity of 93% and the tracer was stable at 4 degree C for 65 days. The assay was of high sensitivity (lower detection limit 5pg/ml), intra-CV, 6.5 %; inter-CV, 11%. The plasma and pineal body tissue contents of melatonin in the rat models were consistently significantly lower than those in control rats both during summer and winter, while the contents of melatonin during winter were always significantly higher than those during summer in both groups of animals. Conclusion: The newly developed assay was of good specificity and sensitivity with stable agents (65 days). The experimental results demonstrated definite correlationship between the depression disorder and melatonin contents in the rat models, however, the disorder was not seasonally affective. The seasonal variation of the melatonin contents in the animals was due to different

  16. An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture.

    Science.gov (United States)

    Bee, J A; von der Mark, K

    1990-07-01

    To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin:collagenase dissociation in the absence of calcium. This aggregation is largely Ca2(+)-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2(+)-free trypsin:collagenase dissociation, aggregation of the surviving population is exclusively Ca2(+)-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K = 10(3) Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5 K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregation-inhibiting antiserum, and is

  17. Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

    Directory of Open Access Journals (Sweden)

    Schaudinn Christoph

    2005-01-01

    Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

  18. Evaluation of the Secretor Status of ABO Blood Group Antigens in Saliva among Southern Rajasthan Population Using Absorption Inhibition Method.

    Science.gov (United States)

    Metgud, Rashmi; Khajuria, Nidhi; Mamta; Ramesh, Gayathri

    2016-02-01

    The ABO blood group system was the significant element for forensic serological examination of blood and body fluids in the past before the wide adaptation of DNA typing. A significant proportion of individuals (80%) are secretors, meaning that antigens present in the blood are also found in other body fluids such as saliva. Absorption inhibition is one such method that works by reducing strength of an antiserum based on type and amount of antigen present in the stains. To check the efficacy of identifying the blood group antigens in saliva and to know the secretor status using absorption inhibition method among southern Rajasthan population. Blood and saliva samples were collected from 80 individuals comprising 20 individuals in each blood group. The absorption inhibition method was used to determine the blood group antigens in the saliva and then the results were correlated with the blood group of the collected blood sample. The compiled data was statistically analysed using chi-square test. Blood groups A & O revealed 100% secretor status for both males and females. While blood groups B and AB revealed 95% secretor status. Secretor status evaluation of the ABO blood group antigen in saliva using absorption inhibition method can be a useful tool in forensic examination.

  19. Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

    DEFF Research Database (Denmark)

    Hønberg, L S; Larsen, B; Koch, C

    1995-01-01

    Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16....... To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M(r) 44,000 and M(r) 12000 polypeptides can be precipitated from both...... difference in the pI of the immunoprecipitable components of red and white cells was observed. All together, this indicates that either the blood group antigen M' is the BoLA-A16 class I antigen or M' and BoLA-A16 are two different class I polypeptides with the same relative mass, sharing identical epitopes...

  20. Fragments of the constant region of immunoglobulin light chains are constituents of AL-amyloid proteins

    DEFF Research Database (Denmark)

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

    1998-01-01

    Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin l...... light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously......Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin...... in the purification process. The significance of the constant part in AL-proteins is unclear, but adds new aspects to the discussion of pre- or post-fibrillogenic cleavage of the immunoglobulin light chains....

  1. Measurement of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Karlin, J.B.; Juhn, D.J.; Fless, G.; Scanu, A.M.; Rubenstein, A.H.

    1978-02-01

    A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The antiserum was raised to LDL (d 1.030 to 1.040 g/ml) and the LDL/sub 2/ (d 1.020 to 1.050 g/ml) was labeled with /sup 125/I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02 to 0.5 ..mu..g of LDL/sub 2/, had an interassay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an average American diet, a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet.

  2. Development and clinical application of human gastrin radioimmunoassay

    International Nuclear Information System (INIS)

    Ginabreda, M.G.P.; Borghi, V.C.; Bettarello, A.

    1988-08-01

    The determination of human gastrin levels in the blood is very important for diagnosis of gastrointestinal disorders. This work describes the radioimmunoassay of gastrin developed according to Russell et al. and its clinical application measuring fasting levels of this hormone in normal subjects, gastrectomized, chagasics, patients with chronic renal failure (CRF), pernicious anemia (PA) and Zollinger-Ellison syndrome (ZES). Synthetic human gastrin was used for radioiodination and as standard, while the specific antibody was raised in rabbits. Gastrin was radioiodinated by a modification of the chloramine T technique and purified by anion exchange chromatography in QAE-Sephadex A-25 to a specific activity around 200 uCi/ug. The assays were performed by incubation of 125 I-gastrin, standard gastrin (zero to 500 pmol/l) or unknown samples with the antiserum for 4 days at 4 0 C. The antibody bound and free 125 I-gastrin was separated by adsorption of the latter to the charcoal. The basal gastrin values of normal subjects ranged from 2 to 74 pmol/l, being these levels higher in the chagasics (from 6 to 261 pmol/l). Higher levels of gastrin were determined in patients with CRF (from 12 to 350 pmol/l), PA (from 160 to 680 pmol/l) and with ZES(1010 pmol/l), while very low levels were confirmed in gastrectomized (from 1 to 8 pmol/l). (author) [pt

  3. Novel Drosophila receptor that binds multiple growth factors

    International Nuclear Information System (INIS)

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-01-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

  4. Gonadotropin-releasing hormone in mulberry cells of Saccoglossus and Ptychodera (Hemichordata: Enteropneusta).

    Science.gov (United States)

    Cameron, C B; Mackie, G O; Powell, J F; Lescheid, D W; Sherwood, N M

    1999-04-01

    Mulberry cells are epidermal gland cells bearing a long basal process resembling a neurite and are tentatively regarded as neurosecretory cells. They occur scattered through the ectoderm of the proboscis, collar, and anterior trunk regions of the acorn worms Saccoglossus, usually in association with concentrations of nervous tissue. They contain secretion granules that appear from electron micrographs to be released to the exterior. The granules are immunoreactive with antisera raised against mammalian and salmon gonadotropin-releasing hormone (GnRH). Similar results were obtained with another enteropneust, Ptychodera bahamensis, using antisera raised against tunicate-1 and mammalian GnRH. Mulberry cells were not found in either Cephalodiscus or Rhabdopleura (Hemichordata: Pterobranchia). Extracts of tissues from 4200 Saccoglossus contain an area of immunoreactive GnRH that is detected by an antiserum raised against lamprey GnRH when characterized by high-performance liquid chromatography and radioimmunoassay. This is the first report of the occurrence of GnRH in hemichordates, probably the most primitive group clearly belonging to the chordate lineage. The physiological function of GnRH in enteropneusts is unknown, but an exocrine function appears more likely than an endocrine or neurotransmitter role. Copyright 1999 Academic Press.

  5. Hypercalcaemia in a patient with fatal adenosquamous carcinoma of the colon

    International Nuclear Information System (INIS)

    Links, M.; Ho, Happy; Clingan, Ph.; Diamond, T.

    1994-01-01

    This article reports a rare manifestation of metastatic adenosquamous carcinoma of the colon in a patient presenting with humoral hypercalcaemia of malignancy mediated by parathyroid hormone related peptide (PTHrP). A 58-year-old man with metastatic adenosquamous carcinoma of the colon presented with hypercalcaemia. A technetium bone scan was performed and excluded osteolytic bone secondaries. A negative parathyroid subtraction scan and a low serum immunoreactive parathyroid hormone level made the diagnosis of primary hyperparathyroidism unlikely. The diagnosis of humoral hypercalcaemia of malignancy was considered on the basis of an elevated serum PTHrP level and positive tumour immunoreactivity to PTHrP antiserum. The hypercalcaemia was effectively treated on two occasions with intravenous administration of aminohydroxy propilidene diphosphonate. Despite interventional chemotherapy, the patient died of progressive carcinomatosis. It is concluded that hypercalcaemia is an extremely rare occurrence in carcinoma of the colon, This being the first documented case of humoral hyper-calcaemia of malignancy associated with adenosquamous carcinoma of the colon mediated by PTHrP. 11 refs., 1 fig

  6. Expression and ontogeny of growth hormone (Gh) in the protogynous hermaphroditic ricefield eel (Monopterus albus).

    Science.gov (United States)

    Chen, Dong; Liu, Jiang; Chen, Wanping; Shi, Shuxia; Zhang, Weimin; Zhang, Lihong

    2015-12-01

    Growth hormone (GH) is a single-chain polypeptide hormone mainly secreted by somatotropes of the anterior pituitary gland and is an important regulator of somatic growth in vertebrates including teleosts. In this study, a polyclonal antiserum against ricefield eel Gh was generated and the expression of Gh at the mRNA and protein levels was analyzed. Both RT-PCR and western blot analysis showed that Gh was predominantly expressed in the pituitary glands of ricefield eels. The immunoreactive Gh signals were localized to the multicellular layers of the adenohypophysis adjacent to the neurohypophysis in ricefield eels. Ontogenetic analysis showed that immunoreactive Gh signals could be detected in the pituitary glands of ricefield eel embryos as early as 3 days post-fertilization. During the sex change from female to male, the levels of the immunoreactive Gh signals in the pituitary glands of the ricefield eels peaked at the intersexual stage. These results suggest that Gh in the pituitary glands may be associated with embryonic development before hatching, as well as with the sex change in the adult ricefield eels, possibly via the classical endocrine manner.

  7. Detection of Met-enkephalin: Development of a RIA and of an extraction method for studies on hypophyseal and brain tissues

    International Nuclear Information System (INIS)

    Holl, R.

    1982-01-01

    The thesis describes the development of a method of detecting Met-enkephalin, and the verification of the method's suitability to measurements in extracted tissue. The tissue extraction method reported has been the first step towards the goal of establishing the method for determining Met-enkephalin in cell and tissue extracts, in culture media, plasma and liquor samples. The difficulties involved in the development of the RIA for Met-enkephalin specifically arose from the following peculiarities of the substance: a) Due to the low molecular weight, (574), ME itself does not act as an antigen, antibodies can only be obtained by means of fixation to carrier molecules. b) Enkephalin very rapidly is decomposed by endogenic proteinases. c) The fact that normal physiological processes will produce peptides very similar in sequence requires the RIA to be extremely specific. The method has first been applied to screening measurements of Met-enkephalin concentrations in various brain sectors. The radioimmunological studies have been intended to supplement and verify the immunocytochemical results obtained, with the latter experiments having been made using the same antiserum, in order to improve the basis of comparison between the immunocytochemical results and the findings on antibody specifity obtained from the RIA. (orig./MG) [de

  8. Characterization of renal hyperemia in portal hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Premen, A.J.; Banchs, V.; Go, V.L.W.; Benoit, J.N.; Granger, D.N.

