scholarly journals Chikungunya E2 Protein Produced in E. coli and HEK293-T Cells—Comparison of Their Performances in ELISA

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 939
Author(s):  
Flávia Fonseca Bagno ◽  
Lara Carvalho Godói ◽  
Maria Marta Figueiredo ◽  
Sarah Aparecida Rodrigues Sérgio ◽  
Thaís de Fátima Silva Moraes ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Indirect Elisa ◽  
Acute Onset ◽  
Diagnostic Tools ◽  
Igg Antibodies ◽  
Post Translational Modifications ◽  
E Coli ◽  
Glycosylation Sites ◽  
Igm Elisa ◽  
Antigen Antibody

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)—a prokaryotic system unable to produce post-translational modifications—or in HEK-293T mammalian cells—a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

scholarly journals Analysis of IgG, IgA, and IgM antibodies against SARS-CoV-2 spike protein S1 in convalescent and vaccinated patients with the Pfizer-BioNTech and CanSinoBio vaccines

2021 ◽  
Author(s):  
Edgar Melgoza-González ◽  
Diana Hinojosa-Trujillo ◽  
Monica Resendiz ◽  
Verónica Mata-Haro ◽  
Sofía Hernández-Valenzuela ◽  
...  
Keyword(s):  
Indirect Elisa ◽  
Natural Infection ◽  
Rapid Development ◽  
Elisa Test ◽  
Antibody Detection ◽  
Diagnostic Tools ◽  
Igg Antibodies ◽  
The World ◽  
The One ◽  
First Time

The SARS-CoV-2 virus was detected for the first time in December 2019 in Wuhan, China. Currently, this virus has spread around the world, and new variants have emerged. This new pandemic virus provoked the rapid development of diagnostic tools, therapies and vaccines to control this new disease called COVID-19. Antibody detection by ELISA has been broadly used to recognize the number of persons infected with this virus or to evaluate the response of vaccinated individuals. As the pandemic spread, new questions arose, such as the prevalence of antibodies after natural infection and the response induced by the different vaccines. In Mexico, as in other countries, mRNA and viral-vectored vaccines have been widely used among the population. In this work, we developed an indirect ELISA test to evaluate S1 antibodies in convalescent and vaccinated individuals. By using this test, we showed that IgG antibodies against the S1 protein of SARS-CoV-2 were detected up to 42 weeks after the onset of the symptoms, in contrast to IgA and IgM, which decreased 14 weeks after the onset of symptoms. The evaluation of the antibody response in individuals vaccinated with Pfizer-BioNTech and CanSinoBio vaccines showed no differences two weeks after vaccination. However, after completing the two doses of Pfizer-BioNTech and the one dose of CanSinoBio, a significantly higher response of IgG antibodies was observed in persons vaccinated with Pfizer-BioNTech than in those vaccinated with CanSinoBio. In conclusion, these results confirm that after natural infection with SARS-CoV-2, it is possible to detect antibodies for up to ten months. Additionally, our results showed that one dose of the CanSinoBio vaccine induces a lower response of IgG antibodies than that induced by the complete scheme of the Pfizer-BioNTech vaccine.

scholarly journals Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes

2000 ◽  
Vol 350 (2) ◽  
pp. 429-441 ◽  
Author(s):  
Stuart M. PITSON ◽  
Richard J. D'ANDREA ◽  
Lucianne VANDELEUR ◽  
Paul A. B. MORETTI ◽  
Pu XIA ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Active Form ◽  
Chemical Properties ◽  
Sphingosine Kinase ◽  
Exchange Chromatography ◽  
Recombinant Enzymes ◽  
Post Translational Modifications ◽  
E Coli ◽  
Physico Chemical

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 106-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

Expression Analysis of Recombinant Equine Chorionic Gonadotropin in Three Host Systems: E. coli BL21C, Sf insect cell lysate and COS-1 mammalian cells

2020 ◽  
Author(s):  
Anuradha Bhardwaj ◽  
Varij Nayan ◽  
Sanjay Kumar ◽  
Parvati Sharma ◽  
Sanjeev Kumar ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Chorionic Gonadotropin ◽  
Insect Cell ◽  
Mammalian Cells ◽  
Reproductive Hormones ◽  
Animal Origin ◽  
Post Translational Modifications ◽  
E Coli ◽  
Cell Lysate ◽  
Equine Chorionic Gonadotropin

Mammalian cells are the recommended host for recombinant eukaryotic protein production aimed at incorporation of post-translational modifications for downstream applications. The bacterial system and insect cells are widely used because of ease of technical methodology, economics of production, purification and yield of final protein. The present research objective was expression of recombinant reproductive hormones of animal origin and study of their immunogenic potential for reproductive applications. The equine Chorionic Gonadotropin (eCG) is one of the most heavily glycosylated protein amongst all glycoprotein hormone family. Hence, experiments were carried out to observe its expression in the three most popular host systems and it led to comparative studies for their post-translational modifications. The Pregnant Mare Serum Gonadotropin (PMSG, also called as eCG) gene was cloned in TOPO-TA vector, pIX 4.0 and pTARGET vectors accordingly and expression analysis in E. coli BL21C, Sf insect cell lysate and COS-1 cells was carried out. We observed diverse sizes of recombinant proteins in SDS-PAGE analysis which indicated post-translational modification in mammalian expression system towards the linking of tags as well as side chains in respective host cells. Basic diagnostic immunogenicity tests showed encouraging results, however, no significant in vivo and in vitro activity was observed for the expressed reCG in all the employed host systems.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

