Case study: Quantifying antibody binding to infected cells
Scientific challenge
Measure cell-surface antibody binding
A biotechnology company developing anti-HCMV antibodies required a quantitative method to measure antibody binding to infected cells expressing viral antigens on their surface.
While neutralisation assays provide essential information on functional antiviral activity, the ability to quantify antibody binding to infected cells provides an additional layer of characterisation, particularly for antibodies where surface antigen recognition may influence downstream immune-mediated mechanisms.
The challenge was to develop an assay capable of accurately measuring cell-surface antibody binding in a biologically relevant infection model. The method needed to generate quantitative, dose-dependent measurements suitable for comparing antibody candidates and identifying the strongest binders.
Virus |
HCMV |
Project |
Development of a quantitative cell-surface antibody binding assay |
Assay |
High-content imaging-based immunofluorescence assay, neutralisation assay |
Client |
Biopharmaceutical company |
Stage |
Antibody discovery and characterisation |
Services |
Bespoke assay development, quantitative immunofluorescence, antibody binding analysis, functional antibody characterisation |
Related expertise |
HCMV biology, antibody characterisation, high-content imaging, assay optimisation, neutralisation assays |
Key considerations
Careful optimisation
The assay required careful optimisation to ensure that measured fluorescence represented antibody binding to viral antigens exposed on the surface of infected cells, rather than non-specific staining or detection of intracellular viral proteins.
Key considerations included:
- selecting infection conditions that generated sufficient surface antigen expression, whilst maintaining biologically relevant infection conditions and avoiding excessive cellular damage,
- ensuring selective detection of surface-exposed viral antigen
- establishing appropriate controls to confirm assay specificity and robustness,
- generating quantitative data suitable for antibody comparison and dose-response analysis.
Our Approach
From the ground up
VRS developed the assay from the ground up, systematically optimising each component of the workflow.
Initial development focused on identifying the optimal infection parameters, including MOI and time point after infection, to achieve sufficient viral antigen expression on the cell surface while maintaining cell viability and physiologically relevant infection conditions.
To specifically measure surface-associated antibody binding, infected cells were fixed using a methanol-free paraformaldehyde (PFA)-based fixation approach, avoiding permeabilisation and enabling selective staining of extracellular viral antigen without detection of intracellular viral proteins.
High-content imaging was then used to quantify fluorescence intensity at the single-cell level, providing a robust and quantitative measure of antibody binding across the infected cell population.
Extensive controls were incorporated throughout assay development, including control antibodies and optimisation experiments designed to confirm that the assay was measuring the intended biological parameter.
Following validation of the workflow, the assay was used to characterise client antibodies in a dose-response format and identify differences in binding performance between candidates.
A complementary HCMV neutralisation assay was also used to provide functional assessment of antiviral activity alongside antibody binding characteristics.
Outcome
Supporting informed antibody selection
VRS successfully developed a quantitative high-content imaging assay capable of measuring antibody binding to the surface of HCMV-infected cells.
The assay provided reproducible dose-response measurements, enabling comparison of antibody candidates based on their ability to recognise infected-cell surface antigens.
Combined with neutralisation testing, the workflow provided the client with complementary information on antibody binding properties and functional antiviral activity, supporting informed antibody selection and characterisation.
VRS INSIGHT
Explore cutting-edge research with our ALI cell culture services
Antibody characterisation against complex viruses often requires multiple complementary approaches. While neutralisation assays remain essential for evaluating functional antiviral activity, understanding antibody interaction with infected cells can provide additional insight into candidate performance.
Through careful optimisation of infection conditions, and leveraging its experience on fixation strategies, imaging parameters and experimental controls, VRS established a robust platform for quantitative antibody characterisation against HCMV-infected cells.
Subscribe