    1986-03-01

    In anesthetized sham-operated control (C) and portal vein stenosed (PVS) rats, renal blood flow (RBF) was measured with radioactive microspheres on days 2, 4, 6, 8, and 10 following surgery. On day 2, only a small increase in RBF (19%) was produced in PVS versus C rats. However, by day 4, a significant increase in RBF (35%) was observed in PVS versus C animals. By day 6, the renal hyperemia in PVS rats reached a maximal value that was 42% higher than in C rats. Thereafter (on days 8 and 10), the renal hyperemia remained at the maximal value. In a separate group of 10-day PVS rats, glucagon antiserum failed to attenuate the 44% increase in RBF observed in PVS versus C rats. Radioimmunoassay of C and PVS plasma (10-day samples) revealed that vasoactive intestinal polypeptide, substance P, cholecystokinin/gastrin, neurotensin, pancreatic polypeptide, beta-endorphin, and peptide histidine-isoleucine amide are not elevated in arterial plasma of PVS rats. These data indicate that the renal hyperemia induced by chronic portal hypertension is manifested within 4 days after the hypertensive insult. Our studies also suggest that at least 9 blood-borne gastrointestinal peptides are not directly involved in the renal response to portal vein stenosis.

  9. Effects of chronic ethanol treatment on the in vitro biosynthesis of pro-opiomelanocortin and its posttranslational processing to beta-endorphin in the intermediate lobe of the rat pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Seizinger, B.R.; Hoellt, V.; Herz, A.

    1984-09-01

    Chronic treatment of rats with 15% (vol/vol) ethanol in tap water as their only source of liquid over a period of 3 weeks resulted in a strong decrease by almost 50% in tissue levels and in vitro release of immunoreactive beta-endorphin of the neurointermediate pituitary. Moreover, the in vitro incorporation of (3H)phenylalanine into peptides of the neurointermediate pituitary, immunoprecipitable with beta-endorphin antiserum, was found to be decreased by more than 30%. Analysis of beta-endorphin-related peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that chronic ethanol treatment reduced the in vitro biosynthesis of the beta-endorphin precursor pro-opiomelanocortin. This ethanol-induced effect was combined with a retardation in the time course of the posttranslational processing of the precursor into beta-endorphin. Thus, chronic ethanol treatment may influence the activity of enzymes which process the opioid peptide precursor pro-opiomelanocortin, leading to a decreased formation of the final secretory product beta-endorphin.

  10. SEED AND POLLEN TTRANSMISSION OF A NEW UNIDENTIFIED MOTTLE DISORDER OF MAIZE IN INDONESIA

    Directory of Open Access Journals (Sweden)

    W. Wakman

    2018-01-01

    Full Text Available A new unidentified mottle disorder of maize Indonesia was found at the Research Institute for Maize and Other Cereals (RIMOC, Maros, South Sulawesi in 1995. Attempts to identify the disorder were made by mechanical inoculation, insect vector (Rhopalosiphum maidis and Peregrinus maidis transmission, seed and pollen transmission, electron microscopy, and serological test. Fifty seeds from each of 22 ears of Arjuna maize plants showing the disorder were planted and symptoms on the seedlings were recorder at 1, 2, and 3 weeks after planting. The percentage of seedlings showing the disorder ranged from 40 to 100. Pollen of affected Arjuna was then used to pollinate four sweet corn female flowers. Hybrid seeds (50 per ear of the crosses were planted and symptoms were recorded at 1, 2, and 3 weeks after planting. The results showed that percentage of seedlings showing the disorder ranged from 22 to 84. Electron microscopy and ELISA tests on 15 viruses and one phytospiroplasma antiserum however, gave negative results. Therefore, maize disorder at Maros was not identical to any known viral disease of maize. It could be a genetical disorder and has been given the name maize mottle.

  11. Synapsin function in GABA-ergic interneurons is required for short-term olfactory habituation.

    Science.gov (United States)

    Sadanandappa, Madhumala K; Blanco Redondo, Beatriz; Michels, Birgit; Rodrigues, Veronica; Gerber, Bertram; VijayRaghavan, K; Buchner, Erich; Ramaswami, Mani

    2013-10-16

    In Drosophila, short-term (STH) and long-term habituation (LTH) of olfactory avoidance behavior are believed to arise from the selective potentiation of GABAergic synapses between multiglomerular local circuit interneurons (LNs) and projection neurons in the antennal lobe. However, the underlying mechanisms remain poorly understood. Here, we show that synapsin (syn) function is necessary for STH and that syn(97)-null mutant defects in STH can be rescued by syn(+) cDNA expression solely in the LN1 subset of GABAergic local interneurons. As synapsin is a synaptic vesicle-clustering phosphoprotein, these observations identify a presynaptic mechanism for STH as well as the inhibitory interneurons in which this mechanism is deployed. Serine residues 6 and/or 533, potential kinase target sites of synapsin, are necessary for synapsin function suggesting that synapsin phosphorylation is essential for STH. Consistently, biochemical analyses using a phospho-synapsin-specific antiserum show that synapsin is a target of Ca(2+) calmodulin-dependent kinase II (CaMKII) phosphorylation in vivo. Additional behavioral and genetic observations demonstrate that CaMKII function is necessary in LNs for STH. Together, these data support a model in which CaMKII-mediated synapsin phosphorylation in LNs induces synaptic vesicle mobilization and thereby presynaptic facilitation of GABA release that underlies olfactory STH. Finally, the striking observation that LTH occurs normally in syn(97) mutants indicates that signaling pathways for STH and LTH diverge upstream of synapsin function in GABAergic interneurons.

  12. Construction of the Coding Sequence of the Transcription Variant 2 of the Human Renalase Gene and Its Expression in the Prokaryotic System

    Directory of Open Access Journals (Sweden)

    Alexei E. Medvedev

    2013-06-01

    Full Text Available Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible genes, their transcript variants, etc.

  13. Prévalence de Acidovorax avenae subsp. avenae, agent des rayures bactériennes du riz dans les semences de base produites au Burkina Faso

    Directory of Open Access Journals (Sweden)

    Dakouo, D.

    2005-01-01

    Full Text Available Prevalence of Bacterial Stripe Organism, Acidovorax avenae subsp. avenae, in Breeder Rice Seed Samples from Burkina Faso. Nine rice seed samples of improved and local varieties were tested at DGISP (Denmark for the incidence of seed-borne bacterial stripe organism, Acidovorax avenae subsp. avenae, using the cassette holder method. Twenty-six suspected bacterial colonies were identified by different methods including colony morphology, pigmentation, biochemical and pathogenicity tests. Using Biolog GN computer identification system, isolates were also identified as A. avenae subsp. avenae (sim 0.51 to 0.9. All the 26 isolates reacted positively in ELISA tests performed with antiserum against A. avenae subsp. avenae. The bacterium was detected in all the samples, except in that of the local variety, indicating that seeds of improved varieties are highly infected by this pathogen. Seedlings raised from infected seed samples showed typical bacterial stripe symptoms with infection rates ranging from 4.7 to 20.1%. Since such seeds are used for production of certified rice seed, it is important to develop an effective control strategy against this disease to reduce the propagation of the bacterial agent in other healthy regions of rice culture in Burkina Faso.

  14. Activation of spinal glucagon-like peptide-1 receptors specifically suppresses pain hypersensitivity.

    Science.gov (United States)

    Gong, Nian; Xiao, Qi; Zhu, Bin; Zhang, Chang-Yue; Wang, Yan-Chao; Fan, Hui; Ma, Ai-Niu; Wang, Yong-Xiang

    2014-04-09

    This study aims to identify the inhibitory role of the spinal glucagon like peptide-1 receptor (GLP-1R) signaling in pain hypersensitivity and its mechanism of action in rats and mice. First, GLP-1Rs were identified to be specifically expressed on microglial cells in the spinal dorsal horn, and profoundly upregulated after peripheral nerve injury. In addition, intrathecal GLP-1R agonists GLP-1(7-36) and exenatide potently alleviated formalin-, peripheral nerve injury-, bone cancer-, and diabetes-induced hypersensitivity states by 60-90%, without affecting acute nociceptive responses. The antihypersensitive effects of exenatide and GLP-1 were completely prevented by GLP-1R antagonism and GLP-1R gene knockdown. Furthermore, exenatide evoked β-endorphin release from both the spinal cord and cultured microglia. Exenatide antiallodynia was completely prevented by the microglial inhibitor minocycline, β-endorphin antiserum, and opioid receptor antagonist naloxone. Our results illustrate a novel spinal dorsal horn microglial GLP-1R/β-endorphin inhibitory pathway in a variety of pain hypersensitivity states.

  15. Radioimmunological dosage of human prolactin

    International Nuclear Information System (INIS)

    Reuter, A; Kennes, F.; Gevaert, Y.; Franchimont, P.

    1976-01-01

    The radioimmunological dosage of human prolactin using the hormone as tracer and reference and an anti-prolactin antiserum is described. The labelling of human prolactin is carried out in the presence of a small quanitity of chloraminet (10 ug). The purification of the labelled hormone is carried out by gel filtration on sephadex G-75 by polyacrylamide gel electrophoresis. The optimal incubation conditions were found using a first incubation of 40-48 hours at room temperature followed by one of 4 to 6 hours with cellulosebound second antibody. The interference of serum proteins is small. There is no cross-reaction with follicle stimulating hormone (FSH), luteinizing hormone (LH), tyroid stimulating hormone (FSH), human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and human growth hormone (HGH). However, cross-reaction with ovine, bovine and rat prolactin is found. These cross-reactions are complete with endogenous human prolactin and hormone of different origin. Serum prolactin was determined in normal subjects and subjects treated with TRH and bromoergocryptine. (G.C.)

  16. Radioimmunoassay of human urinary kallikrein

    International Nuclear Information System (INIS)

    Goering, W.

    1980-01-01

    Using a human urinary kallikrein, purified by means of Trasylol sepharose, it has been possible to develop a radioimmunoassay of kallikrein capable of detecting the substance down to a concentration of 0.5 ng/ml. The specific activity of the tracer labelled with 125-iodine using the Chloramine-T method was 30-70 nCi/ng of kallikrein. The antiserum titres for the antikallikrein serum were 1:20.000 up to 1:50.000. Human urine, submandibular and parotid salivae as well as pancreatic secretion in this RIA reacted in the same manner as the kallikrein standard solution. The kallikrein content in urine, as determined by the RIA was between 0 and 300 ng/ml, in the saliva between 400 and 2.000 ng/ml, and in the pancreatic juice between 300 and 12.000 ng/ml. Using human serum, only an incomplete immunological cross-reaction could be achieved. In human liquor as well as in animal preparation, no cross-reacting substances could be detected. (orig.) [de

  17. Sex differences in the photoperiodic regulation of RF-Amide related peptide (RFRP) and its receptor GPR147 in the syrian hamster

    DEFF Research Database (Denmark)

    Henningsen, Jo B; Poirel, Vincent-Joseph; Mikkelsen, Jens D

    2016-01-01

    RF-(Arg-Phe) related peptides (RFRP-1 and -3) are considered to play a role in the seasonal regulation of reproduction; however, the effect of the peptides depends on species and gender. This study aimed at comparing the RFRP system in male and female Syrian hamsters over long and short photoperi......RF-(Arg-Phe) related peptides (RFRP-1 and -3) are considered to play a role in the seasonal regulation of reproduction; however, the effect of the peptides depends on species and gender. This study aimed at comparing the RFRP system in male and female Syrian hamsters over long and short...... photoperiods to investigate the neuroanatomical basis of these differential effects. The neuroanatomical distribution of RFRP neurons and fibers, revealed using an antiserum recognizing RFRP-1 and -3, as well as GPR147 mRNA, are similar in male and female Syrian hamsters. RFRP neurons are mainly found...... in the anteroventral-periventricular nucleus is higher only in females adjusted to a short photoperiod. Our results suggest that the RFRP system, which is strongly regulated by photoperiod in both male and female Syrian hamsters, is particularly important in females, with a distinct role in the anteroventral...

