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Vaccine Research

High throughput development of new vaccines represents only a small part of what can be achieved in the field of virus research with SYNENTEC's automated cell imagers. Amplify your work with the wide range of SYNENTEC's imaging solutions and reduce time to the clinical phase.

High throughput focus forming assay (FFA) for virus particle quantification.

SYNENTEC's profound experience in the field of virus research and vaccine development enables us to provide a wide range of applications in this area for our customers worldwide.

The high-throughput imaging systems CELLAVISTA® and NYONE® accelerate your work in various areas of virological research. Our automated cell imagers support your research with high-resolution imaging in brightfield and diverse fluorescence channels in combination with automated YT-software® for image cytometry.

We support the vaccine research & development (Influenca, Noro, SARS-CoV-2, Malaria, HIV...) for human and veterenary vaccines as well as the CLD upstream processes for production purpose (therefore please refer to CLD-section):

  • Viral titer quantification & infectivity determination
    • Label-free plaque assay
    • Endpoint dilution assays (TCID50)
    • Plaque reduction neutralization test (PRNT) (virus neutralization assay)
    • Focus forming assay (FFA)
    • Immunofluorescence foci assays (IFA)
    • Measurement of host cell viability & viral cytopathic effects (cell confluence, Live/Dead, apoptosis...)
    • Viral Pathogenesis (morphological change in infected host cells)
  • Vaccine technology
    • in vitro immune responses to vaccines
    • Vaccine vector testing in transduction efficiency
    • Immunologic in vitro studies
    • Reverse vaccinology (ELISpot and FluoroSpot [upside-down detection or transparent substrate])

 

Take a look below at a selection of high-throughput assay options with integrated automated image processing:

Focus Forming Assay – Virus Titer Quantification

[BF Dark spots]

virus_titer_quantification_focus_forming_assay.jpg

This focus forming assay (FFA) is based on an immunostaining technique and a variation of the viral plaque assay. Instead of detecting the plaque formation after virus-induced cell lysis these assays detect infected host cells and infectious virus particles before a plaque is formed.

Different cell monolayer samples are infected with a serial dilution of the virus of interest. After incubating the samples overnight the host cells built clusters of infected cells which could be visualized with specific horse radish peroxidase (HRP) coupled antibodies against a viral antigen.

The HRP can oxidize with the 3-3’diaminobenzidine tetrahydrochloride (DAB) which results in  brown/black insoluble precipitates. These foci were visualized with the brightfield illumination and the number of foci was quantified with our YT-image analysis software®.

Extract from the software readout (for further information look at ShortNote BF Dark Spots)

  • # of Foci
  • mean Foci Size
  • total Confluence
  • % Foci covered Area

Immunofluorescence Foci Assay (IFA) – Viral Infectivity

[Nuclei Colony Count; FL Cluster Diffusion]

Automated high throughput focus forming assay based on fluorescent staining.

This type of focus forming assay (FFA) is based on an immunofluorescence technique and a variation of the viral plaque assay. Instead of detecting the plaque formation after virus-induced cell lysis these assays detect infected host cells and infectious virus particles before a plaque or a noticeable infection site is formed. Especially viruses with little or no cytopathic effect can be robustly quantified using immunofluorescence. This assay is also applicable with virus mediated fluorophore expression and thus live cell imaging (no endpoint dilution assays (TCID50) needed).

Different cell monolayer samples are infected with a serial dilution of the virus of interest. After incubating the samples, the host cells show clusters of infection which can be visualized with fluorophore coupled antibodies against a viral antigen.

These foci are visualized with the appropriate fluorescence configuration and the number of foci is quantified with our YT-image analysis software®.

Extract from the software readout (for further information look at ShortNote-Section)

  • # of Foci
  • mean Foci Size
  • % Foci covered Area

Viral Plaque Assay – Even Label-free Assay-setups

[Plaque Count]

Automated Label-free viral plaque assay - high-throughput

The viral plaque assay is a method for the detection and quantitation of infectious cytopathic virus particles which are added in different dilutions across a multiwell plate containing confluent cell layers.