Viability of Edwardsiella tarda and Esherichia coli preserved with glycerol-tryptone soy broth (TSB) kept at freezing temperature

2013 ◽  
Vol 1 (2) ◽  
pp. 154
Author(s):  
Abdur Rohman ◽  
Frans Ijong ◽  
I K Suwetja
Keyword(s):  
Research And Development ◽  
Germ Plasm ◽  
Freezing Temperature ◽  
Experimental Methods ◽  
Edwardsiella Tarda ◽  
Diagnostic Tools ◽  
Glycerol Concentration ◽  
E Coli ◽  
Esherichia Coli ◽  
Escherchia Coli

Preservation of bacteria carried out in relation to the collection and preservation of germ plasm microbe is useful for research and development or for the establishment of diagnostic tools. Glycerol is a good preservation media but it is not known what doses should be used for effective preservation.  This research used two experimental  methods consisting of 2 factors and 3 treatments. This study aimed to find the best glycerol concentration that can be used to preserve Edwarsiella tarda and Escherchia coli in the -20ºC environment, to understand the viability of bacteria after being preserved and to describe the characteristics of the preserved bacteria. Treatments applied were 10%, 15% and 20%  glycerol in TSB. Viability of the bacteria was analyzed after 7, 14, 28, 35, and 42 days of preservation. Results showed that E.coli bacteria preserved in 15%  glycerol had the highest viability, i.e. 84% and preserved in 10% glycerol had the lowest viability, i.e. 80%. But for E. tarda bacteria preserved in 10% glycerol had the highest viability, i.e. 1.83% and preseved in 15% glycerol had the lowest viability, i.e. 0,55%. Preservasi bakteri dilakukan dalam kaitannya dengan koleksi dan konservasi plasma nutfah mikroba yang berguna untuk penelitian dan pengembangan atau untuk pembentukan alat diagnosa. Gliserol merupakan bahan preservasi yang baik, tetapi belum diketahui dosis yang baik dan efektif untuk preservasi bakteri Edwarsiella tarda dan Escherchia coli pada suhu -20ºC. Penelitian ini dilakukan dengan metode eksperimen yang terdiri dari 2 faktor dan 3 taraf perlakuan, masing-masing perlakuan dengan 3 kali ulangan, media preservasi yang digunakan adalah TSB dan gliserol dengan konsentrasi 10%, 15% dan 20%. Parameter yang diukur adalah viabilitas dan kecocokan/penyimpangan karakteristik biokimia. Penelitian ini dilaksanakan di Laboratorium Balai Karantina Ikan Pengendalian Mutu dan Keamanan Hasil Perikanan Manado, dari bulan September sampai dengan November 2013. Tujuan Penelitian ini adalah untuk menentukan konsentrasi gliserol dalam TSB sebagai media preservasi yang efektif dan efisien pada bakteri  Edwarsiella tarda dan Escherchia coli yang dipreservasi dengan suhu -20ºC dan disimpan selama 42  hari. Hasil penelitian menunjukkan adanya penurunan laju pertumbuhan bakteri selama preservasi. Persentase viabilitas  bakteri E. coli yang tertinggi selama preservasi diperoleh dengan penggunaan gliserol konsentrasi 15% dengan jumlah 84% dan yang terendah adalah dengan penggunaan konsentrasi 10% yakni sebesar 80%, sedangkan untuk E. tarda persentase viabilitas  bakteri yang tertinggi selama preservasi diperoleh dengan penggunaan gliserol konsentrasi 10% dengan jumlah 1,83% dan yang terendah adalah dengan penggunaan konsentrasi 15% yakni sebesar 0,55%. Berdasarkan uji statistik analisis variasi (ANAVA) didapat hasil F hitung E. tarda dan E. coli yang lebih besar  dari FTabel dengan tingkat kepercayaan 95 %.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Analysis of a Routinely Used Commercial Anti-Chikungunya IgM ELISA Reveals Cross-Reactivities with Dengue in Brazil: A New Challenge for Differential Diagnosis?