  18. Sandwich radioimmunolabeling for the study of surface properties of bone marrow lymphocytes

    International Nuclear Information System (INIS)

    Yoshida, Y.; Uchino, H.; Kuribayashi, K.; Shimizu, S.; Konda, S.

    1980-01-01

    A modification of sandwich radioautographic method was applied to the study of surface immunoglobulin and/or specific antigens on small lymphocytes in mouse and human bone marrow. After incubation of marrow cell suspensions at 37 0 C, cells were reacted at 0 0 C for 30 min with graded dilutions of rabbit anti-mouse or anti-human immunoglobulin followed by further reaction with a sheep anti-rabbit immunoglobulin labeled with 125 I. Detectable surface immunoglobulin was demonstrated in approximately one-third of mouse marrow lymphocytes and 20-25% of human marrow lymphocytes. The densities of surface immunoglobulin as assessed by grain counts on individual labeled lymphocytes tended to be lower in the marrow than in spleen or peripheral blood. When the same rabbit antiserum was used to compare the sensitivity of the sandwich method with that of the direct radioautography, the former was found sufficiently sensitive to give a plateau level of labeling without seriously increasing background grains. The advantages of the method are discussed with reference to studies on T and B cell specific antigens on human bone marrow lymphocytes. (Auth.)

  19. Development and evaluation of drug-antibody conjugates for the treatment of human myelogenous leukemia

    International Nuclear Information System (INIS)

    Latif, Z.A.

    1978-01-01

    An immune serum to the Ph 1+ human myelogenous leukemia cell line, K-562, was developed in goats. Following exhaustive absorptions, the antiserum and its immunoglobulin (Ig) fraction were highly cytotoxic for the homologenous cells in vitro in the presence of complement. In a nude mouse-human myelogenous leukemia model system, the Ig inhibits the growth and proliferation of myelosarcomas made up of K-562 cells. At the concentration of 6 mg or more, and beginning at 7 days after transplantation of myelosarcomas in nude mice, the administration of immunoglobulins resulted in the total suppression and subsequent elimination of the tumors. The dose-response relationship between the amount of Ig injected and the growth of myelosarcomas was demonstrated to be linear i.e., the extent of inhibition of tumor growth was directly dependent upon the dose of Ig given. Also, the uptake of 125 I-labelled immunoglobulins by the K-562 myelosarcomas was at least 6-fold higher than that of the corresponding preimmune globulins

  20. Uptake and localization of 131I-labeled anti-calcitonin immunoglobulins in rat medullary thyroid carcinoma tissue

    International Nuclear Information System (INIS)

    Gautvik, K.M.; Svindahl, K.; Skretting, A.; Stenberg, B.; Myhre, L.; Ekeland, A.; Johannesen, J.V.

    1982-01-01

    A medullary carcinoma of the thyroid gland (MCT) which has been transplanted repeatedly under the kidney capsule of Wag/Rij rats secretes calcitonin (CT) spontaneously. From 10-20 weeks after transplantation, immunoreactive serum calcitonin (iCT) is abnormally elevated and continues to rise parallel to tumor growth. The immunoglobulin fraction of the rabbit anti-CT antiserum raised against intact synthetic hormone, was purified and iodinated electrolytically. Specific activities of 131 I-labeled immunoglobulin of 0.008-0.014 mCi/μg protein were obtained with 80% preservation of CT binding activity. Wag/Rij rats with MCT tumor and increased serum iCT concentrations received intravenous injections of 131 I-labeled immunoglobulins (0.54-0.811 mCi). The distribution of radioactivity in the rats was followed for 14 days using external scintigraphy in combination with radioactivity measurements of blood and different organs at the end of the observation period. The distribution of /sup 113m/In was used as a marker for blood distribution. When the radioactivity ratios ( 131 I//sup 113m/In) in tumor and different organs were related to that of blood which was equal to unity, tumor tissue contained 3-6 times higher activity. Nonhyperimmune rabbit immunoglobulins or rabbit antirat prolactin immunoglobulins were not concentrated in MCT tissue, nor did anti-CT immunoglobulins localize in rat prolactin adenomas

  1. Uptake and localization of 131I-labeled anti-calcitonin immunoglobulins in rat medullary thyroid carcinoma tissue

    International Nuclear Information System (INIS)

    Gautvik, K.M.; Svindahl, K.; Skretting, A.; Stenberg, B.; Myhre, L.; Ekeland, A.; Johannesen, J.V.

    1982-01-01

    A medullary carcinoma of the thyroid gland (MCT) which has been transplanted repeatedly under the kidney capsule of Wag/Rij rats secretes calcitonin (CT) spontaneously. From 10--20 weeks after transplantation, immunoreactive serum calcitonin (iCT) is abnormally elevated and continues to rise parallel to tumor growth. The immunoglobulin fraction of the rabbit anti-CT antiserum raised against intact synthetic hormone, was purified and iodinated electrolytically. Specific activities of 131 I-labeled immunoglobulin of 0.008--0.014 mCi/microgram protein were obtained with 80% preservation of CT binding activity. Wag/Rig rats with MCT tumor and increased serum iCT concentrations received intravenous injections of 131 I-labeled immunoglobulins (0.054--0.811 mCi). The distribution of radioactivity in the rats was followed for 14 days using external scintigraphy in combination with radioactivity measurements of blood and different organs at the end of the observation period. The distribution of 113 mIn was used as a marker for blood distribution. When the radioactivity ratios ( 131 I/ 113 mIn) in tumor and different organs were related to that of blood which was set equal to unity, tumor tissue contained 3--6 times higher activity. Nonhyperimmune rabbit immunoglobulins or rabbit antirat prolactin immunoglobulins were not concentrated in MCT tissue, nor did anti-CT immunoglobulins localize in rat prolactin adenomas

  2. [Subcloning of M1 gene fragment of H5N1 influenza virus and its expression in Escherichia coli].

    Science.gov (United States)

    Chen, Ai-jun; Guo, Jian-qiang; Yao, Li-hong; Cheng, Cong-sheng; Liu, Xiao-yu; Fu, Jin-qi; Xu, Peng-wei; Zhang, Zhi-qing

    2011-08-01

    To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein. M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1. The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals. These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.

  3. IDENTIFIKASI ESCHERICHIA COLI O157:H7 SERTA DETEKSI GEN SHIGA LIKE TOXIN 1 DAN 2 ASAL FESES HEWAN, DAGING, DAN FESES MANUSIA

    Directory of Open Access Journals (Sweden)

    I Wayan Suardana

    2010-12-01

    Full Text Available Escherichia coli O157:H7 with the ability to produce shiga-like toxin was isolated from beef, cattle, chicken, and human feces. Due to its importance to human health, it is necessary to identify the genes encoding the production of shiga-like toxin, stx1 and stx2 respectively to further understand the pathogenesis. Isolation of E. coli was done on Eosin Methylene Blue Agar (EMBA, followed by identification on Sorbitol MacConkey Agar (SMAC, latex agglutination test, and H7 antiserum test, respectivelly. The existence of genes stx1 and stx2 in E. coli O157:H7 was confirmed molecularly using PCR method with specific primers LP 30/31 and LP 43/44, Stx2 (F/Stx2 (R respectively. Escherichia coli O157:H7 was isolated from 22 out of 344 samples (6,4%. Some isolates showed gene stx1 and stx2 was detected in two isolates as indicated by a 384 bp band (stx1 gene, 584 bp and 1588 bp bands (stx2 gene respectivelly. The results indicated that local isolates E. coli O157:H7 are potential as a zoonoses agent.

  4. ISOLASI DAN IDENTIFIKASI ESCHERICHIA COLI O157:H7 PADA DAGING SAPI DI KABUPATEN BADUNG PROVINSI BALI. ISOLATION AND IDENTIFICATION ESCHERICHIA COLI O157:H7 ON BEEF AT BADUNG REGENCY PROVINCE OF BALI

    Directory of Open Access Journals (Sweden)

    I Wayan Suardana

    2007-03-01

    Full Text Available Keamanan bahan pangan merupakan masalah yang amat penting bagi konsumen dan industri pangan. Cemaran bakteri Escherichia coli dan Coliform dianggap sebagai indikator sanitasi dalam proses pengolahan bahan pangan. Pelacakan bakteri patogen dalam pangan juga telah dilakukan secara rutin, termasuk yang bersifat zoonosis seperti Escherichia coli O157:H7. Bakteri ini menghasilkan toksin yang dikenal dengan Shiga toxin. Toksin ini dapat menimbulkan diare berdarah, colitis haemorrhagi dan hemolytic uremic syndrome (HUS pada manusia. Dalam penelitian ini dipelajari hubungan antara tingkat cemaran dan insidensi Coliform, E.coli, E.coli O157 dan E.coli O157:H7 pada daging sapi. Bakteri pertama ditumbuhkan pada media EMBA, selanjutnya dipupuk pada media SMAC dan diakhiri dengan uji aglutinasi lateks untuk memastikan keberadaan bakteri E.coli O157 dan uji antiserum H7 untuk memastikan isolat yang diisolasi merupakan isolat E.coli O157:H7. Hasil isolasi dan identifikasi terhadap 89 sampel daging sapi diperoleh hasil rata-rata tingkat cemran coliform dan E.coli sebesar 93,01+ 2,64x103 cfu/g dan

  5. Identifikasi Escherichia coli O157:H7 serta Deteksi Gen Shiga Like Toxin 1 dan 2 Asal Feses Hewan, Daging, dan Feses Manusia

    Directory of Open Access Journals (Sweden)

    I Wayan Suardana

    2016-04-01

    Full Text Available Escherichia coli O157:H7 with the ability to produce shiga-like toxin was isolated from beef, cattle,chicken, and human feces. Due to its importance to human health, it is necessary to identify the genesencoding the production of shiga-like toxin, stx1 and stx2 respectively to further understand the pathogenesis.Isolation of E. coli was done on Eosin Methylene Blue Agar (EMBA, followed by identification on SorbitolMacConkey Agar (SMAC, latex agglutination test, and H7 antiserum test, respectivelly. The existence ofgenes stx1 and stx2 in E. coli O157:H7 was confirmed molecularly using PCR method with specific primersLP 30/31 and LP 43/44, Stx2 (F/Stx2 (R respectively. Escherichia coli O157:H7 was isolated from 22 outof 344 samples (6,4%. Some isolates showed gene stx1 and stx2 was detected in two isolates as indicatedby a 384 bp band (stx1 gene, 584 bp and 1588 bp bands (stx2 gene respectivelly. The results indicatedthat local isolates E. coli O157:H7 are potential as a zoonoses agent.

