scholarly journals A Sensitive Immunoblotting Technique for the Serodiagnosis of Brucella Ovis Infections

1994 ◽  
Vol 6 (2) ◽  
pp. 188-194 ◽  
Author(s):  
Reinhold Kittelberger ◽  
Mike Hansen ◽  
Gail P. Ross ◽  
Frans Hilbink
Keyword(s):  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Brucella Ovis ◽  
Specific Pathogen ◽  
History Of ◽  
Molecular Masses ◽  
Culture Positive ◽  
Gel Diffusion ◽  
Disease Free ◽  
Immunoblotting Technique

A simplified electrophoretic immunoblotting technique based on antigen extracted from Brucella ovis cells with sodium dodecyl sulfate/mercaptoethanol was compared with the complement fixation test (CFT), the enzyme-linked immunosorbent assay, and the gel diffusion test. Sera from 89 chronically infected, semen culture-positive rams, 378 sera from B. ovis-infected flocks, 300 sera from accredited disease-free flocks, and 29 sera from specific-pathogen-free sheep were used. The immunoblotting technique had sensitivity and specificity comparable to those of the standard tests and was able to identify several CFT-negative or -borderline sera as positive. The major immunoreactive antigens of B. ovis had molecular masses of 63,29, 19 kD (proteins) and 8-12 kD (rough lipopolysaccharide). Antibodies against these antigens were present in 96% of CFT-positive sera from infected flocks and in 100% of sera from semen culture-positive rams. However, immunoblotting also identified antibodies to components other than the major antigens in 1% of CFT-negative sera from infected flocks and in 7.7% of the sera from flocks with a history of freedom from the disease. These reactions probably represent cross-reactivities with other microorganisms and were distinguishable from truly positive reactions.

scholarly journals Characterization of Serological Responses to Pertussis

2006 ◽  
Vol 13 (3) ◽  
pp. 341-348 ◽  
Author(s):  
Mineo Watanabe ◽  
Beverly Connelly ◽  
Alison A. Weiss
Keyword(s):  
Adenylate Cyclase ◽  
Serological Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vaccine Antigens ◽  
History Of ◽  
Antibody Titers ◽  
Iga Antibody ◽  
Conventional Vaccine ◽  
Culture Positive

ABSTRACT We have compared the use of five nonvaccine antigens to the use of conventional vaccine antigens, pertussis toxin (PT), and filamentous hemagglutinin (FHA) for the serological diagnosis of pertussis by enzyme-linked immunosorbent assay (ELISA). The nonvaccine antigens included the catalytic region of adenylate cyclase toxin (CatACT), the C-terminal region of FHA (C-FHA), lipooligosaccharide (LOS), the peptidoglycan-associated lipoprotein (PAL), and the BrkA protein. The serological responses of individuals with culture-confirmed pertussis were compared to those of adults with no recent history of a coughing disease. An immunoglobulin G (IgG) ELISA for PT was the most sensitive (92.2%) test for the serodiagnosis of pertussis. Of the nonvaccine antigens, ELISA for IgG responses to CatACT (sensitivity, 62.8%), C-FHA (sensitivity, 39.2%), and LOS IgA (sensitivity, 29.4%) were less sensitive but could also distinguish culture-positive individuals from control individuals. The use of a combination of multiple ELISA targets improved the sensitivity of the assay for serological diagnosis. Elevated IgG and IgA antibody titers persisted for more than a year in the individuals with culture-confirmed pertussis.

scholarly journals An Opsonizing Monoclonal Antibody That Recognizes a Noncapsular Epitope Expressed on Cryptococcus neoformans

1999 ◽  
Vol 67 (10) ◽  
pp. 4994-5000 ◽  
Author(s):  
Glenn J. Merkel ◽  
Barbara A. Scofield
Keyword(s):  
Monoclonal Antibody ◽  
Cell Walls ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immune Electron Microscopy ◽  
Phagocytic Cells ◽  
Molecular Masses ◽  
Culture Supernatants ◽  
Secreted Antigens

