scholarly journals A Cell-Based Capture Assay for Rapid Virus Detection

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1165
Author(s):  
Elad Milrot ◽  
Efi Makdasi ◽  
Boaz Politi ◽  
Tomer Israely ◽  
Orly Laskar
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Virus Detection ◽  
Vero Cells ◽  
Detection Sensitivity ◽  
Clinical Samples ◽  
Response Analysis ◽  
Proof Of Concept ◽  
Virus Identification ◽  
Capture Assay

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

scholarly journals Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry

2019 ◽  
Vol 20 (21) ◽  
pp. 5307 ◽  
Author(s):  
Valentina Puca ◽  
Eva Ercolino ◽  
Christian Celia ◽  
Giuseppina Bologna ◽  
Luisa Di Marzio ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Biofilm Formation ◽  
Lactobacillus Reuteri ◽  
Bacterial Species ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Extracellular Dna ◽  
Clinical Samples ◽  
H Pylori

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

scholarly journals Improved Mg2+-Based Reverse Transcriptase Assay for Detection of Primate Retroviruses

1999 ◽  
Vol 37 (6) ◽  
pp. 1704-1708 ◽  
Author(s):  
Johnna F. Sears ◽  
Roy Repaske ◽  
Arifa S. Khan
Keyword(s):  
Reverse Transcriptase ◽  
Virus Detection ◽  
Distinct Type ◽  
Detection Sensitivity ◽  
Clinical Samples ◽  
Potential Health ◽  
Type D ◽  
Immunodeficiency Virus ◽  
Type D Retroviruses ◽  
Hiv 1

The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10−5 U, which is equivalent to 4.25 × 104 virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans.

scholarly journals A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 791
Author(s):  
Alba Folgueiras-González ◽  
Robin van den Braak ◽  
Martin Deijs ◽  
Lia van der Hoek ◽  
Ad de Groof
Keyword(s):  
High Throughput ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Genetic Material ◽  
Virus Detection ◽  
Detection Sensitivity ◽  
Clinical Samples ◽  
Tissue Samples ◽  
Sequencing Technologies ◽  
Organic Extraction

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

PAICA: A Method for Characterizing Platelet-Associated Antibodies - Its Application to the Study of Idiopathic Thrombocytopenic Purpura and to the Detection of Platelet-bound c7E3

1996 ◽  
Vol 76 (06) ◽  
pp. 1020-1029 ◽  
Author(s):  
Laurent Macchi ◽  
Gisèle Clofent-Sanchez ◽  
Gérald Marit ◽  
Claude Bihour ◽  
Catherine Durrieu-Jais ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Antithrombotic Therapy ◽  
Membrane Glycoproteins ◽  
Serum Antibodies ◽  
Platelet Destruction ◽  
Thrombocytopenic Purpura ◽  
Capture Assay ◽  
Specific Membrane

SummaryIn idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA (“Platelet-Associated IgG Characterization Assay”), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient’s own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA (“Monoclonal Antibody-specific Immobilization of Platelet Antigens”). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP Ilb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP Ilb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.

scholarly journals Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 365
Author(s):  
Fengli Liu ◽  
Yanxin Cao ◽  
Maokai Yan ◽  
Mengxu Sun ◽  
Qingshui Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colloidal Gold ◽  
Preventive Intervention ◽  
Virus Detection ◽  
Cross Reactivity ◽  
Duck Enteritis Virus ◽  
Detection Sensitivity ◽  
Immunochromatographic Assay ◽  
Efficient Detection ◽  
Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

Approaching stability challenges for flow cytometry in a regulated bioanalytical environment

Bioanalysis ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 1845-1858 ◽  
Author(s):  
Anamica Muruganandham ◽  
Carolyne Dumont ◽  
Kuiyi Xing
Keyword(s):  
Flow Cytometry ◽  
Critical Parameter ◽  
Storage Period ◽  
Reliable Data ◽  
Clinical Samples ◽  
Sample Collection ◽  
Testing Laboratory ◽  
Stability Parameters ◽  
Stability Issues ◽  
Limited Period