  6. Faktor Resiko Infeksi Escherichia coli O157:H7 pada Ternak Sapi Bali di Abiansemal, Badung, Bali.

    Directory of Open Access Journals (Sweden)

    Eva Damayanti

    2015-08-01

    Full Text Available Penelitian ini bertujuan untuk mengetahui dan menganalisis faktor resiko yang memengaruhi infeksi E. coli O157:H7 pada ternak sapi bali di Kecamatan Abiansemal. Penelitian diawali dengan pengambilan data epidemiologi yang meliputi umur sapi, jenis kelamin, sistem pemeliharaan, sumber air minum, keadaan cuaca, ketinggian daerah, jenis lantai kandang, kebersihan lantai kandang, kemiringan lantai kandang, dan kebersihan sapi. Selanjutnya dilakukan isolasi dan  identifikasi keberadaan E. coli dengan pengujian pada media eosin methylene blue agar (EMBA, uji biokimia indol, methyl red, voges proskauer, dan citrate serta uji pewarnaan Gram. Isolasi dan identifikasi E. coli O157:H7 dilakukan dengan uji Sorbitol Mac Conkey Agar (SMAC, uji lateks aglutinasi O157, dan diakhiri dengan uji antiserum H7. Hasil penelitian menunjukan nilai Odds Ratio dari faktor resiko yang paling dominan berkontribusi menyebabkan kejadian infeksi E. coli O157:H7 pada ternak sapi bali di Kecamatan Abiansemal adalah faktor kebersihan sapi, umur sapi, dan ketinggian tempat dari permukaan laut, dengan Odds Ratio masing-masing sebesar  2,90; 1,18; dan 1,16. Kesimpulan dari penelitian ini menunjukan bahwa kebersihan sapi, umur sapi, dan ketinggian tempat dari permukaan laut berkontribusi menyebabkan kejadian infeksi E. coli O157:H7 pada ternak sapi di Kecamatan Abiansemal.

  7. Methane producing bacteria: Immunological characterization: Progress report, April 1, 1984--June 30, 1988

    Energy Technology Data Exchange (ETDEWEB)

    Conway de Macario, E.; Macario, A.J.L.; Wolin, M.J.

    1988-01-01

    A major contribution of this research has been a significant advance of the immunology of methanogens and other archaebacteria (e.g., extreme halophiles). The foundations have been laid to begin the immunologic study of microbes which are non-methanogens themselves but are important for the fermentation process. This work helped to make clear that bacterial immunology goes beyond the study of pathogens for man, animals, or plants. Immunology can be applied successfully to the study of isolates of importance to understand evolution, phylogeny, ecology, bio-conversion systems, and to advance methanogenic biotechnology. Immunology holds considerable potential to aid in genetic and genetic engineering manipulations as well as in in situ handling of microbes relevant to methanogenesis. Thus, antibodies can help in the discovery of useful microbes, the generation of improved stains, the selection of desirable microorganisms, and in the monitoring and controlling of bioreactors. Immunogolic work in this new field should generate knowledge and devices relevant to areas such as Biological Energy Research, Ecology of Microorganisms, and Environmental (Sanitary) Engineering. In this regard, this work has contributed a comprehensive antiserum bank, a large panel of calibrated polyclonal antibody probes, and techniques for producing and utilizing these probes in the study of methanogens and related bacteria. 67 refs.

  8. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    L.M.P. Passaglia

    1998-11-01

    Full Text Available NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  9. A novel method for detection of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Zagariya, Alexander M., E-mail: zagariya@uic.edu

    2012-04-15

    There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

  10. Occurrence of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India.

    Science.gov (United States)

    Madhubala, R; Bhadramurthy, V; Bhat, A I; Hareesh, P S; Retheesh, S T; Bhai, R S

    2005-06-01

    Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.

  11. Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

    Science.gov (United States)

    Nieberl, P; Ehrl, C; Pommerrenig, B; Graus, D; Marten, I; Jung, B; Ludewig, F; Koch, W; Harms, K; Flügge, U-I; Neuhaus, H E; Hedrich, R; Sauer, N

    2017-05-01

    Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  12. Isolation and properties of a lectin from sainfoin (Onobrychis viciifolia, Scop.).

    Science.gov (United States)

    Hapner, K D; Robbins, J E

    1979-09-29

    A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.

  13. Tick histamine release factor is critical for Ixodes scapularis engorgement and transmission of the lyme disease agent.

    Directory of Open Access Journals (Sweden)

    Jianfeng Dai

    2010-11-01

    Full Text Available Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF from Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens.

  14. A specific and sensitive radioimmunoassay for human choriogonadotropin

    International Nuclear Information System (INIS)

    Muralidhar, K.; Chaudhuri, G.; Lippes, J.; Bahl, O.P.

    1983-01-01

    A specific and sensitive radioimmunoassay for human choriogonadotropin (hCG) has been developed using rabbit antiserum to chemical analogs of beta subunit of human chorionic gonadotropin prepared by controlled reduction and S-alkylation of its disulfied linkages. The assay was highly specific for hCG as the binding of [ 125 1]-hCG to the antibody was not affected by standard human lutropin, by human male serum, postpartum serum from women, serum from post-menopausal women and human menopausal gonadotropin (Pergonal). The assay was highly sensitive, the minimal detection limit in terms of highly purified hCG (L-129, 12.500 IU/mg) being 1 ng/ml or 0.2 ng/tube (or 12 mlU/ml in terms of WHO 2nd international reference preparation of hCG). Using this assay we were unable to detect any immunoreactive hCG in human tissues like lung, liver and colon. The high specificity, sensitivity, accuracy and reproducibility of the assay make this a highly desirable radioimmunoassay for human choriogonadotropin. (orig.)

  15. Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice

    International Nuclear Information System (INIS)

    Champion, K.M.; Tollaksen, S.L.; Giometti, C.S.; Cook, R.J.

    1994-01-01

    During the analysis of liver protein expression in the offspring of male mice irradiated with fission-spectrum neutrons, one offspring displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacking detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10-formyl-THF DH; 10-formyltetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.6). NH 2 -terminal sequence analysis demonstrated that both proteins share identical sequences in the first 25 residues, and this sequence matches (96% identity) that of rat and human 10-formyl-THF DH. In addition, these proteins showed cross-reactivity to polyclonal antiserum raised against purified rat 10-formyl-THF DH. Southern (DNA) blot analysis revealed a restriction fragment length polymorphism consistent with a deletion mutation in the 10-formyl-THF DH structural gene in homozygous mice. Results of Northern (RNA) blot analysis demonstrated the absence of 10-formyl-THF DH mRNA in mice lacking 10-formyl-THF DH protein. Furthermore, liver cytosolic 10-formyl-THF DH enzymatic activity was undetectable in homozygotes. Measurement of hepatic folate pools showed that in homozygotes the total folate pool is decreased and the level of tetrahydrofolate is markedly depleted. 26 refs., 4 figs., 1 tab

  16. Folic acid in the monkey brain: an immunocytochemical study.

    Science.gov (United States)

    Mangas, A; Coveñas, R; Geffard, K; Geffard, M; Marcos, P; Insausti, R; Dabadie, M P

    2004-05-27

    The present report describes the first visualization of folic acid-immunoreactive fibers in the mammalian central nervous system using a highly specific antiserum directed against this vitamin. The distribution of folic acid-immunoreactive structures was studied in the brainstem and thalamus of the monkey using an indirect immunoperoxidase technique. We observed fibers containing folic acid, but no folic acid-immunoreactive cell bodies were found. In the brainstem, no immunoreactive structures were visualized in the medulla oblongata, pons, or in the medial-caudal mesencephalon, since at this location immunoreactive fibers containing folic acid were only found at the rostral level in the dorsolateral mesencephalon (in the mesencephalic-diencephalic junction). In the thalamus, the distribution of folic acid-immunoreactive structures was more widespread. Thus, we found immunoreactive fibers in the midline, in nuclei close to the midline (dorsomedial nucleus, centrum medianum/parafascicular complex), in the ventral region of the thalamus (ventral posteroinferior nucleus, ventral posteromedial nucleus), in the ventrolateral thalamus (medial geniculate nucleus, lateral geniculate nucleus, inferior pulvinar nucleus) and in the dorsolateral thalamus (lateral posterior nucleus, pulvinar nucleus). The highest density of fibers containing folic acid was observed in the dorsolateral mesencephalon and in the pulvinar nucleus. The distribution of folic acid-immunoreactive structures in the monkey brain suggests that this vitamin could be involved in several mechanisms, such as visual, auditory, motor and somatosensorial functions. Copyright 2004 Elsevier Ireland Ltd.

  17. Immunological development in nestling American kestrels Falco sparverius: post-hatching ontogeny of the antibody response.

    Science.gov (United States)

    Smits, Judit E G; Bortolotti, Gary R

    2008-12-01

    Avian research involving examination of immune function or testing of immunocompetence in wild birds has been based upon information on Galliforms, (chicken and quail) even though they are precocial, whereas most wild species with which ecologists, biologists and toxicologists work are altricial; blind, naked and completely dependent at hatching. Here we begin to address this gap in knowledge, offering insight into the early, post-hatching, humoral immune response in an altricial bird, the American kestrel (Falco sparverius). Over two breeding seasons, nestling kestrels were immunized with a non-pathogenic antigen, dinitrophenol keyhole limpet hemocyanin (DNP-KLH), between 3 and 9 days post-hatching and boostered 6 days later. Background levels, primary and secondary immune responses were measured using an enzyme linked immunosorbent assay. The specificity of our laboratory produced rabbit, anti-kestrel antibody was determined using a double immunodiffusion assay. Results showed the rabbit antiserum to have specific anti-kestrel IgG activity. Birds as young as three days old could successfully mount an antibody response, the magnitude of which increased with age at first vaccination. Early immunization did not compromise growth rate, nor did it affect the maximum secondary response. Comparatively, adult kestrels immunized during the same season and following the same protocol, had antibody levels four times higher than those of the nestlings.

  18. Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

    International Nuclear Information System (INIS)

    Tallant, E.A.; Wallace, R.W.

    1987-01-01

    The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca 2+ /calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125 I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis

  19. Insulin radioimmunoassay kit (125I) using polyethyleneglycol (PEG) and a double antibody separation method

    International Nuclear Information System (INIS)

    Borza, Virginia; Chariton, Delfina; Neacsu, Elena

    1997-01-01

    Insulin is a polypeptide hormone formed from proinsulin in the b-cells of the islets of Langerhans in the pancreas. It has a widespread effect on carbohydrate, lipid and protein metabolism. Diabetes mellitus is the result of an insulin deficiency brought about either by insufficient insulin secretion or by rapid insulin catabolism. The determination of the insulin level is important for differential etiologic diagnosis and subsequent therapy and prognosis. Insulin radioimmunoassay kit provides a sensitive, precise and specific assay for insulin concentration in serum. Standard and insulin in the patient sample compete with tracer for binding sites on an insulin antibody. The antigen-antibody combination, which forms during incubation time, will be separated from free insulin by different methods. The separation technique using the double antibody technique combined with Polyethyleneglycol (PEG) is presented. The results are compared with the separation method using PEG alone and with double antibody technique. Antiserum to insulin was produced in rats immunized with porcine insulin, while rabbits immunized with rat-g globulin were used as a source for the second antibody.The tested PEG was PEG 6000. The best results were obtained using the double antibody at a 1/50 dilution combined with 7.5 PEG solutions. The time for precipitating the antibody bound fraction by this technique was established to be 30 minutes. The results obtained using this method as separation technique for insulin - antibody complex were better than those obtained using the double antibody techniques or PEG as precipitating agent alone. (authors)

  20. Cloning and Immunocytochemical Localization of a Cyclic Nucleotide–Gated Channel α-Subunit to All Cone Photoreceptors in the Mouse Retina

    Science.gov (United States)

    HIRANO, ARLENE A.; HACK, IRIS; WÄSSLE, HEINZ; DUVOISIN, ROBERT M.

