Neutralizing Antibody Detection & Quantification

The plaque reduction neutralization test (PRNT) is considered the gold standard for detecting and measuring the ability of antibodies to neutralize a virus by preventing the formation of a plaque. While the   xCELLigence Real Time Cell Analysis principle is similar to a standard PRNT, the automated data acquisition provided by xCELLigence RTCA dramatically reduces the required hands-on time.  The xCELLigence system’s ability to track virus-induced cytopathic effects in real-time allows for the accurate detection and quantification of neutralizing antibodies. It also eliminates the subjectivity in data analysis exhibited by the visual interpretations of PRNT assays for more precise and reproducible assays.


Vaccine & Virology Handbook
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Typical methods vs xCELLigence RTCA

Plaque Reduction Neutralization TestxCELLigence RTCA Antibody Neutralization Assay
Considered the gold standard for detecting and measuring the ability of antibodies to neutralize a virus by preventing the formation of a plaque. With this method, virus and serum are diluted and plated onto cells. Plaques are manually counted to determine plaque forming units Direct, real-time kinetics to quantify amount of neutralizing antibody. Cells are pre-incubated with neutralizing antibody and plated, infected with virus, and xCELLigence RTCA automatically monitors virus neutralization by antibody in real-time.
Viral plaque formation can take days to weeks to be detectable.Quantitative monitoring of both fast (hours) and slow (days to weeks) CPE. 
PRNT assay provides no information about the kinetics of the virus neutralization by antibodies. xCELLigence provides assessment and quantification of the kinetics of virus neutralization by antibodies, monitor the efficacy of vaccination or the kinetics of virus resistance emergence.
The manual counting of plaques by visual inspection can be highly subjective and inconsistent since plaque formation rates and sizes can vary dramatically. Plotting CIT50 (time required for the Cell Index to decrease by 50%) as a function of the reciprocal of the antibody titer generates a standard curve, used for quantifying the amount of neutralizing antibody present in the sample


The rapid, sensitive, and high throughput xCELLigence Real-Time Cell Analysis Assay is ideal for neutralizing antibody detection & quantification

Example Data: Quantifying West Nile Virus neutralizing antibody in wild birds

Figure reprinted from Journal of Virological Methods, volume 173(2), Fang, Y. et al., “Real-Time Monitoring of Flavivirus Induced Cytopathogenesis Using Cell Electric Impedance Technology,” pages 251–8. Copyright 2011, with permission from Elsevier.

Quantifying WNV neutralizing antibody titer using xCELLigence RTCA. (A) Vero cells were infected with 106 plaque forming units of WNV that had been pre-incubated with different dilutions of neutralizing antibody.  Ctrl = Vero cells that were not infected with virus; WNV only = Vero cells infected with virus that was not pre-exposed to antibody.  The horizontal line denotes the point at which Cell Index dropped to 50% of its initial value (i.e. before virus addition). The time required to reach this point was referred to as “CIT50”.  (B) By plotting CIT50 as a function of the reciprocal of antibody titer, a standard curve was produced which can be used for assessing the antibody concentrations in wild avian sera.


Example Data: Quantifying Influenza A H1N1 neutralizing antibody in vaccinated human

Figure adapted from Asia Pacific Biotech News, volume 14(10), Lu, H. et al. “Label-free Real-time Cell Based Assay System for Evaluating H1N1 Vaccination Success,” pages 31–32. Copyright 2010, with permission from Asia Pacific Biotech News.

Measuring neutralizing antibody activity in H1N1-vaccinated human subjects using xCELLigence. Serum samples from two human patients were collected prior to vaccination (day 0), and then 7 and 21 days post vaccination. These serum samples were incubated with purified H1N1 virus before adding the virus/serum mixture to cells growing in an E-Plate. For both patients, serum from day zero (purple and blue curves) provided no protection and the virus killed the cells with kinetics very similar to the positive control (red curve). For patient #2, serum from day 7 showed significant delay of H1N1-induced CPE (brown curve), indicating the presence of specific neutralizing antibodies against H1N1 virus. In contrast, patient #1’s serum at day 7 showed no prophylactic effect (pink curve), indicating that an H1N1 neutralizing antibody activity was not yet present. However, 21 days post-vaccination the serum from both patients displayed robust neutralizing antibody activity against H1N1, rendering the virus completely incapable of inducing a cytopathic effect dark green and aqua curves). This assay makes it possible to quantitatively assess the efficacy of a given vaccine, as well as the kinetics of virus resistance emergence.


New Techniques in Viral CPE Assessment using Real Time Cell Analysis
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Neutralizing Antibody Detection & Quantification Application Notes


Neutralizing Antibody Detection & Quantification Publications


Featured xCELLigence RTCA Systems for Viral CPE Assays

Dual PurposeSingle PlateMulti PlateHigh Throughput
3×16 wells1×96 wells6×96 wellsUp to 4×384 wells