Stability of samples for flow cytometry is a critical parameter since storage period of samples is restricted to only a limited period after collection. For most studies, clinical samples have to be shipped to a testing laboratory, in contrast to preclinical samples, which can be analyzed on-site or off-site. Therefore, evaluating stability is critical to provide flexibility on testing of samples to obtain reliable data. A wide variety of factors contributes to establishing stability from sample collection through acquisition. We provided suggestions for experimental and stability parameters to be taken into consideration when designing a flow cytometry method. The case studies presented represent how certain stability issues were overcome to perform flow cytometry assays in a regulated bioanalytical environment.

scholarly journals Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

2021 ◽  
Vol 22 (5) ◽  
pp. 2723
Author(s):  
Linhua Tian ◽  
Elzafir B. Elsheikh ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
Adolfas K. Gaigalas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Receptor Binding Domain ◽  
Proof Of Concept ◽  
Reference Standard ◽  
Improve Measurement ◽  
Antibody Titers

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

LYMPHOCYTE PHENOTYPING BY FLUORESCENCE MICROSCOPY AND FLOW CYTOMETRY: RESULTS IN HOMOSEXUAL MEN AND HETEROSEXUAL CONTROLS

Aids Research ◽  
1983 ◽  
Vol 1 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Joseph T. Newman ◽  
Donna S. Nicodemus ◽  
Guido A. Ordonez ◽  
Marvin J. Stone
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Homosexual Men ◽  
Lymphocyte Phenotyping

scholarly journals Sandwich-Type DNA Micro-Optode Based on Gold–Latex Spheres Label for Reflectance Dengue Virus Detection

Sensors ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 1820
Author(s):  
Jeningsih ◽  
Ling Ling Tan ◽  
Alizar Ulianas ◽  
Lee Yook Heng ◽  
Nur-Fadhilah Mazlan ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Limit Of Detection ◽  
Virus Detection ◽  
Average Diameter ◽  
Optical Biosensor ◽  
Clinical Samples ◽  
Sandwich Hybridization ◽  
Latex Spheres ◽  
Sandwich Type ◽  
Thiol Binding

A DNA micro-optode for dengue virus detection was developed based on the sandwich hybridization strategy of DNAs on succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS)) microspheres. Gold nanoparticles (AuNPs) with an average diameter of ~20 nm were synthesized using a centrifugation-based method and adsorbed on the submicrometer-sized polyelectrolyte-coated poly(styrene-co-acrylic acid) (PSA) latex particles via an electrostatic method. The AuNP–latex spheres were attached to the thiolated reporter probe (rDNA) by Au–thiol binding to functionalize as an optical gold–latex–rDNA label. The one-step sandwich hybridization recognition involved a pair of a DNA probe, i.e., capture probe (pDNA), and AuNP–PSA reporter label that flanked the target DNA (complementary DNA (cDNA)). The concentration of dengue virus cDNA was optically transduced by immobilized AuNP–PSA–rDNA conjugates as the DNA micro-optode exhibited a violet hue upon the DNA sandwich hybridization reaction, which could be monitored by a fiber-optic reflectance spectrophotometer at 637 nm. The optical genosensor showed a linear reflectance response over a wide cDNA concentration range from 1.0 × 10−21 M to 1.0 × 10−12 M cDNA (R2 = 0.9807) with a limit of detection (LOD) of 1 × 10−29 M. The DNA biosensor was reusable for three consecutive applications after regeneration with mild sodium hydroxide. The sandwich-type optical biosensor was well validated with a molecular reverse transcription polymerase chain reaction (RT-PCR) technique for screening of dengue virus in clinical samples, e.g., serum, urine, and saliva from dengue virus-infected patients under informed consent.

Flow cytometry inmunophenotyping can identify and characterize solid tumour cells in clinical samples: A preliminary study

2019 ◽  
Vol 493 ◽  
pp. S148-S149
Author(s):  
C. Quirós ◽  
L. Fernández ◽  
H. Torres ◽  
F. Domínguez ◽  
F.V. Álvarez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Solid Tumour ◽  
Tumour Cells ◽  
Clinical Samples ◽  
Preliminary Study
Sign in / Sign up

Export Citation Format

Share Document