scholarly journals A simple, sensitive and quantitative FACS-based test for SARS-CoV-2 serology in humans and animals

2021 ◽  
Author(s):  
Agnes Maurel Ribes ◽  
Pierre Bessiere ◽  
Jean-Charles Guery ◽  
Eloise Joly Featherstone ◽  
Timothee Bruel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tissue Culture ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Elisa Test ◽  
Negative Control ◽  
Serological Tests ◽  
S Protein ◽  
Flow Test ◽  
Better Than

Serological tests are important for understanding the physiopathology and following the evolution of the Covid-19 pandemic. Assays based on flow cytometry (FACS) of tissue culture cells expressing the spike (S) protein of SARS-CoV-2 have repeatedly proven to perform slightly better than the plate-based assays ELISA and CLIA (chemiluminescent immuno-assay), and markedly better than lateral flow immuno-assays (LFIA). Here, we describe an optimized and very simple FACS assay based on staining a mix of two Jurkat cell lines, expressing either high levels of the S protein (Jurkat-S) or the mCherry fluorescent protein (Jurkat-R, which serve as an internal negative control). We show that this Jurkat-S&R-flow test has a much broader dynamic range than a commercial ELISA test and performs at least as well in terms of sensitivity and specificity. Also, it is more sensitive and quantitative than the hemagglutination-based test HAT, which we described recently. The Jurkat-R&S-flow test requires only a few microliters of blood; thus, it can be used to quantify various Ig isotypes in capillary blood collected from a finger prick. It can be used also to evaluate serological responses in mice, cats and dogs. FACS tests offer a very attractive solution for laboratories with access to tissue culture and flow cytometry who want to monitor serological responses in humans or in animals, and how these relate to susceptibility to infection, or re-infection, by the virus, and to protection against Covid-19.

scholarly journals FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN

2020 ◽  
Author(s):  
Lydia Horndler ◽  
Pilar Delgado ◽  
Ivaylo Balabanov ◽  
Georgina Cornish ◽  
Miguel Angel Llamas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Correct Identification ◽  
Truncated Form ◽  
Serological Tests ◽  
S Protein ◽  
Human Antibodies ◽  
Degree Of Protection ◽  
Immunoglobulin Isotypes ◽  
Jurkat T Cell ◽  
The Mean

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.

Antiproliferative and Genotoxic Action of an Underexploited Organoteluran Derivative on Sarcoma 180 Cells

2020 ◽  
Vol 20 ◽  
Author(s):  
Maria L.L. Barreto do Nascimento ◽  
Antonielly Campinho dos Reis ◽  
José V.O. Santos ◽  
Helber A. Negreiros ◽  
Felipe C. Carneiro da Silva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nuclear Division ◽  
Micronucleus Test ◽  
Mutagenic Activity ◽  
Chemical Compounds ◽  
Negative Control ◽  
Nuclear Division Index ◽  
Apoptosis And Necrosis ◽  
Genotoxicity Tests ◽  
Genotoxic Action

Background: The search for novel metallic chemical compounds with toxicogenic effects have been of great importance for more efficient cancer treatment. Objective: The study evaluated the cytotoxic, genotoxic and mutagenic activity of organoteluran RF07 in S-180 cell line. Methods: The bioassays used were cell viability with 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, evaluation of apoptosis and necrosis using fluorescence and flow cytometry, cytokinesis-block micronucleus test and comet assay. The compound was tested at 1; 2.5 and 5 µM. Results: The results showed the cytotoxicity of RF07 at concentrations of 2.5, 5, 10 and 20 µM when compared to the negative control. For genotoxicity tests, RF07 showed effects in all concentrations assessed by increased index and frequencies of damage and mutagenic alterations. The compound was also cytotoxic due to the significant decrease in nuclear division index, with significant values of apoptosis and necrosis. The results of fluorescence and flow cytometry showed apoptosis as the main type of cell death caused by RF07 at 5 µM, which is thought to avoid an aggressive immune response of the organism. Conclusion: In addition to cytotoxic and genotoxic effects, RF07 creates good perspectives for future antitumor formulations.

scholarly journals Analytical Validation and Clinical Application of Rapid Serological Tests for SARS-CoV-2 Suitable for Large-Scale Screening

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 869
Author(s):  
Amedeo De Nicolò ◽  
Valeria Avataneo ◽  
Jessica Cusato ◽  
Alice Palermiti ◽  
Jacopo Mula ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time Pcr ◽  
Large Scale ◽  
High Sensitivity ◽  
Analytical Validation ◽  
Pcr Assay ◽  
Serological Tests ◽  
Flow Test ◽  
Lateral Flow Test ◽  
Large Scale Screening