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 819
Author(s):  
Monique da Rocha Queiroz Lima ◽  
Raquel Curtinhas de Lima ◽  
Elzinandes Leal de Azeredo ◽  
Flavia Barreto dos Santos
Keyword(s):  
Disease Surveillance ◽  
Endemic Area ◽  
Chikungunya Virus ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Immunoglobulin M ◽  
Diagnostic Tools ◽  
Healthy Individuals ◽  
Denv Serotypes ◽  
Igm Elisa

In Brazil, chikungunya emerged in 2014, and by 2016, co-circulated with other arbovirosis, such as dengue and zika. ELISAs (Enzyme-Linked Immunosorbent Assays) are the most widely used approach for arboviruses diagnosis. However, some limitations include antibody cross reactivities when viruses belong to the same genus, and sensitivity variations in distinct epidemiological scenarios. As chikungunya virus (CHIKV) is an alphavirus, no serological cross reactivity with dengue virus (DENV) should be observed. Here, we evaluated a routinely used chikungunya commercial IgM (Immunoglobulin M) ELISA test (Anti-Chikungunya IgM ELISA, Euroimmun) to assess its performance in confirming chikungunya in a dengue endemic area. Samples (n = 340) representative of all four DENV serotypes, healthy individuals and controls were tested. The Anti-CHIKV IgM ELISA test had a sensitivity of 100% and a specificity of 25.3% due to the cross reactivities observed with dengue. In dengue acute cases, the chikungunya test showed an overall cross-reactivity of 31.6%, with a higher cross-reactivity with DENV-4. In dengue IgM positive cases, the assay showed a cross-reactivity of 46.7%. Serological diagnosis may be challenging and, despite the results observed here, more evaluations shall be performed. Because distinct arboviruses co-circulate in Brazil, reliable diagnostic tools are essential for disease surveillance and patient management.

scholarly journals POS0384 A NOVEL MECHANISM LINKING MUCOSAL BACTERIA WITH AUTOANTIBODY RESPONSES IN RA: ACETYLATED BACTERIAL LYSATE AS A MODEL ANTIGEN

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 422.1-422
Author(s):  
M. Volkov ◽  
A. S. B. Kampstra ◽  
K. van Schie ◽  
J. Kwekkeboom ◽  
T. Huizinga ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Activation ◽  
Positive Control ◽  
Negative Control ◽  
B Cell Activation ◽  
Human Fibrinogen ◽  
Post Translational Modifications ◽  
E Coli ◽  
Bacterial Proteins ◽  
Citrullinated Proteins

Background:Rheumatoid arthritis (RA) is characterized by autoantibodies against post-translationally modified proteins (AMPA) such as citrullinated, carbamylated and acetylated proteins. Importantly, these antibodies are highly multireactive, as they often recognize more than one of these post-translational modifications. Despite extensive research, the antigens inducing the breach of tolerance remain unknown, although microbial antigens are often suspected. Various bacteria are known to be capable of acetylation, therefore, it is intriguing to know what mechanisms can underlie the breach of tolerance towards acetylated proteins and development of anti-acetylated protein antibodies (AAPA).Objectives:To investigate whether acetylated proteins of bacterial origin (1) are recognized by human derived AMPA and AMPA expressing B cells; and (2) can induce AMPA development when used to immunize mice.Methods:Acetylated E. coli proteins were acquired with two separate methods (Figure 1A): by culturing E. coli in a condition promoting auto-acetylation (intrinsically acetylated bacterial proteins, IABP), or by directly acetylating lysate-derived proteins via a chemical reaction (extrinsically acetylated BP, EABP). Acetylated ovalbumin (AcOVA) served as positive control for AAPA induction in mice, non-acetylated BP (NABP) and phosphate buffer saline (PBS) served as negative control. Mice were immunized with these proteins and the resulting antibody response was studied by ELISA. Furthermore, EABP/IABP/NABP were investigated for recognition by human-derived AAPA with ELISA and AAPA-expressing B cells with spleen tyrosine kinase (Syk) phosphorylation assay; acetylated human fibrinogen and native fibrinogen served as positive and negative control.Results:Repetitive immunization of mice with EABP resulted in an AMPA response recognizing acetylated, carbamylated and citrullinated proteins. AMPA titers in these mice exceeded the titers in the positive control mice immunized with AcOVA and were substantially higher than in the NABP-immunized mice (Figure 1B). Human-derived monoclonal AAPA recognized EABP and IABP (not shown). B cell activation (measured by Syk phosphorylation) assay indicated that AAPA expressing B cells recognized EABP and (to a lesser extent) IABP, but not NABP (Figure 1C).Conclusion:Acetylated bacterial proteins are potent antigens that can induce cross-reactive AMPA responses in mice and they are recognized by human AAPA. This suggests that acetylated bacterial proteins could possibly be involved in the breach of tolerance in RA.Acknowledgements:We thank Dr. Can Araman and Prof. Chunaram Choudhary for their advice regarding optimization of bacterial auto-acetylation.Disclosure of Interests:None declared

scholarly journals Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 843
Author(s):  
Balagra Kasim Sumabe ◽  
Synnøve Brandt Ræder ◽  
Lisa Marie Røst ◽  
Animesh Sharma ◽  
Eric S. Donkor ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutation Frequency ◽  
Nucleoside Analogues ◽  
Cancer Therapies ◽  
E Coli ◽  
Replicative Stress ◽  
Pol V ◽  
Dna And Rna ◽  
Sos Induction ◽  
Anti Cancer

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

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