Neutralizing Antibody Detection & Quantification

The ability of the xCELLigence system to track virus-induced CPEs in real-time can be employed for detecting and quantifying neutralizing antibodies. The assay principle is similar to a standard plaque reduction neutralization test (PRNT), but the automated data acquisition of the xCELLigence instrument dramatically reduces the hands-on time that is required, and completely eliminates the subjectivity of data analysis due to visual interpretation. As a result, the data obtained is much more precise and reproducible. The two examples shown below demonstrate the utility of this approach for both research and clinical applications.

 

After growing Vero cells to confluence in E-Plate wells, Fang and coworkers infected each well with 106 plaque forming units of West Nile Virus (WNV) that had been pre-incubated with serially diluted House finch antisera with known neutralizing antibody concentration1. As seen in Figure A below, the onset of WNV-induced CPE was delayed by the neutralizing antisera incubation in a manner that was directly dependent on antibody concentration. By plotting the CIT50 (time required for the Cell Index to decrease by 50%) as a function of the reciprocal of the antibody titer, a standard curve was generated (Figure B) which could be used for quantifying the amount of neutralizing antibody present in avian specimens. Importantly, using this xCELLigence-based standard curve to determine antibody concentrations in infected birds out in the wild gave values that correlated very well with values determined by a traditional plaque reduction neutralization test1.

 

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 has dropped to 50% of its initial value (i.e. before virus addition). The time required to reach this point is referred to as “CIT50”.  (B) By plotting CIT50 as a function of the reciprocal of antibody titer, a standard curve is produced which can be used for assessing the antibody concentrations in wild avian sera.  Figure adapted from reference 1.

The xCELLigence RTCA technology has also been used for quantifying the amount of neutralizing antibody against influenza A H1N1 virus that is present in human sera. In short, within E-Plate wells confluent cells were infected with purified H1N1 virus that either had or had not been pre-treated with patient serum. These serum samples had been collected before vaccination, or 7 or 21 days post vaccination, making it possible to track the emergence of an H1N1-specific neutralizing response over time. As expected, the robustness of the H1N1 neutralizing antibody activity increases progressively over the first 21 days post vaccination, evidenced by the delayed onset or complete block of the cytopathic effect.
With its simple, automated workflow and objective and quantitative readout, xCELLigence clearly offers advantages over traditional assays for detecting and quantifying neutralizing antibodies. This approach provides a simple means for monitoring the efficacy of vaccination, and for elucidating the kinetics of virus resistance emergence.

 

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) provides no protection and the virus kills 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 particular vaccine, as well as the kinetics of virus resistance emergence. Figure adapted from reference 2.

Vaccine handbook

Handbook: Explore Viral Cytopathic Effect Assays for Virology and Vaccine Research

  • Viral Titer Determination
  • Detection and Quantification of Neutralizing Antibodies
  • Studying Anti-viral Drugs
  • Testing Virucides
  • Oncolytic Viruses
  • Assessing virus quality/fitness

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Virus webinar

Webinar on Demand: A Groundbreaking Technology for Vaccine Development: New Techniques in Viral CPE Assessment using Real Time Cell Analysis

On Thursday, Nov 29, 2018 at 10am EST, Loic Benair (Sanofi Pasteur, France) and Brandon Lamarche (ACEA Biosciences) will describe new approach aimed at quantifying viral cytopathic effects during vaccine development.

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App note 18

Application Note 18: A New Way to Monitor Virus-Mediated Cytopathogenicity

A demonstration on the experimental workflow and the power of real-time impedance-based technology to evaluate viral cytopathic effects. 

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xCELLigence instruments that are compatible with virology/vaccine applications:

Dual PurposeSingle PlateMulti PlateHigh Throughput
RTCA DPRTCA SPRTCA MPRTCA HT
3×16 wells1×96 wells6×96 wellsUp to 4×384 wells
  1. Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology. J Virol Methods. 2011 May;173(2):251-8.
  2. Label-free Real-time Cell Based Assay System for Evaluating H1N1 Vaccination Success.  Asiabiotech 2010, 14(10): 31-32.
  3. Inhibition of metastasis of rhabdomyosarcoma by a novel neutralizing antibody to CXC chemokine receptor-4.  Cancer Sci. 2014 Oct;105(10):1343-50.
  4. Real-time cell analysis–a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.  J Virol Methods. 2013 Nov;193(2):364-70.
  5. Novel, real-time cell analysis for measuring viral cytopathogenesis and the efficacy of neutralizing antibodies to the 2009 influenza A (H1N1) virus.  PLoS One. 2012;7(2):e31965.