    2010-01-01

    Cyclic nucleotide–gated channels (CNGC) are ligand-gated ion channels that open and close in response to changes in the intracellular concentration of the second messengers, 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic guanosine monophosphate. Most notably, they transduce the chemical signal produced by the absorption of light in photoreceptors into a membrane potential change, which is then transmitted to the ascending visual pathway. CNGCs have also been implicated in the signal transduction of other neurons downstream of the photoreceptors, in particular the ON-bipolar cells, as well as in other areas of the central nervous system. We therefore undertook a search for additional cyclic nucleotide–gated channels expressed in the retina. Following a degenerate reverse transcription polymerase chain reaction approach to amplify low-copy number messages, a cDNA encoding a new splice variant of CNGC α-subunit was isolated from mouse retina and classified as mCNG3. An antiserum raised against the carboxy-terminal sequence identified the retinal cell type expressing mCNG3 as cone photoreceptors. Preembedding immunoelectron microscopy demonstrated its membrane localization in the outer segments, consistent with its role in phototransduction. Double-labeling experiments with cone-specific markers indicated that all cone photoreceptors in the murid retina use the same or a highly conserved cyclic nucleotide–gated channel. Therefore, defects in this channel would be predicted to severely impair photopic vision. PMID:10813773

  1. Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Gotlieb, A.I.; Spector, W.

    1981-05-01

    The purpose of this study was to compare the group-cell migration characteristics of endothelial cells (ECs) and smooth muscle cells (SMCs) derived from the same source, the porcine thoracic aorta, as they moved into an experimental in vitro wound. The authors characterized migration by measuring two aspects of the migrating cells: the number of free cells in the wound and the distance of migration of the sheet of cells at the wound edge. The quantitative data showed that ECs migrated into the wound as a sheet of cells, while SMCs migrated as free single cells. In addition, since irradiated cells have been used to study cell migration and since the irradiated cells do undergo some shape changes, the distribution of the cytoskeletal microfilament fibres was compared in migrating irradiated and nonirradiated cells in order to see whether this feature of cell migration was different. Irradiated and nonirradiated migrating ECs showed a strikingly different pattern in the orientation of microfilament bundles when studied by immunofluorescence microscopy with antiserums to myosin and tropomyosin.

  2. Characterization of the chicken muscle insulin receptor

    International Nuclear Information System (INIS)

    Adamo, M.; Simon, J.; Rosebrough, R.W.; McMurtry, J.P.; Steele, N.C.; LeRoith, D.

    1987-01-01

    Insulin receptors are present in chicken skeletal muscle. Crude membrane preparations demonstrated specific 125 I-insulin binding. The nonspecific binding was high (36-55% of total binding) and slightly lower affinity receptors were found than are typically observed for crude membrane insulin binding in other chicken tissues. Affinity crosslinking of 125 I-insulin to crude membranes revealed insulin receptor alpha-subunits of Mr 128K, intermediate between those of liver (134K) and brain (124K). When solubilized and partially purified on wheat germ agglutinin (WGA) affinity columns, chicken muscle insulin receptors exhibited typical high affinity binding, with approximately 10(-10) M unlabeled insulin producing 50% inhibition of the specific 125 I-insulin binding. WGA purified chicken muscle insulin receptors also exhibited insulin-stimulated autophosphorylation of the beta-subunit, which appeared as phosphorylated bands of 92- and 81K. Both bands were immunoprecipitated by anti-receptor antiserum (B10). WGA purified membranes also demonstrated dose-dependent insulin-stimulated phosphorylation of the exogenous substrate poly(Glu,Tyr)4:1. However, unlike chicken liver, chicken muscle insulin receptor number and tyrosine kinase activity were unaltered by 48 hr of fasting or 48 hr of fasting and 24 hr of refeeding. Thus, despite the presence of insulin receptors in chicken muscle showing normal coupling to receptor tyrosine kinase activity, nutritional alterations modulate these parameters in a tissue-specific manner in chickens

  3. Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFlP and DNA sequencing methods

    Directory of Open Access Journals (Sweden)

    Monique Ribeiro Tiba

    Full Text Available Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

  4. Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea.

    Science.gov (United States)

    Baader, C D; Schuchert, P; Schmid, V; Heiermann, Reinhard; Plickert, Günter

    1995-01-01

    An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a λ gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.

  5. Outer membrane vesicles of Pasteurella multocida contain virulence factors.

    Science.gov (United States)

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-10-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50-300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Amp(r) (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  6. Enzymatic and immunological properties of Bungarus flaviceps (red-headed krait venom

    Directory of Open Access Journals (Sweden)

    NH Tan

    2010-01-01

    Full Text Available Bungarus flaviceps (red-headed krait venom presents an intravenous LD50 of 0.32 μg/g and exhibits enzymatic activities similar to other Bungarus toxins. ELISA cross-reactions between anti-Bungarus flaviceps and a variety of elapid and viperid venoms were observed in the current study. Double-sandwich ELISA was highly specific, since anti-B. flaviceps serum did not cross-react with any tested venom, indicating that this assay can be used for species diagnosis in B. flaviceps bites. In the indirect ELISA, anti-B. flaviceps serum cross-reacted moderately with three different Bungarus venoms (9-18% and Notechis scutatus venom, but minimally with other elapid and viperid toxins. The results indicated that B. flaviceps venom shares common epitopes with other Bungarus species as well as with N. scutatus. The lethality of the B. flaviceps venom was neutralized effectively by antiserum prepared against B. candidus and B. flaviceps toxins and a commercial bivalent elapid antivenom prepared against B. multicinctus and Naja naja atra venoms, but was not neutralized by commercial antivenoms prepared against Thai cobra, king cobra and banded krait. These data also suggested that the major lethal toxins of B. flaviceps venom are similar to those found in B. multicinctus and B. candidus venoms.

  7. Development and clinical application of a highly sensitive hCG-β radioimmuno assay with a critical view to cross-reaction with LH

    International Nuclear Information System (INIS)

    Schuessler, B.

    1979-01-01

    A highly sensitive hCG-β-RIA with a lower detection limit of 1 mIU/ml was developed with an hCG-β-antiserum. Despite the crossreaction with LH-concentrations of 30 mIU/ml - 150mIU/ml ocurring between 1 mIU/ml and 5 mIU/ml, also in this region a definite statement on the hCG-concentration could be made, by comparison with the values developed in LH-RIA. The assay for the hCG-determination was applied in carcinomas of the uterine cervix (47%), ovaries (25%), body of the uterus (25%), mamma (17,6B), and prostate (5,8%). Another assay with a sensitivity of 5 mIU/ml was arranged for clinical routine examinations of chorionepithelioma and testis-carcinoma. In contrast to the first two systems which had a reaction time of appr. 100 hrs, we obtained safe results with another hCG-β-RIA with low antibodytiters after 110 min. The sensitivity was 15 mIU/ml. The procedure was found to be valuable for the exclusion of EU-gravidities. (orig.) [de

  8. Properties of human gonadotrophins eluted from human corpus luteum and mouse luteoma LH-HCG receptors

    International Nuclear Information System (INIS)

    Cole, F.E.; Arquembourg, P.C.; Rice, B.F.

    1977-01-01

    Studies were performed to try to determine if gonadotrophins are altered during their interaction with tissue receptors. Immunologic, electrophoretic and binding properties of lactoperoxidase labelled [ 125 I]HLH and [ 125 I]HCG were examined before and after elution from mouse lutomea and human corpora lutea receptor preparations. The anti-HCG used in these studies at a 1:10 000 dilution precipitated 92% of a freshly iodinated [ 125 I]HCG preparation. Receptor eluted [ 125 I]HCG, derived from the same batch of labelled ligand, was virtually quantitatively precipitated by the same dilution of anti-HCG. [ 125 I]HCG eluted from the human corpus luteum was electrophoretically more homogeneous when compared to its heterogenous parent labelled preparation and migrated to a position similar to that of native HCG. In Ouchterlony double diffusion experiments against anti-HCG antiserum, corpus lutheum eluted [ 125 I]HCG and [ 125 I]HLH showed immunologic identity with each other as well as with native HCG and HLH. Receptor eluted [ 125 I]HCG from the mouse luteoma, following in vivo administration via tail vein injection or after incubation in vitro with labelled hormones, was immunologically indistinguishable from native HCG. The electrophoretic mobility of HCG was retarded when HCG was added to extracts of mouse luteoma, liver and kidney. Eluates of mouse luteoma, applied to Bio-Gel columns previously equilibrated with [ 125 I]HCG showed the ability to concentrate [ 125 I]HCG in the high molecular weight column fractions. (author)

  9. Migration of the Fungal Protein Cryptogein within Tobacco Plants 1

    Science.gov (United States)

    Devergne, Jean-Claude; Bonnet, Philippe; Panabières, Frank; Blein, Jean-Pierre; Ricci, Pierre

    1992-01-01

    Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea. ImagesFigure 1Figure 2Figure 4 PMID:16669010

  10. Molecular characterization and immunolocalization of the olfactory co-recepter Orco from two blood-feeding muscid flies, the stable fly (Stomoxys calcitrans, L.) and the horn fly (Haematobia irritans irritans, L.)

    Science.gov (United States)

    Olafson, Pia Untalan

    2012-01-01

    Biting flies are economically important, blood-feeding pests of medical and veterinary significance. Chemosensory-based biting fly behaviors, such as host/nutrient source localization and ovipositional site selection, are intriguing targets for the development of supplemental control strategies. In an effort to expand our understanding of biting fly chemosensory pathways, transcripts encoding the highly conserved insect odorant co-receptor (Orco) were isolated from two representative biting fly species, the stable fly (Scal\\Orco) and the horn fly (Hirr\\Orco). Orco forms a complex with an odor-specific odorant receptor to form an odor-gated ion channel. The biting fly transcripts were predicted to encode proteins with 87% – 94% amino acid similarity to published insect Orco sequences and were detected in various immature stages as well as in adult structures associated with olfaction, i.e. antennae and maxillary palps, and gustation, i.e. proboscis. Further, the relevant proteins were immunolocalized to specific antennal sensilla using anti-serum raised against a peptide sequence conserved between the two fly species. Results from this study provide a basis for functional evaluation of repellent/attractant effects on as yet uncharacterized stable fly and horn fly conventional odorant receptors. PMID:23278866

  11. Molecular characterization and immunolocalization of the olfactory co-receptor Orco from two blood-feeding muscid flies, the stable fly (Stomoxys calcitrans, L.) and the horn fly (Haematobia irritans irritans, L.).