ABSTRACT A mouse hybridoma secreting a monoclonal antibody (MAb) that bound a noncapsular epitope expressed on C. neoformans was developed by immunizing BALB/c mice with formalin-killed serotype A yeasts. The hybridoma, designated CSFi, secreted an immunoglobulin G2b MAb that reacted with all C. neoformans serotypes tested, including the acapsular mutant ATCC 52817 (Cap67). Postsectioned immune electron microscopy revealed extensive binding of the MAb to the cell walls of both encapsulated and acapsular yeasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of secreted antigens recovered from concentrated culture supernatants from both encapsulated and acapsular strains was conducted. The results showed that this MAb bound predominantly to antigens with molecular masses of approximately 75 and 100 kDa. A competitive enzyme-linked immunosorbent assay was used to demonstrate that the MAb was not cross-reactive with purified glucuronoxylomannan derived from either serotypes A or D. Experiments conducted with mouse peritoneal phagocytes and the mouse phagocyte-like cell line, J774A.1, demonstrated that the CSFi MAb opsonized the yeasts and increased their adherence to both types of phagocytic cells. We conclude, therefore, that antibodies directed at noncapsular epitopes can serve as opsonins and may have a role in modulating cryptococcal infection.

scholarly journals A Novel Automated Immunoassay Platform to Evaluate the Association of Adiponectin and Leptin Levels with Breast Cancer Risk

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3303
Author(s):  
Debora Macis ◽  
Valentina Aristarco ◽  
Harriet Johansson ◽  
Aliana Guerrieri-Gonzaga ◽  
Sara Raimondi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Risk ◽  
Cancer Risk ◽  
Cox Model ◽  
Disease Free Survival ◽  
Postmenopausal Breast Cancer ◽  
Enzyme Linked Immunosorbent Assay ◽  
Baseline Serum ◽  
Free Survival ◽  
Disease Free

Adiponectin and leptin are adipokines secreted by the adipose tissue that are associated with several chronic diseases including cancer. We aimed to compare the immunoassay platform ELLA with an enzyme-linked immunosorbent assay (ELISA) kit and to assess whether the results of the association analyses with breast cancer risk were dependent on the assay used. We measured adiponectin and leptin with ELLA and ELISA on baseline serum samples of 116 Italian postmenopausal women enrolled in two international breast cancer prevention trials. Results were compared with Deming, Passing–Bablok regression and Bland–Altman plots. Disease-free survival was analyzed with the Cox model. There was a good correlation between the methods for adiponectin and leptin (r > 0.96). We found an increased breast cancer risk for very low adiponectin levels (HR for ELLA = 3.75; 95% CI: 1.37;10.25, p = 0.01), whereas no significant association was found for leptin levels. The disease-free survival curves were almost identical for values obtained with the two methods, for both biomarkers. The ELLA platform showed a good concordance with ELISA for adiponectin and leptin measurements. Our results support the association of very low adiponectin levels with postmenopausal breast cancer risk, irrespective of the method used. The ELLA platform is a time-saving system with high reproducibility, therefore we recommend its use for biomarker assessment.

scholarly journals Comparison of pregnenolone sulfate, pregnanolone and estradiol levels between patients with menstrually-related migraine and controls: an exploratory study

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Cecilia Rustichelli ◽  
Elisa Bellei ◽  
Stefania Bergamini ◽  
Emanuela Monari ◽  
Flavia Lo Castro ◽  
...  
Keyword(s):  
Exploratory Study ◽  
Serum Levels ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Allosteric Modulator ◽  
Positive Allosteric Modulator ◽  
Pregnenolone Sulfate ◽  
Menstrually Related Migraine ◽  
History Of ◽  
Estradiol Levels

