Chapter 34
Viral Infectivity Quantification and Neutralization Assays
Using Laser Force Cytology
Colin G. Hebert, Keya L. Rodrigues, Nicole DiNardo,
and Anna-Barbara Hachmann
Abstract
Despite the widespread need to assess cell-based viral infectivity during vaccine development and production, as well as viral clearance monitoring and adventitious agent testing for viral safety, traditional methods,
including the end-point dilution assay (TCID50) and viral plaque assay, are slow, labor-intensive, and can
vary depending upon the skill and experience of the user. LumaCyte’s Radiance
® instrument uses Laser
Force Cytology
TM (LFC), a combination of advanced optics and microfluidics, to rapidly analyze the viral
infectivity of cell populations in a quantitative fashion. LFC applies optical and fluidic forces to single cells in
order to measure their intrinsic biophysical and biochemical properties without the use of stains, antibodies
or fluorescent labels. These properties, including refractive index, change with a wide variety of biological
phenomena, including viral infection, cell differentiation, activation, size, and cytoskeletal stiffness. Here,
we present the experimental design and methods to use LFC data to facilitate rapid and robust infectivity
measurements for a variety of applications including initial titer measurement (TCID50 replacement),
in-process infectivity (e.g., bioreactor monitoring), and viral neutralization (PRNT replacement).
Key words Viral infectivity, Viral titration, TCID50, Plaque assay, Laser force cytology, Vaccines,
Neutralization assay
1 Introduction
The quantification of infectious virus is an important analytical
need across the biopharmaceutical industry, whether it be for vaccine research and development, vaccine production [1, 2] or the
detection of adventitious viruses to ensure the safety of biologics,
(e.g., monoclonal antibodies) [3]. Widely accepted methods
include the plaque and TCID50 assays as well as antibody-based
methods [4–6]. However, plaque and TCID50 assays are labor
intensive, lengthy, and subjective as they often rely on individual
interpretation of results [7, 8]. As such, a large number of dilutions
and replicates are needed in order to obtain statistically relevant
data [9, 10].
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_34, © Springer Science+Business Media, LLC, part of Springer Nature 2021
575
Viral Infectivity Quantification and Neutralization Assays
Using Laser Force Cytology
Colin G. Hebert, Keya L. Rodrigues, Nicole DiNardo,
and Anna-Barbara Hachmann
Abstract
Despite the widespread need to assess cell-based viral infectivity during vaccine development and production, as well as viral clearance monitoring and adventitious agent testing for viral safety, traditional methods,
including the end-point dilution assay (TCID50) and viral plaque assay, are slow, labor-intensive, and can
vary depending upon the skill and experience of the user. LumaCyte’s Radiance
® instrument uses Laser
Force Cytology
TM (LFC), a combination of advanced optics and microfluidics, to rapidly analyze the viral
infectivity of cell populations in a quantitative fashion. LFC applies optical and fluidic forces to single cells in
order to measure their intrinsic biophysical and biochemical properties without the use of stains, antibodies
or fluorescent labels. These properties, including refractive index, change with a wide variety of biological
phenomena, including viral infection, cell differentiation, activation, size, and cytoskeletal stiffness. Here,
we present the experimental design and methods to use LFC data to facilitate rapid and robust infectivity
measurements for a variety of applications including initial titer measurement (TCID50 replacement),
in-process infectivity (e.g., bioreactor monitoring), and viral neutralization (PRNT replacement).
Key words Viral infectivity, Viral titration, TCID50, Plaque assay, Laser force cytology, Vaccines,
Neutralization assay
1 Introduction
The quantification of infectious virus is an important analytical
need across the biopharmaceutical industry, whether it be for vaccine research and development, vaccine production [1, 2] or the
detection of adventitious viruses to ensure the safety of biologics,
(e.g., monoclonal antibodies) [3]. Widely accepted methods
include the plaque and TCID50 assays as well as antibody-based
methods [4–6]. However, plaque and TCID50 assays are labor
intensive, lengthy, and subjective as they often rely on individual
interpretation of results [7, 8]. As such, a large number of dilutions
and replicates are needed in order to obtain statistically relevant
data [9, 10].
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_34, © Springer Science+Business Media, LLC, part of Springer Nature 2021
575