    Science.gov (United States)

    Olafson, P U

    2013-04-01

    Biting flies are economically important blood-feeding pests of medical and veterinary significance. Chemosensory-based biting fly behaviours, such as host/nutrient source localization and ovipositional site selection, are intriguing targets for the development of supplemental control strategies. In an effort to expand our understanding of biting fly chemosensory pathways, transcripts encoding the highly conserved insect odorant co-receptor (Orco) were isolated from two representative biting fly species, the stable fly (Scal\\Orco) and the horn fly (Hirr\\Orco). Orco forms a complex with an odour-specific odorant receptor to form an odour-gated ion channel. The biting fly transcripts were predicted to encode proteins with 87-94% amino acid similarity to published insect Orco sequences and were detected in various immature stages as well as in adult structures associated with olfaction, i.e. the antennae and maxillary palps, and gustation, i.e. the proboscis. Further, the relevant proteins were immunolocalized to specific antennal sensilla using anti-serum raised against a peptide sequence conserved between the two fly species. Results from the present study provide a basis for functional evaluation of repellent/attractant effects on as yet uncharacterized stable fly and horn fly conventional odorant receptors. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  12. Abscess-forming inflammatory granulation tissue with Gram-positive cocci and prominent eosinophil infiltration in cats: possible infection of methicillin-resistant Staphylococcus.

    Science.gov (United States)

    Ozaki, K; Yamagami, T; Nomura, K; Haritani, M; Tsutsumi, Y; Narama, I

    2003-05-01

    We occasionally encounter feline cervical or mesenteric lesions diagnosed histopathologically as abscess or inflammatory granulation tissue with eosinophil infiltration. Gram-positive cocci accompany the lesions. In the present study, such lesions obtained from 27 cats were examined to evaluate the histopathologic features and the nature of the causative bacteria. The average age was 7.3 +/- 3.5 years. No sex predilection was observed. Most frequent locations of the lesions included the abdominal cavity with/without mesenteric lymph nodes (11/27, 41%) and subcutaneous tissue or lymph nodes of the neck (9/27, 33%). Common clinical presentation was a localized mass. Grossly, the lesions contained abscesses in the center and were surrounded by fibrous tissue. Microscopically, the necrotic zone contained bacterial colonies. Large numbers of eosinophils and macrophages infiltrated the area surrounding the necrotic tissue. The surrounding connective fiber-rich granulation tissue demarcated the eosinophilic abscess. The bacteria were Gram-positive cocci in 23 of the 27 cats and were positive for anti-staphylococcus antiserum in 19 of the 23 cats. In 15 out of 17 lesions, the colonies expressed immunoreactivity to penicillin-binding protein 2', which is a drug-resistance gene product of methicillin-resistant Staphylococcus (MRS) species. These findings suggest strongly that MRS causes this type of infectious lesion.

  13. The Experimental Study on Anti-bacterial Potency of Soyum Herbal-acupuncture, Hwangryunhaedoktang & Coptidis rhizoma on Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hyung-Sik Seo

    2006-06-01

    Full Text Available Objectives : The aim of this study was to observe prevention of allergic reactions of Sweet Bee Venom (removing enzyme components from Bee Venom. Methods: Content analysis of Sweet Bee Venom and Bee Venom was rendered using HPLC method and characterization of Anti-Sweet Bee Venom in Rabbit Serum. Clinical observation was conducted for inducement of allergic responses to Sweet BV. Results : 1. Analyzing melittin content using HPLC, Sweet BV contained 34.9% more melittin than Bee venom pharmacopuncture at same concentration. 2. Observing chromatogram of HPLC, removal of the enzyme was successfully rendered on Sweet BV. 3. The anti-serum of Sweet BV showed high titers against melittin and bee venom and relatively low titer against phospholipase A2. 4. After conducting approximately 3,000 cases of Sweet BV administration, not a single case of generalized anaphylatic reaction occurred in clinical observation. 5. Mild compared to the bee venom pharmacopuncture, Sweet BV showed some acute hypersensitive reactions of edema, itchiness, and aching locally. 6. Sweet BV was administered on six patients with previous history of suffering from generalized acute hypersensitive reactions with the bee venom. None of the patients showed allergic reactions with Sweet BV, suggesting it can effectively prevent anaphylatic shock which may occur after the bee venom pharmacopuncture procedure. Conclusion : Summarizing above results, Sweet Bee Venom appears to be an effective measurement against allergic reactions from the bee venom pharmacopuncture especially against anaphylatic shock.

  14. Oral DNA Vaccine in Chickens

    Directory of Open Access Journals (Sweden)

    Seyed Davoud Jazayeri

    2012-01-01

    Full Text Available Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2% and MCF-10A (0.5% human breast cancer cells. Newly hatched specific-pathogen-free (SPF chicks were inoculated once by oral gavage with 109 colony-forming unit (CFU of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH and polymerase chain reaction (PCR were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

  15. Post-embedding tem signal-to-noise ratio of S-100

    Science.gov (United States)

    Fermin, C. D.; Lee, D. H.; Martin, D.

    1994-01-01

    We assessed the reactivity of purified S-100 antiserum in immuno-electron microscopy by counting the number of gold particles per microns 2 over inner ear tissues embedded in different media. Sections containing predominantly Schwann's cell cytoplasm and nucleus, afferent fiber axoplasm and myelin sheath of chick cochleae were reacted with anti-S-100 IgG, an antibody to a calcium binding protein of neuronal tissues, then labeled with anti-IgG-gold conjugate. This investigation was conducted because previously published procedures, unmodified, did not yield acceptable results. Preparation of all specimens was identical. Only the medium (PolyBed 812, Araldite or Spurr epoxies; and LR White, LR Gold or Lowicryl plastics) was changed. The medium was made the changing variable because antigens available in post-embedding immuno-electron microscopy are decreased by heat, either used and/or released during polymerization of the embedding medium. The results indicate that: (a) none of the embedding media above provided optimal signal-to-noise ratio for all parts of the nerve stained in the same section; (b) aggregation of gold particles over cells was highest in embedding media with high background labeling over areas devoid of tissue (noise); (c) aggregation occurred randomly throughout both cellular and acellular regions; and (d) particles aggregated less and were distributed more evenly in tissues from media yielding good ultrastructural integrity.

  16. Ovarian and placental morphology and endocrine functions in the pregnant giraffe (Giraffa camelopardalis).

    Science.gov (United States)

    Wilsher, S; Stansfield, F; Greenwood, R E S; Trethowan, P D; Anderson, R A; Wooding, F B W; Allen, W R

    2013-06-01

    Gross, histological and immunocytochemical examinations carried out on maternal and fetal reproductive tissues from two pregnant giraffes at an estimated 8 and 13.5 months of gestation (term=15 months) revealed a typically ruminant macrocotyledonary placenta with binucleate trophoblast cells scattered sparsely in the placentome where they stained intensely with a prolactin antiserum. Binucleate cells were present in greater numbers in the intercotyledonary allantochorion where they did not stain for prolactin whereas the uninucleate trophoblast still did. A single large corpus luteum of pregnancy and several small luteinised follicles were present in the maternal ovaries while the fetal ovaries at 13.5 months gestation showed an assortment of enlarging antral follicles and partially and completely lutenised follicles, the granulosa and luteal cells of which stained positively for 3β-hydroxysteroid dehydrogenase (3β-HSD), 17,20 lyase, prolactin, progesterone receptor and androgen receptor, but negatively for aromatase. The uninucleate trophoblast of the placentome and intercotyledonary allantochorion, the epithelium of the maternal endometrial glands, the seminiferous epithelium in the fetal testis at 8 months of gestation and the zonae fasciculata and reticularis of the fetal adrenal at 13.5 months also stained positively for 3β-HSD and negatively for aromatase. Endocrinologically, it appears that the giraffe placenta is more similar to that of the sheep than the cow with a placental lactogen as the likely driver of the considerable degree of luteinisation seen in both the maternal and the fetal ovaries.

  17. Pathogenic characteristics of a novel triple-reasserted H1N2 swine influenza virus.

    Science.gov (United States)

    Liu, Huili; Tao, Jie; Zhang, Pengchao; Yin, Xiuchen; Ha, Zhuo; Zhang, Chunling

    2016-07-01

    A novel triple reasserted H1N2 virus A/swine/Shanghai/1/2007 (SH07) was isolated from nasal swabs of weaned pig showing clinical symptoms of coughing and sneezing. To explore the virus characteristics, mice, chickens and pigs were selected for pathogenicity study. Pigs inoculated intranasally with 10(6) TCID50 SH07 showed clinical symptoms with coughing and sneezing, but no death. The virus nuclear acid was detected in many tissues using real-time PCR, which was mainly distributed in respiratory system particularly in the lungs. The virus was low-pathogenic to chickens with 10(6) TCID50 dose inoculation either via intramuscular or intranasal routes. However virus nuclear acid detection and virus isolation confirmed that the virus can also be found in nasal and rectum. When virus was inoculated into mice by intramuscular or intranasal routes we observed 100% and 80% lethality respectively. The third generation of samples passaged on MDCK cell were SIV positive in indirect immunofluorescence assay (IFA) using antiserum against H1N2 SIV. Furthermore, the lungs of mice showed obvious lesion with interstitial pneumonia. Data in our study suggest that SH07 is preferentially pathogenic to mammals rather than birds although it is a reasserting virus with the fragments from swine, human and avian origin. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  18. Expression, characterisation and antigenicity of a truncated Hendra virus attachment protein expressed in the protozoan host Leishmania tarentolae.

    Science.gov (United States)

    Fischer, Kerstin; dos Reis, Vinicius Pinho; Finke, Stefan; Sauerhering, Lucie; Stroh, Eileen; Karger, Axel; Maisner, Andrea; Groschup, Martin H; Diederich, Sandra; Balkema-Buschmann, Anne

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Enteropathogenic Escherichia coli associated with diarrhea in children in Cairo, Egypt.

    Science.gov (United States)

    Behiry, Iman K; Abada, Emad A; Ahmed, Entsar A; Labeeb, Rania S

    2011-01-01

    In this study we isolate and identify the Enteropathogenic Escherichia coli (EPEC) causing diarrhea in children less than five years in Cairo, Egypt, during different seasons. Children younger than five years with diarrhea, attending the Pediatric Gastroenterology Intensive Care Unit of the Cairo University Pediatric Hospital in one year period were our group of study. Our control group was age and sex matched concurrent healthy children. The identified E. coli isolates were subjected to antimicrobial disc diffusion susceptibility test and further identified for EPEC serotype by slide agglutination test, using antiserum E. coli somatic trivalent I (O111, O55, O26) according to the instructions of the manufacturer. Out of 134 patients 5.2% of them revealed EPEC in the fecal sample, while the 20 children control group showed no EPEC isolates in their samples. Our EPEC frequency showed variations from the compared results of other studies. Higher rate of EPEC (18.7%) was found in patients between 2 to 3 years, while EPEC rate was (7.5%) in patients less than 6 months old, with P chronic diarrhea in children. EPEC were detected in all seasons except in winter, and was predominant in summer season. Four (57%) EPEC isolates were resistant to ampicillin, ticarcillin, and cotrimoxazole, and (14.3%) to the third generation cephalosporins.