Abstract Background Neurosteroids affect the balance between neuroexcitation and neuroinhibition but have been little studied in migraine. We compared the serum levels of pregnenolone sulfate, pregnanolone and estradiol in women with menstrually-related migraine and controls and analysed if a correlation existed between the levels of the three hormones and history of migraine and age. Methods Thirty women (mean age ± SD: 33.5 ± 7.1) with menstrually-related migraine (MM group) and 30 aged- matched controls (mean age ± SD: 30.9 ± 7.9) participated in the exploratory study. Pregnenolone sulfate and pregnanolone serum levels were analysed by liquid chromatography-tandem mass spectrometry, while estradiol levels by enzyme-linked immunosorbent assay. Results Serum levels of pregnenolone sulfate and pregnanolone were significantly lower in the MM group than in controls (pregnenolone sulfate: P = 0.0328; pregnanolone: P = 0.0271, Student’s t-test), while estradiol levels were similar. In MM group, pregnenolone sulfate serum levels were negatively correlated with history of migraine (R2 = 0.1369; P = 0.0482) and age (R2 = 0.2826, P = 0.0025) while pregnenolone sulfate levels were not age-related in the control group (R2 = 0.04436, P = 0.4337, linear regression analysis). Conclusion Low levels of both pregnanolone, a positive allosteric modulator of the GABAA receptor, and pregnenolone sulfate, a positive allosteric modulator of the NMDA receptor, involved in memory and learning, could contribute either to headache pain or the cognitive dysfunctions reported in migraine patients. Overall, our results agree with the hypothesis that migraine is a disorder associated with a loss of neurohormonal integrity, thus supporting the therapeutic potential of restoring low neurosteroid levels in migraine treatment.

scholarly journals Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Nayoun Hong ◽  
Seockmo Ku ◽  
Kyungjin Yuk ◽  
Tony V. Johnston ◽  
Geun Eog Ji ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Shuttle Vector ◽  
Sodium Dodecyl ◽  
Interleukin 10 ◽  
Biologically Active ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Evaluation ◽  
Ht 29 ◽  
Cell Free Culture Supernatant

Abstract Background Bifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines. Results The vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473 ± 300 ng/mL (n = 4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n = 6, p < 0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n = 6, p < 0.01) or TNFα-induced HT-29 cells (n = 6, p < 0.001). Conclusion B. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.

Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Phage Display ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Library ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Phage Display Antibody

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

scholarly journals An epidemic of cholera due toVibrio choleraeO139 in Dhaka, Bangladesh: clinical and epidemiological features

1994 ◽  
Vol 112 (3) ◽  
pp. 463-471 ◽  
Author(s):  
D. Mahalanabis ◽  
A. S. G. Faruque ◽  
M. J. Albert ◽  
M. A. Salam ◽  
S. S. Hoque
Keyword(s):  
Oral Rehydration Therapy ◽  
Watery Diarrhoea ◽  
Large Hospital ◽  
Oral Rehydration ◽  
Hygiene Practices ◽  
Disease Spectrum ◽  
Intravenous Rehydration ◽  
Epidemiological Features ◽  
History Of ◽  
Culture Positive

SUMMARYWe describe the disease spectrum and socio-demographic and epidemiological features of an epidemic of cholera due to a new pathogen.Vibrio choleraeO139, in patients attending a very large hospital in the metropolitan city of Dhaka, Bangladesh.This hospital treats 70000–90000 patients a year with diarrhoeal diseases. A 4% systematic sample of 1854 patients attending from January to April 1993 were studied.Five hundred and two (27%) of the 1854 patients were culture positive forV. choleraeO139 and 63 (3%) were culture positive forV. choleraeO1 biotype El Tor. Patients withV. choleraeO139 were mainly adults with a short history of watery diarrhoea. Eight-three percent of patients had moderate to severe dehydration. All recovered except one 80-year-old man with compromised renal function who died. Seventy-eight percent of patients required initial intravenous rehydration followed by oral rehydration therapy with rice ORS; they also received tetracycline to reduce diarrhoea severity. Most patients were from urban slums with inadequate sanitation facilities and hygiene practices.The newly recognizedV. choleraeO139 infection produced an epidemic of severe dehydrating diarrhoea indistinguishable from clinical cholera in a population which experiences two epidemic peaks of cholera in a year due toV. choleraeO1. Infection with the latter does not appear to confer any cross-protection fromV. choleraeO139. The new pathogen suppressed, albeit temporarily,V. choleraeO1. Unlike other non-O1 serogroups ofV. choleraethis new serogroup appears to have epidemic potential.