Recently, large-scale screening for COVID-19 has presented a major challenge, limiting timely countermeasures. Therefore, the application of suitable rapid serological tests could provide useful information, however, little evidence regarding their robustness is currently available. In this work, we evaluated and compared the analytical performance of a rapid lateral-flow test (LFA) and a fast semiquantitative fluorescent immunoassay (FIA) for anti-nucleocapsid (anti-NC) antibodies, with the reverse transcriptase real-time PCR assay as the reference. In 222 patients, LFA showed poor sensitivity (55.9%) within two weeks from PCR, while later testing was more reliable (sensitivity of 85.7% and specificity of 93.1%). Moreover, in a subset of 100 patients, FIA showed high sensitivity (89.1%) and specificity (94.1%) after two weeks from PCR. The coupled application for the screening of 183 patients showed satisfactory concordance (K = 0.858). In conclusion, rapid serological tests were largely not useful for early diagnosis, but they showed good performance in later stages of infection. These could be useful for back-tracing and/or to identify potentially immune subjects.

Automated method for the isolation of collecting ducts

2006 ◽  
Vol 291 (1) ◽  
pp. F236-F245 ◽  
Author(s):  
R. Lance Miller ◽  
Ping Zhang ◽  
Tong Chen ◽  
Andreas Rohrwasser ◽  
Raoul D. Nelson
Keyword(s):  
Flow Cytometry ◽  
Large Particle ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Complex Object ◽  
Cortical Collecting Duct ◽  
Fluorescent Intensity ◽  
Automated Method ◽  
Collecting Ducts ◽  
Time Required

The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68 ± 0.44–14.5 ± 0.66 cm or 9.2 ± 0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.

scholarly journals Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

2012 ◽  
Vol 45 (4) ◽  
pp. 510-513 ◽  
Author(s):  
Teiliane Rodrigues Carneiro ◽  
Marta Cristhiany Cunha Pinheiro ◽  
Sara Menezes de Oliveira ◽  
Ana Lúcia de Paula Hanemann ◽  
José Ajax Nogueira Queiroz ◽  
...  
Keyword(s):  
Serological Test ◽  
Laboratory Diagnosis ◽  
Elisa Test ◽  
Northeastern Brazil ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Predictive Values ◽  
Transmission Area ◽  
Elisa Technique ◽  
Endemic Areas

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

scholarly journals Devising a conceptual method for generating cryptocompression codograms of images without loss of information quality

2021 ◽  
Vol 4 (2(112)) ◽  
pp. 6-17
Author(s):  
Vladimir Barannik ◽  
Serhii Sidchenko ◽  
Dmitriy Barannik ◽  
Sergii Shulgin ◽  
Valeriy Barannik ◽  
...  
Keyword(s):  
Information Quality ◽  
Dynamic Range ◽  
Video Data ◽  
Service Component ◽  
Loss Of Information ◽  
Video Information ◽  
Information Components ◽  
Service Data ◽  
Processing Steps ◽  
Better Than

Along with the widespread use of digital images, an urgent scientific and applied issue arose regarding the need to reduce the volume of video information provided it is confidential and reliable. To resolve this issue, cryptocompression coding methods could be used. However, there is no method that summarizes all processing steps. This paper reports the development of a conceptual method for the cryptocompression coding of images on a differentiated basis without loss of information quality. It involves a three-stage technology for the generation of cryptocompression codograms. The first two cascades provide for the generation of code structures for information components while ensuring their confidentiality and key elements as a service component. On the third cascade of processing, it is proposed to manage the confidentiality of the service component. The code values for the information components of nondeterministic length are derived out on the basis of a non-deterministic number of elements of the source video data in a reduced dynamic range. The generation of service data is proposed to be organized in blocks of initial images with a dimension of 16×16 elements. The method ensures a decrease in the volume of source images during the generation of cryptocompression codograms, by 1.14–1.58 times (12–37 %), depending on the degree of their saturation. This is 12.7‒23.4 % better than TIFF technology and is 9.6‒17.9 % better than PNG technology. The volume of the service component of cryptocompression codograms is 1.563 % of the volume of the source video data or no more than 2.5 % of the total code stream. That reduces the amount of data for encryption by up to 40 times compared to TIFF and PNG technologies. The devised method does not introduce errors into the data in the coding process and refers to methods without loss of information quality.

scholarly journals Comparison between the efficiency of serological tests for Identification of Brucellosis

2010 ◽  
Vol 7 (2) ◽  
pp. 901-909
Author(s):  
Baghdad Science Journal
Keyword(s):  
Rose Bengal ◽  
Serological Test ◽  
Elisa Test ◽  
Health Centers ◽  
Confirmatory Test ◽  
Igg Antibodies ◽  
Serological Tests ◽  
Primary Screening ◽  
Low Concentrations ◽  
Immune Assay