  20. Enteropathogenic Escherichia coli Associated with Diarrhea in Children in Cairo, Egypt

    Directory of Open Access Journals (Sweden)

    Iman K. Behiry

    2011-01-01

    Full Text Available In this study we isolate and identify the Enteropathogenic Escherichia coli (EPEC causing diarrhea in children less than five years in Cairo, Egypt, during different seasons. Children younger than five years with diarrhea, attending the Pediatric Gastroenterology Intensive Care Unit of the Cairo University Pediatric Hospital in one year period were our group of study. Our control group was age and sex matched concurrent healthy children. The identified E. coli isolates were subjected to antimicrobial disc diffusion susceptibility test and further identified for EPEC serotype by slide agglutination test, using antiserum E. coli somatic trivalent I (O111, O55, O26 according to the instructions of the manufacturer. Out of 134 patients 5.2% of them revealed EPEC in the fecal sample, while the 20 children control group showed no EPEC isolates in their samples. Our EPEC frequency showed variations from the compared results of other studies. Higher rate of EPEC (18.7% was found in patients between 2 to 3 years, while EPEC rate was (7.5% in patients less than 6 months old, with <0.05. EPEC was identified from fecal specimens as a unique pathogen or associated with other pathogens in acute and chronic diarrhea in children. EPEC were detected in all seasons except in winter, and was predominant in summer season. Four (57% EPEC isolates were resistant to ampicillin, ticarcillin, and cotrimoxazole, and (14.3% to the third generation cephalosporins.

  1. Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein.

    Science.gov (United States)

    Babij, Konrad; Bajzert, Joanna; Dąbrowska, Anna; Szołtysik, Marek; Zambrowicz, Aleksandra; Lubec, Gert; Stefaniak, Tadeusz; Willak-Janc, Ewa; Chrzanowska, Józefa

    2015-11-01

    In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

  2. A coproantigen diagnostic test for Strongyloides infection.

    Directory of Open Access Journals (Sweden)

    Alex M Sykes

    Full Text Available Accurate diagnosis of infection with the parasite Strongyloides stercoralis is hampered by the low concentration of larvae in stool, rendering parasitological diagnosis insensitive. Even if the more sensitive agar plate culture method is used repeated stool sampling is necessary to achieve satisfactory sensitivity. In this manuscript we describe the development of a coproantigen ELISA for diagnosis of infection. Polyclonal rabbit antiserum was raised against Strongyloides ratti excretory/secretory (E/S antigen and utilized to develop an antigen capture ELISA. The assay enabled detection of subpatent rodent S. ratti and human S. stercoralis infection. No cross-reactivity was observed with purified E/S from Schistosoma japonicum, the hookworms Ancylostoma caninum, A. ceylanicum, nor with fecal samples collected from rodents harboring Trichuris muris or S. mansoni infection. Strongyloides coproantigens that appear stable when frozen as formalin-extracted fecal supernatants stored at -20 °C remained positive up to 270 days of storage, whereas supernatants stored at 4 °C tested negative. These results indicate that diagnosis of human strongyloidiasis by detection of coproantigen is an approach worthy of further development.

  3. 3D-reconstructions and virtual 4D-visualization to study metamorphic brain development in the sphinx moth Manduca sexta

    Directory of Open Access Journals (Sweden)

    Wolf Huetteroth

    2010-03-01

    Full Text Available During metamorphosis, the transition from the larva to the adult, the insect brain undergoes considerable remodeling: New neurons are integrated while larval neurons are remodeled or eliminated. One well acknowledged model to study metamorphic brain development is the sphinx moth Manduca sexta. To further understand mechanisms involved in the metamorphic transition of the brain we generated a 3D standard brain based on selected brain areas of adult females and 3D reconstructed the same areas during defined stages of pupal development. Selected brain areas include for example mushroom bodies, central complex, antennal- and optic lobes. With this approach we eventually want to quantify developmental changes in neuropilar architecture, but also quantify changes in the neuronal complement and monitor the development of selected neuronal populations. Furthermore, we used a modeling software (Cinema 4D to create a virtual 4D brain, morphing through its developmental stages. Thus the didactical advantages of 3D visualization are expanded to better comprehend complex processes of neuropil formation and remodeling during development. To obtain datasets of the M. sexta brain areas, we stained whole brains with an antiserum against the synaptic vesicle protein synapsin. Such labeled brains were then scanned with a confocal laser scanning microscope and selected neuropils were reconstructed with the 3D software AMIRA 4.1.

  4. Establishment of a method of murine obestatin RIA and its primary application

    International Nuclear Information System (INIS)

    Yan Guangtao Lin Ji; Hao Xiuhua; Xue Hui; Den Zihui; Di Dongdong

    2009-01-01

    Objective: To develop a method of murine obestatin RIA and study the role obestatin played in traumatic stress responses. Methods: New Zealand white rabbits were immunized with synthetic murine obestatin to obtain anti-serum, while chloramines-T method was used to iodinate obestatin antigen and a method of RIA for obestatin was established. A mouse sepsis model made with cecal ligation and puncture was established. Serum obestatin levels in the sacrificed models were determined with this method of RIA at 3h, 6h, 9h after the cecum puncture respectively (5 animals each time) as well as in 5 animals after sham operation. Results: Both the shape of standard curve and metrical results of the obestatin RIA were satisfactory. Serum obstatin levels at 6h after injury were significantly higher than those in animals with sham operation out the levels at 3h and 9h were not much different from those in sham operation animals. Meanwhile, the levels at 6h after injury expressed a trend to be significantly higher than that at 3h or 9h after injury. Conclusion: The established method for murine obestatin RIA is highly sensitive, simple and reliable, and it can be used to detect samples from rats and mice. Obestatin may be a traumatic stress factor participating in the modulation of homeostasis after sepsis. (authors)

  5. Modulation of immunoglobulin production by invariant Vα19-Jα33 TCR-bearing cells.

    Directory of Open Access Journals (Sweden)

    Michio Shimamura

    Full Text Available We have previously shown that invariant Vα19-Jα33 TCR(+ (Vα19i T cells suppress the disease progress in some models for organ specific autoimmune diseases and type IV allergy that deteriorate along with decline to excess in Th1- or Th17- immunity. In this study, we examined the effects of over-generation of Vα19i T cells on the Th2-controlled immunoglobulin isotype production in the models for type I allergy. IgE production by invariant Vα19-Jα33 TCR transgenic (Tg mice was suppressed compared with that by non-Tg controls following administration with goat anti-mouse IgD antiserum or OVA, while IgG2a production was not influenced by the introduction of the transgene into the recipients. IgE production by wild type mice was similarly reduced when they were subjected to adoptive transfer with invariant Vα19-Jα33 TCR Tg(+ but not Tg(- cells prior to immunization. Furthermore, the suppression of IgE production by these recipients was enhanced when they were previously administered with a Vα19i T cell activator, one of the modified α-mannosyl ceramides. In summary, it is suggested that Vα19i T cells have potential to participate in the homeostasis of immunity and that they suppress disease progression resulting from not only Th1- but also Th2- immunity excess.

  6. Radioimmunoassay of free urinary 18-hydroxydeoxycorticosterone (18-OH-DOC) in patients with essential hypertension

    International Nuclear Information System (INIS)

    Hornung, J.; Gless, K.H.; Abdelhamid, S.; Vielhauer, W.; Vecsei, P.

    1978-01-01

    Specific antiserum was raised in white New Zealand rabbits using 18-hydroxydeoxycorticosterone-3-oxime-BSA complex as antigen. The urinary free 18-OH-DOC was estimated after dichloromethane extraction and separation in one paper chromatographic system (propylene glycol/toluene). The mean 18-OH-DOC excretion value (+-S.D.) in normal subjects was 0.861+-0.527 μg/24 h (n=23). ACTH produced a 25-fold increase in the excretion of free 18-OH-DOC. Dexamethasone suppressed the values to the lower range of sensitivity. 32% of patients of essential hypertension showed a moderate increase in the free urinary 18-OH-DOC values. The mean value (+-S.D.) in the low renin hypertension group was 2.50 +- 1.49 μg/24 h (n=19), in the normal renin patient group 1.84 +- 1.32 μg/24 h (n=38), and in the group with elevated plasma renin 1.61 +- 1.22 μg/24 h (n=8). The difference between controls and the hypertensive groups was statistically significant. Among the different hypertensive groups significant differences could not be calculated. (Auth.)

  7. Radioimmunoassay of plasma 18-hydroxy-11-deoxycorticosterone and its response to ACTH

    International Nuclear Information System (INIS)

    May, C.N.; Lewis, P.S.; Horth, C.E.

    1979-01-01

    A radioimmunoassay for 18-OH-DOC in plasma was developed using an antiserum raised against the γ-lactone derivative. The steroids with the greatest degree of cross reaction were 18-OH-corticosterone-γ-lactone and aldosterone-γ-lactone which showed cross-reactivities of 1.96% and 0.47% respectively. These and other interfering steroids were eliminated by chromatography of the extracts on columns of Sephadex LH-20. The lowest limit of detection of 18-OH-DOC in 1 ml of plasma corresponded to 33 pmol -1 . The intra-assay precision was 9.7, 4.8 and 2.6% at 102.0, 316.1 and 1144.0 pmol l -1 respectively and the interassay precision was 15.3 and 5.4% at 71.3 and 404.7 pmol l -1 respectively. The concentration of 18-OH-DOC in ten normal subjects was 172.1 +- 39.1 pmol l -1 at 09.00 h, 100.9 +- 16.9 pmol l -1 at 12.00 h and 95.8 +-33.3 pmol l -1 at 16.30 h. Plasma 18-OH-DOC and cortisol levels were measured after various intravenous doses of ACTH in three patients with essential hypertension. Lower doses of ACTH caused considerably greater increases in 18-OH-DOC. These results confirm the ACTH dependency of 18-OH-DOC secretion. (author)

  8. Short communication: ELISA system for screening of bovine mastitis caused by Prototheca zopfii.

    Science.gov (United States)

    Kano, Rui; Sato, Ayano; Sobukawa, Hideto; Sato, Yuko; Ito, Takaaki; Suzuki, Kazuyuki; Hasegawa, Atsuhiko; Kamata, Hiroshi

    2016-08-01

    Prototheca zopfii is an achlorophyllic alga that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. This study evaluated the use of an ELISA system for distinguishing cows with mastitis due to P. zopfii genotype 2 from healthy cows and cows with chronic candidal mastitis. We also investigated the transitional changes of specific antibody titers in healthy cows injected with inactivated P. zopfii genotype 2 cells. The ELISA system exhibited the highest sensitivity (94%) and specificity (100%) for chronic protothecal mastitis when the positive cutoff value was set at 43.4 ELISA units. Anti-protothecal IgG titers were positive in all cows after they were inoculated with inactivated P. zopfii genotype 2 cells. These results indicated that ELISA detection of anti-protothecal IgG in serum provided specificity and sensitivity sufficient for diagnosing protothecal mastitis. Thus, an ELISA system incorporating this specific antiserum is expected to be valuable for definitive field-based diagnosis of bovine mastitis due to P. zopfii genotype 2. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Isolation of Aureimonas altamirensis, a Brucella canis-like bacterium, from an edematous canine testicle.