scholarly journals STUDY THE EFFECT OF MYCOPLASMA CONTAMINATION OF EGGS USED IN VIRUS TITRATION AND EFFICACY OF SOME LIVE ATTENUATED POULTRY VIRAL VACCINES

2016 ◽  
Vol 10 (1) ◽  
pp. 216 ◽  
Author(s):  
Marwa Fathy ◽  
Mounir M. El-safty ◽  
Jakeen K. El-jakee ◽  
Howaida I. Abd-alla ◽  
Hala Mahmoud
Keyword(s):  
Disease Virus ◽  
Newcastle Disease ◽  
Disease Process ◽  
Mycoplasma Gallisepticum ◽  
Infectious Bursal Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infectious Bronchitis ◽  
Bursal Disease ◽  
Specific Pathogen ◽  
Viral Vaccines

ABSTRACTObjective: The study of Mycoplasma gallisepticum (MG) infection is needed, not only to understand the disease process but also to understand theinterference with the evaluation of some live viral poultry vaccines. This study aims to investigate the titration and potency of some live attenuatedpoultry viral vaccines; Newcastle disease, infectious bronchitis, infectious bursal disease, and Reo in both specific pathogen-free (SPF) embryonatedchicken eggs (ECEs) and chickens.Methods: Titration of live attenuated viral poultry vaccines in ECEs was carried out by dividing the inoculated eggs into four groups; the pre-,simultaneously-, post-, and non-MG contaminated. MG effect on the potency test was carried out using seventeen groups of SPF chickens (25 chicken/group) placed into separate isolators. Each live attenuated viral poultry vaccine was inoculated into 4 groups.Results: The highest titer of these vaccines that appeared in MG pre- contaminated ECEs were 1011, 107.5, 107.9, and 10, respectively. The lowest vaccinetiters that appeared in non-MG contaminated ECEs were 108, 106, 106.8, and 1067.5, respectively. Although the potency of these previous vaccines indicated thatthe highest antibodies titer that appeared in MG pre-infected vaccinated chickens were 7.5 log, 36 enzyme-linked immunosorbent assay unit (EU), and42 EU, respectively; the lowest antibodies titer that appeared in non-MG infected vaccinated chickens were 6.5 log22, 12 EU, 17 EU, and 10 EU, respectively.Conclusion: The present study findings underline the importance of using Mycoplasma -free eggs or chicken for the production of virus vaccines.Keywords: Mycoplasma gallisepticum, Newcastle disease virus, Infectious bronchitis virus, Infectious bursal disease virus, Reo virus, Chicken, Specificpathogen-free eggs.

scholarly journals The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2, in vitro

2021 ◽  
Author(s):  
Riho Tateyama-Makino ◽  
Mari Abe-Yutori ◽  
Taku Iwamoto ◽  
Kota Tsutsumi ◽  
Motonori Tsuji ◽  
...  
Keyword(s):  
Serine Protease ◽  
Protease Activity ◽  
Oral Care ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Spike Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibitory Effects ◽  
Docking Simulations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the mucosa of the tongue and gingiva, the oral cavity seems like it is an entry point for SARS-CoV-2. Daily oral care using mouthwash seems to play an important role in preventing SARS-CoV-2 infection. However, the relationship between daily oral care and the mechanisms of virus entry into host cells is unclear. In this study, we evaluated the inhibitory effects of ingredients that are generally contained in toothpaste and mouthwash on the interaction between the spike protein and ACE2 and on the serine protease activity of TMPRSS2 using an enzyme-linked immunosorbent assay and in vitro enzyme assay, respectively. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to the inhibitor-binding site of ACE2. In addition, tranexamic acid and 6-aminohexanoic acid, which act as serine protease inhibitors, exerted inhibitory effects on TMPRSS2 protease activity. Further experimental and clinical studies are needed to further elucidate these mechanisms. Our findings support the possibility that toothpaste and mouthwash contain ingredients that inhibit SARS-CoV-2 infection.

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