Analysis of glycosylation heterogeneity in antibody production by Pichia pastoris

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Generalized models from Beta(p,2) densities with strong allee effect: dynamical approach

A dynamical approach to study the behaviour of generalized populational growth models from Bets(p, 2) densities, with strong Allee effect, is presented. The dynamical analysis of the respective unimodal maps is performed using symbolic dynamics techniques. The complexity of the correspondent discrete dynamical systems is measured in terms of topological entropy. Different populational dynamics regimes are obtained when the intrinsic growth rates are modified: extinction, bistability, chaotic semistability and essential extinction.

Acute Hepatitis B in a patient previously positive for antibody to surface antigen (anti-HBs) determined by radioimmunoassay: case report and review of the literature

The determination of anti-HBs as a screening test before vaccination has been advisable in order to encounter immune individuals that don't need to receive vaccine protection. A case-report is presented and three other cases are reviewed from the literature. Anti-HBs was positive in these health-care personnels that developped typical acute B hepatitis. Different subtyping involving the d/y determinants were found in the first case, but false-positive anti-HBs even with high titres, determined by RIA, were found in the other cases. Concomitant determination of anti-HBc or absence of screening tests seem to be more reasonable policies until a low-cost and risk-free vaccine is produced.

Prevalence of antibody to hepatitis B virus in an urban population of northeast Brazil

A sample of 1,288 inhabitants of Salvador, Bahia, Brazil, were submitted to the determination of anti-HBs using radioimmunoassay procedure, and analysed according to age, sex and income. Overall prevalence of anti-HBs was 11,8%. ranging from 6,7% among children aged less than three years old to 26,1% among those aged 30 years and older. Males presented prevalence of anti-HBs similar to female individuals, and those with a higher income showed frequencies of anti-HBs greater than those with a lower income level. The following conclusions were drawn: The high prevalence of anti-HBs observed among children suggests early contact with hepatitis B virus, possibly due to vertical transmission and intrafamiliar dissemination of the disease; the frequency of anti-HBs increases with age; the lower prevalence of anti-HBs among those with low income suggests that this group may present higher prevalence of carriers of the hepatitis B virus surface antigen.

Antibody response to heat-inactivated hepatitis B vaccine (CLB-3mg) in hemodialysis patients and occupational risk personnel: a one year follow-up

Immune response against hepatitis B vaccine (CLB 3mg) was evaluated in 59 hemodialysis patients and 20 occupational risk personnel. Seroconversion was induced in 52.5% and 70.0% respectively. Twelve months after the first dose, 37.5% of patients and 60.0% of occupational risk personnel had detectable anti-HBs level. Antibody level was expressed in sample ratio units (SRU). Considering only the responders, in the patients group 38.7% had a low anti-HBs response (2.1-9.9 SRU) 32.3% a medium response (10-99.9 SRU) and 29.0% a high response (>100 SRU) while in occupational risk personnel these values were 14.3%, 64.3% and 21.4% respectively. The authors suggest the use of HBV vaccines with more elevated HBsAg concentration or a reinforced immunization schedule to improve the anti-HBs response not only for patients but also for healthy persons.

Immunofluorescent antibody to rotavirus and antigen excretion in infantile acute diarrhoea

A serological study was carried out to evaluate the causal relationship of acute diarrhoea and rotavirus in children under five years of age. The rotaviral infection was demonstrated in paired sera by determining the antibody titers by immunofluorescence test (IF). Bovine rotavirus-infected MA-104 cell culture was used as substratum for IF. Out of 80 paired sera it was shown that 23 (28.75%) presented seroconversion, 19 (23.75%) samples showed a twofold increase in their titers and 38 (47.5%) had no increase in rotavirus antibody. This result is discussed on the light of previously obtained results on viral antigen detection by counterimmunoelectrosmophoresis (CIEOP).

Peroxidase antibody test for mucocutaneous leishmaniasis serology performance indexes and comparison with a fluorescent antibody test: Comparação com o desempenho da reação de imunofluorescência

Performance indexes of the peroxidase antibody test were compared to that of the fluorescent antibody test. The peroxidase antibody test had a statistically higher sensitivity and negative predictive value and a higher efficiency than the fluorescent antibody test but its specificity and positive predictive value were within the 95% confidence limits for the values found for the fluorescent antibody test. Such differences did not change when Chagas' disease and visceral leishmaniasis sera were included in index calculations. Statistical analysis showed that the two tests have a substantial degree of agreement but the immunofluorescent test had a specificity index and a positive predictive value equal to 100.0% when Chagas' disease and visceral leishmaniasis sera were not included in the calculations of the performance index; in this instance, a positive test result equals a disclosure of the disease attribute due to the inexistence of false positive results. The enzyme/ protein ratio of the peroxidase conjugate, resulting in heavy or light-labeled conjugates may pose technical problems to its use in serology tests.

The prevalence of antibody to human parvovirus B19 in Rio de Janeiro, Brazil

During 1985 and 1986 serum samples were collected from the Rio de Janeiro population and examined for the presence of IgG antibody to human parvovirus B19. No difference in prevalence was found between males and females. Antibody prevalence rose from 35% in children less than five years old to almost 80% in children aged eleven to fifteen years. The antibody prevalence in individuals over 50 years old was over 90%.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.

Antibody to human T-lymphotropic virus in a patient with Guillain-Barré syndrome (case report)

Serum sample obtained from a male, 12 year old patient suffering from Guillain-Barré syndrome (GBS) was positive for human T-lymphotropic virus (HTLV-I) antibody by the enzyme-linked immunosorbent assay (ELISA) and the Western Blot analysis (WB). Attempts to isolate enteroviruses (including poliovirus) from faecal material in both tissue culture and suckling mice were unsuccessful; in addition, acute and convalescent paired serum samples did not show any evidence of recent poliovirus infection when tested against the three serotypes. Specific tests for detection of Epstein-Barr virus infection were not performed; however, the Paul-Bunnel test yielded negative results. ELISA for detection of anti-cytomegalovirus IgM was also negative. The concomitant occurrence of either adult T cell leukemia (ATL) or lymphoma was not recorded in this case.

Comparative study of adenoviruses with monoclonal antibodies

The obtainment of monoclonal antibodies for adenovirus species 4(Ad4) is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA), in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C), 4 (subgenus E), 7 and 16 (subgenus B) and 9 (subgenus D). Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII) and one monoclonal antibody that cross reacted with adenovirus species 4 and 7 (2HIII) were obtained.

A high throughput colorimetric assay of beta-1,3-D-glucans by Congo red dye

Mushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, β-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for β-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and β-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (> 20 nm) in UV–Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 μg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. β-d-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of β-1,3-d-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of β-1,3-d-glucans was investigated in several mushroom species.

A novel colorimetric assay of beta-D-glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter plate

Basidiomycete strains synthesize several types of beta-D-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these beta-D-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these beta-D-glucans present some disadvantages, a novel colorimetric assay method for beta-D-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (similar to 14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of beta-D-glucans in the range of 0-0.8 mu g, with a slope of 44.15 x 10(-2) and a limit of detection of 0.017 mu g/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for beta-1,3-D-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red beta-D-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify beta-D-glucans from other biological sources. (C) 2015 American Institute of Chemical Engineers

Use of MAC-ELISA for evaluation of yellow fever vaccination

An evaluation of the IgM antibody immune response against yellow fever using strain 17D was carried out by MAC-ELISA and PRNT. The results showed an agreement of 97% between both tests and the authors conclude that MAC-ELISA can be used as a specific and sensitive asssay to replace the PRNT for detecting yellow fever antibodies in human sera, after vaccination programs.