    Science.gov (United States)

    Reilly, Thomas J; Calcutt, Michael J; Wennerdahl, Laura A; Williams, Fred; Evans, Tim J; Ganjam, Irene K; Bowman, Jesse W; Fales, William H

    2014-11-01

    Microbiological and histological analysis of a sample from a swollen testicle of a 2-year-old Border Collie dog revealed a mixed infection of the fungus Blastomyces dermatitidis and the Gram-negative bacterium Aureimonas altamirensis. When subjected to an automated microbial identification system, the latter isolate was provisionally identified as Psychrobacter phenylpyruvicus, but the organism shared several biochemical features with Brucella canis and exhibited agglutination, albeit weakly, with anti-B. canis antiserum. Unequivocal identification of the organism was only achieved by 16S ribosomal RNA gene sequencing, ultimately establishing the identity as A. altamirensis. Since its first description in 2006, this organism has been isolated infrequently from human clinical samples, but, to the authors' knowledge, has not been reported from a veterinary clinical sample. While of unknown clinical significance with respect to the pathology observed for the polymicrobial infection described herein, it highlights the critical importance to unambiguously identify the microbe for diagnostic, epidemiological, infection control, and public health purposes. © 2014 The Author(s).

  10. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats.

    Directory of Open Access Journals (Sweden)

    Gloria E Hoffman

    Full Text Available A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC, would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness.

  11. Identification of a Unique Ganglioside Binding Loop within Botulinum Neurotoxins C and D-SA

    Energy Technology Data Exchange (ETDEWEB)

    Karalewitz, Andrew P.-A.; Kroken, Abby R.; Fu, Zhuji; Baldwin, Michael R.; Kim, Jung-Ja P.; Barbieri, Joseph T. (MCW); (Missouri)

    2010-09-22

    The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

  12. Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa.

    Science.gov (United States)

    Nuss, Andrew B; Brown, Mark R

    2018-03-01

    Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Feeding Patterns of Mosquitoes (Diptera: Culicidae) in the Atlantic Forest, Rio de Janeiro, Brazil.

    Science.gov (United States)

    Alencar, Jeronimo; Mello, Cecília Ferreira de; Gil-Santana, Hélcio R; Giupponi, Alessandro Ponce de Leão; Araújo, Andressa Nunes; Lorosa, Elias Seixas; Guimarães, Anthony Érico; Silva, Júlia Dos Santos

    2015-09-01

    The stomach contents of culicids from the Atlantic Forest in Rio de Janeiro state, Brazil, were analyzed using the precipitin technique to evaluate the feeding patterns of the species. Sampling was performed from February 2012 to December 2013, using CO2-baited Centers for Disease Control and Prevention traps to catch mosquitoes from 15 00 to 07 00 hours. The following antisera were used: bird, rodent, opossum, human, horse, capybara, lizard, and frog. Of the 325 adult bloodfed females caught and analyzed, 273 (84.0%) reacted in the precipitin test. The percentage of specimens with a positive reaction to a single antiserum included bird (39.2%), rodent (22.5%), opossum (13.2%), capybara (6.6%), horse (5.7%), frog (6.2%), human (4.0%), and lizard (2.6%). The specimens that reacted positively against more than one blood source (46) most frequently presented the following combinations: bird + rodent and bird + frog (17.4%), followed by bird + human (13.0%). The predominance of positive results for birds suggested that the avian-rich environment might have influenced the feeding behavior of the culicids. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Feeding patterns of Haemagogus capricornii and Haemagogus leucocelaenus (Diptera: Culicidae) in two Brazilian states (Rio de Janeiro and Goiás).

    Science.gov (United States)

    Alencar, Jeronimo; Marcondes, Carlos Brisola; Serra-Freire, Nicolau Maués; Lorosa, Elias Seixas; Pacheco, Juliana Barreto; Guimarães, Anthony Erico

    2008-09-01

    We present the identification of bloodfeeding sources of Haemagogus (Haemagogus) capricornii Lutz and Haemagogus (Conopostegus) leucocelaenus (Dyar & Shannon) (Diptera: Culicidae) from different regions of Brazil, as analyzed by precipitin tests. Anti-sera for bird, bovine, equine, human, opossum, and rodent were used. Two hundred one mosquitoes were examined (147 Hg. leucocelaenus and 54 Hg. capricornii), of which 177 reacted for some anti-serum. For Hg. leucocelaenus, 86 (68.3%) reacted to one blood source, 38 (30.2%) to two sources, and two (1.6%) to three sources; the combinations of bird + human (18.4%), bird + rodent (15.8%), and bird + marsupial (15.8%) were the most frequent. For Hg. capricornii, 34 (66.7%) reacted to one blood source; combinations bird + rodent (37.5%) and bird + marsupial (25%) were the most frequent combinations. Mosquito preference for bloodfeeding sources was different in these areas, possibly because of the availability of sources. This diversity of sources can have important epidemiological implications.

  15. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.

    Science.gov (United States)

    Farci, P; Shimoda, A; Wong, D; Cabezon, T; De Gioannis, D; Strazzera, A; Shimizu, Y; Shapiro, M; Alter, H J; Purcell, R H

    1996-12-24

    The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

  16. Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli

    Directory of Open Access Journals (Sweden)

    Rajib Deb

    2010-01-01

    Full Text Available Mycobacterium avium subsp. paratuberculosis (Map contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9 kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into Escherichia coli vector pQE30 UA. The recombinant plasmid designated as pQPPE was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 37.1 kDa His-fusion protein (34.9 kDa PPE and 2.2 kDa His-tag, which was confirmed by immunoblotting. Recombinant PPE protein was then purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant PPE protein reacted with expressed 37.1 kDa His-fusion protein as well as with Map sonicate. The protein elicited significant delayed type hypersensitivity (DTH skin reaction in mice sensitized with Map. The results indicated that the recombinant PPE protein of Map was associated with cellular immune response.

  17. r-Sm14 - pRSETA efficacy in experimental animals

    Directory of Open Access Journals (Sweden)

    Ramos Celso Raul Romero

    2001-01-01

    Full Text Available Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14 produced in Escherichia coli by the pGEMEX system (Promega. The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

  18. Oral beta-stimulants can inhibit passive cutaneous anaphylaxis in rats through an indirect inhibitory mechanism: possible involvement of afferent and efferent nervous system via gastric beta2-adrenoceptor stimulation.

    Science.gov (United States)

    Shibata, H; Minami, E; Hirata, R; Nabe, T; Kohno, S

    2000-12-01

    We previously demonstrated that oral l-ephedrine exerts an extremely rapid (within 20 s) inhibition of 48-h passive cutaneous anaphylaxis reaction (PCA) in rats by a possibly unidentified mode of action. In the present experiments, we elucidated the mechanism of the PCA inhibition by l-ephedrine using adrenoceptor agonists and antagonists. Rat antiserum was prepared with dinitrophenylated Ascaris suum extract + Bordetella pertussis. Passively skin-sensitised Wistar rats were mainly used. l-Ephedrine, and adrenoceptor agonists and antagonists were orally administered immediately before PCA provocation. Catecholamine depleting (6-hydroxydopamine, 6-OHDA), amine depleting (reserpine) or ganglion blocking (hexamethonium) agent was intraperitoneally or intravenously administered before the provocation. The effects of the drugs on PCA were assessed by inhibition of the dye leakage. beta-(propranolol) and beta2-(butoxamine) blocking agents reduced the inhibition of PCA by l-ephedrine, while the inhibition was not altered by either an a-blocking agent (phentolamine) or a beta1-(atenolol) selective antagonist. On the other hand, beta-(isoproterenol) and beta2-selective (salbutamol) agonists showed extremely rapid inhibition of PCA. However, the beta-selective agonist (dobutamine) had no effect on the reaction. The pretreatment with hexamethonium, reserpine or 6-OH-DA substantially attenuated the inhibitory effect of l-ephedrine on PCA. The results strongly suggest that beta2-adrenoceptors locate in the stomach and that their receptor excitement finally may lead to the inhibition of PCA via the stimulation of the central and peripheral nervous systems.

  19. Radioimmunological determination of parathormone peptides in the plasma before and after gel filtration in hyperparathyroidic patients

    International Nuclear Information System (INIS)

    Juengst, U.

    1983-01-01

    Plasmas from different patients suffering from secondary renal hyperparathyroidism and from one patient suffering from primary hyperparathyroidism owing to a parathyroid adenoma, were studied and parathormone concentrations (total IPTH) were determined radio-immunologically. Plasmas were gel-filtered subsequently. Gelfiltration allows to separate intact PTH from PTH fragments which also occur in the plasma. These fragments also react with the antisera used in the PTH-radio-immuno-assay; thus it was possible to determine only the concentration of intact PTH after gel filtration. In patients in whose unfiltered plasma strongly increased PTH values had been measured (more than 100 pmol hPTH/l plasma), only 10 to 40% of total immunoreactivity were taken up by intact parathormone after gel filtration. Histological findings of iliac crest biopsies revealed marked changes corresponding to the increased concentration of biologically active hormone. Whereas unfiltered plasma revealed slightly increased PTH measurement values (below 100 pmol hPTH/l), 60 to 75% of total immuno reactivity was taken up by the intact hormone after gel filtration. Correspondingly, histological changes of the skeleton were less marked. Apparently, there is a correlation - though not a linear one - between total IPTH and the absolute concentration of intact PTH. If increased PTH values are measured with antiserum S478, it may be assumed in general that the concentration of intact and biologically active PTH has increased as well. (orig./MG) [de

  20. Immunoprecipitation of the parathyroid hormone receptor

    International Nuclear Information System (INIS)

    Wright, B.S.; Tyler, G.A.; O'Brien, R.; Caporale, L.H.; Rosenblatt, M.

    1987-01-01

    An 125 I-labeled synthetic analog of bovine parathyroid hormone, [8-norleucine,18-norleucine,34-tyrosine]PTH-(1-34) amide ([Nle]PTH-(1-34)-NH 2 ), purified by high-pressure liquid chromatography (HPLC), was employed to label the parathyroid hormone (PTH) receptor in cell lines derived from PTH target tissues: the ROS 17/2.8 rat osteosarcoma of bone and the CV1 and COS monkey kidney lines. After incubation of the radioligand with intact cultured cells, the hormone was covalently attached to receptors by using either a photoaffinity technique or chemical (affinity) crosslinking. In each case, covalent labeling was specific, as evidenced by a reduction of labeling when excess competing nonradioactive ligand was present. After covalent attachment of radioligand, membranes were prepared form the cells and solubilized in the nonionic detergent Nonidet P-40 or octyl glucoside. Analysis of the immunoprecipitate on NaDod-SO 4 /polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of a doublet of apparent molecular mass 69-70 kDa. Specifically labeled bands of approximate molecular mass 95 and 28 kDa were also observed. The anti-PTH IgG was affinity purified by passage over a PTH-Sepharose column and used to made an immunoaffinity column. These studies suggest that the use of an anti-PTH antiserum that binds receptor-bound hormone is likely to be a useful step in the further physicochemical characterization and purification of the PTH receptor