Although the plaque reduction neutralization test (PRNT) is considered the "gold-standard" assay for measuring neutralizing antibodies for mumps, it is technically demanding, slow and requires large serum volumes, which limits its use for investigating mumps vaccine efficacy and population susceptibility. Therefore, an immunocolourimetric-based focus reduction neutralization test (FRNT) was developed and validated against PRNT using 30 blood donor plasma samples (16 positive, 5 equivocal, and 9 negative for mumps IgG by EIA). The samples were tested in triplicate by FRNT and PRNT in 10 and 4 separate assay runs, respectively, and 50% neutralizing antibody titres calculated using the Kärber formula. There was good correlation between the two neutralization assays (R2 = 0.88). Inter-assay variation for FRNT titres was 2-fold, compared to a 3-fold variation for PRNT titres. From the distribution of results, a positive cut-off for FRNT was defined as 1:4. In conclusion, FRNT has similar sensitivity to the PRNT and offers the advantage of speed (2 days vs. 7 days), reduced sample volume (40 μL vs. 150 μL), and the possibility of automation using 96-well plates. FRNT appears to be a good substitute for PRNT for characterising the immune response to mumps and for vaccine efficacy studies.
Bibliographical noteFunding Information:
This work has been done as part of SRV's PhD studies for which he has received a National Overseas PhD scholarship award from Ministry of Social Justice and Empowerment, Government of India. The authors would like to thank Drs. Xavier Martin and Marc McLoughlin from FMC Biopolymer, Europe Division for the gift of Avicel RC-581 and Dr. Annedore Tischer from Robert Koch Institute, Berlin for providing the European Surveillance Network standard (REF-II) serum.
- Assay validation
- Focus reduction neutralization test (FRNT)
- Mumps neutralizing antibody