The difficulty is to find lysed cell areas between a confluent cell monolayer - the plaque center may miss out cells due to virus-induced lysis.

The high-resolution, precise optics of SYNENTEC's cell imagers are very well suited for this type of virus analysis - even plaques in an unstained cell layer are reliably detected. SYNENTEC has developed the viral plaque operator specifically for the standardised and ultra-fast evaluation of this assay.

Extract from the software readout (for further information look at ShortNote Plaque Assay)

  • # of Plaques
  • Plaque Area [%]
  • Average Plaque size [µm2]

Viral Pathogenesis - Quantify Morphological Changes

[Viral Infectivity]

Automated High-throughput in vitro viral pathogenesis, Cell morphology study; FFA

This focus forming assay (FFA) is based on morphological changes of a cell layer upon virus infection. It is a variation of the viral plaque assay. Instead of detecting plaque formation after virus-induced cell lysis or by immunological assays, this assay detects infected host cells and infectious virus particles before a plaque is formed. Some viruses with little or no cytopathic effect can be robustly quantified using just an immobilizing layer and high speed brightfield imaging.

Different cell monolayer samples are infected with a serial dilution of the virus of interest. After incubating the samples, the host cells show clusters of infected cells which can be visualized using brightfield imaging in 10x magnification.

These foci can quantified with the appropriate image processing to calculate the number of foci with YT-image analysis software®.

Extract from the software readout (for further information look at ShortNote-Section)

  • # of Foci
  • mean Foci Size
  • Foci Roughness
  • % Foci covered Area

Transduction Efficiency - Fluorescence Coupled Gene Expression

[Confluence 1F; Virtual Cytoplasm 1F]

02_phallo647_hoe_golgi_40x-001.jpg

Viral vectors include specifically modified virus particles used in genetic engineering to introduce genetic material into target cells. These can be cells of a living organism or cells of a cell culture. Viral transduction is used in basic research and gene therapy, and even vaccines based on viral vectors are already in use.

In traditional workflows, it is a challenging and time-consuming task to establish an ideal transfection or transduction protocol. Even if the desired vector is designed and cloned, the process of achieving a reliable transduction could still be a long challenging task!

With the high-throughput imaging systems CELLAVISTA® and NYONE® and the automated image analysis YT-software® SYNENTEC offers a standardized and extremely fast method for the development of transduction protocols.

Exemplary extract from the software readout (application dependend; for further please refer to the Download-Section)

  • Confluent Area (adherent) or # of Cells (suspension)
  • % Fluo Area vs. BF Area
  • # of Fluorescent Spots

Cytopathic Effect Quantification - Viral CPE

[Confluence – Suspension Cell Count – Apoptosis- & Viability Assays]

viral_cytopathic_effect_cpe_quantification_in_brightfield.png

Some viruses cause a cytopathic effect to cells. This effect can be quantified using different applications in YT-Software. Non-Invasive confluence monitoring is an extremely rapid and robust tool to determine various properties of virus infected cells. SYNENTEC’s confluence image processing analysis is capable to solve a vast range of different questions – e.g. to monitor CPE under various conditions or compare CPE of different engineered infectious particles and viruses. 

With SYNENTEC’s automated cell culture microscopes NYONE® and CELLAVISTA® and the included YT-image analysis software® the proliferation analysis is possible for a variety of cells. Adherent host cells can be detected as well as suspension cells. In addition, it is very easy to quantify additional fluorescent labels and their ratio to the total growth area.

Extract from the software readout for adherent cells (for further information look at ShortNote Confluence / Confluence 1F)

  • % Confluence BF
  • % Confluence FL
  • % Ratio of Confluence BF/FL
  • Time Chart of Proliferation
  • Growth Curves in Detail