Five serological methods for detection of Brucella were compaired in this study, Four of the methods are commonely used in the detections:- 1-Rose-Bengal: as primary screening test which depends on detecting antibodies in the blood serum. 2-IFAT: which detects IgG and IgM antibodies in the serum. 3-ELISA test: which detects IgG antibodies in the serum. 4-2ME test: which detects IgG antibodies The fifth methods. It was developed by a reasercher in one of the health centers in Baghdad. It was given the name of spot Immune Assay (SIA). Results declares that among (100) samples of patients blood, 76, 49, 49, 37, and 28. samples were positive to Rose Bengal, ELISA, SIA, 2ME and IFAT tests, respectively. When efficiency, sensitivity and specificity of the serological methods were compaired, the Following results were obtained: a) ELISA and SIA were superiors among the other confirming methods (2ME and IFAT) in detecting the highest cases (49 cases); 46 of them were from the (76) cases positive to Rose Bengal The confirmatory test 2ME was not efficient in detecting low concentrations of IgG antibodies when less than half (37) of the total positive cases (76) were detected by this test. b) IFAT test was the least efficient confirmatory test among all other test. c) As a new confirmatory test, SIA proved to be an efficient and serological test for Brucella detection in comparison with other tests. It is an easy to use test, rapid and could be performed without need to the expensive equipment .

Cellular investigations

2013 ◽  
pp. 555-578
Author(s):  
Gavin P Spickett
Keyword(s):  
Flow Cytometry ◽  
Tissue Culture ◽  
T Cells ◽  
Bronchoalveolar Lavage ◽  
Cytotoxic T Cells ◽  
Cd40 Ligand ◽  
Regulatory Factors ◽  
Ligand Expression ◽  
Proliferation Assays ◽  
Protein Assays

Introduction Flow cytometry Tissue culture Proliferation assays Immunohistology Cytokine, chemokine, soluble protein assays Apoptosis assays Adhesion markers Bronchoalveolar lavage (BAL) studies CD40 ligand expression Complement membrane regulatory factors Cytokine and cytokine receptor measurement Cytotoxic T cells FOXP3 (regulatory T cells—IPEX syndrome) Genetic and protein studies...

scholarly journals Comparative study of egg contamination with Salmonella Heidelberg and Salmonella Typhimurium

2019 ◽  
Vol 56 (1) ◽  
pp. e150479
Author(s):  
Germana Vizzotto Osowski ◽  
Lana Flávia Baron ◽  
Arlei Codebella ◽  
Francisco Noé Fonseca ◽  
Sandra Camile Almeida Mota ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Negative Control ◽  
Infection Model ◽  
Hostile Environment ◽  
Specific Pathogen Free ◽  
Foreign Markets ◽  
Serological Tests ◽  
Statistical Software ◽  
Bacterial Penetration ◽  
Specific Pathogen

Cases of salmonellosis in humans have been associated with consumption of eggs contaminated with this bacterial pathogen due to insufficient heat treatment. The most prevalent serotypes of Salmonella in Brazil include serotypes Enteritidis, Typhimurium, and Heidelberg. The first two serotypes are major causes for eggs to be withheld from sale and for recalls over Salmonella contamination concerns in both domestic and foreign markets. Eggs may be contaminated through transovarian infection (transovarial transmission) due to the presence of the microorganism in the hen’s oviduct and bacterial penetration of the eggshell. There is little data in the literature on the susceptibility of egg contamination and eggshell penetration by Brazilian serotypes of Salmonella. The present study aimed to evaluate the ability of S. Heidelberg and S. Typhimurium serotypes to penetrate through the eggshell and detect these bacteria in the albumen and yolk according to the thickness of the eggshell. SPF (specific-pathogen-free) eggs were artificially contaminated by contact with moist cotton containing Salmonella (15 x 108 CFU/ml). Eggs were divided into the following groups: negative control (not contaminated), S. Heidelberg, and S. Typhimurium. Subsequently, these eggs were incubated at 37°C, and their contents analyzed after 4 h and 24 h of incubation. The evaluation (assessment) of the contamination was performed by traditional bacteriology and confirmed by biochemical and serological tests. Treatments were compared with Fisher’s test using a SAS statistical software. For S. Heidelberg, the percentage of positivity (positive cases) was lower in both albumen and yolk at 4 h and 24 h intervals (33.33% and 3.7%, and 3.7% and 3.7%, respectively) compared to S. Typhimurium (26.63% and 7.41%, and 33.33% and 33.33%, respectively). These findings suggest that the former strain (S. Heidelberg) was unable to survive in the hostile environment of the albumen. In contrast, eggshell thickness had no significant correlation with the number of positive samples. In conclusion, the results obtained in the egg infection model show that the Salmonella strains tested were able to penetrate the eggshell and multiply in both the albumen and yolk and that S. Typhimurium proved to be the most efficient to grow within these portions of the egg.

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