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Rapid fluorescent focus inhibition test optimization and validation: Improved detection of neutralizing antibodies to

Tatyana M Timiryasova1, Ping Luo1, Lingyi Zheng1

  • 1Sanofi Pasteur, Discovery Drive, Swiftwater, PA 18370, USA.

Journal of Immunological Methods
|June 23, 2019
PubMed

Insights

This study optimized the rabies rapid fluorescent focus inhibition test (RFFIT) for detecting rabies virus neutralizing antibodies (RVNA). The enhanced assay offers improved accuracy and sensitivity, simplifying RVNA quantification in human serum.

Area of Science:

  • Virology
  • Immunology
  • Assay Development

Background:

  • The rabies rapid fluorescent focus inhibition test (RFFIT) is the standard cell-based assay for quantifying rabies virus neutralizing antibodies (RVNA).
  • RFFIT is a manual, labor-intensive assay susceptible to variability from reagents, cell quality, and analyst expertise.

Purpose of the Study:

  • To identify and evaluate parameters impacting RFFIT performance and RVNA detection.
  • To optimize the RFFIT assay for improved accuracy, linearity, and sensitivity.

Main Methods:

  • Systematic evaluation of serum dilution schemes, virus dose, complement, cell density, DEAE-dextran concentration, and incubation periods.
  • Comparison of manual fluorescent focus counting with automated image analysis using a cell imaging reader.
  • Assay validation according to International Conference on Harmonization (ICH) and Good Clinical Laboratory Practice (GCLP) guidelines.

Main Results:

  • Optimization of key assay parameters led to improved RFFIT performance.
  • Automated image analysis provided a viable alternative for focus counting, enhancing data record-keeping.
  • The optimized RFFIT demonstrated enhanced accuracy, linearity, and sensitivity when tested with WHO Standard Rabies Immune Globulins (SRIGs).

Conclusions:

  • The optimized RFFIT protocol provides a more robust and potentially less subjective method for RVNA quantification.
  • This improved assay facilitates reliable detection and measurement of rabies-specific antibodies.
  • The validated assay meets GCLP standards for clinical laboratory practice.

Keywords:
AntibodiesBHK-21Biological assayNeutralizingRFFITRVNARabies vaccinesRabies virusSRIGValidation studies

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