Development of a focus reduction neutralization test (FRNT) for detection of mumps virus neutralizing antibodies

Dynamics of the serologic response in vaccinated and unvaccinated mumps cases during an epidemic

“…RUBI-1-94 has a mumps-specific IgG concentration of 4384.512 RU/ml and was selected as alternative (internationally-available) standard for mumps, thus enabling comparison and bridging of our serological data, expressed in RU/ml, to other studies. 31 Mumps virus-neutralizing (VN) antibodies were detected by focus reduction neutralization test (FRNT), partly based on the protocol described by Vaidya et al 34 Mumps vaccine virus (JL strain; stored at ¡80 C) was thawed and mixed with heatinactivated (45 min 56 C) plasma samples (both 37.5 ml) to be incubated for 2 hat 37 C. Culture medium (Dulbecco's modified eagle medium (Life Technologies) supplemented with 5% fetal calf serum, penicillin, streptomycin, and L-glutamine) was removed from Vero cells (2 £ 10 4 cells/mL) and 50 ml of virus/plasma mixture was added to each well of a 96 wells plate (i.e., > 20 plaques mumps virus per well). Plates were incubated for 4 hat 36 C, wells were emptied and 200 ml of 0.8% carboxymethylcellulose medium was added to each well.…”

Development of a Focus-Reduction Neutralizing Test for Detecting Equine Herpesvirus Type-1-Neutralizing Antibodies

“…However, the PRNT is laborious and has limitations for screening the large numbers of serum samples needed for epidemiological investigations, because it uses 24-well plates (one and a half plates for testing one sample). Furthermore, it takes 4 or 5 days of culture to see plaques, as is the case with the TCID 50 method.A focus-reduction neutralization test (FRNT)-an alternative VN test that can be performed in 96-well plates-has been introduced for influenza virus, mumps virus, several kinds of flavivirus and some other viruses [2,12,14,15]. Although the principle of the FRNT is the same as that of the PRNT, they differ in the surface area of the culture wells, the volumes of reagents added and the visualization steps.…”

“…However, the high viscosity of the MC makes it difficult to apply the overlay to 96-well plates. Therefore, instead of the MC, we employed Avicel (FMC Biopolymer, Philadelphia, PA, U.S.A.)-a microcrystalline cellulose with high gravity and low viscos- ity-because Avicel has been used in the FRNT for mumps virus and in focus assays for influenza virus on 96-well plates and for coronavirus on 6-well plates [7,9,14].First, we stained the foci after incubation times ranging from 24 to 48 hr to determine the optimum time point for staining after virus inoculation. MDBK cells were seeded on flat-bottomed 96-well plates (Asahi Glass Co., Ltd., Tokyo, Japan) at a concentration of 2 × 10 4 cells/well.…”

“…A focus-reduction neutralization test (FRNT)-an alternative VN test that can be performed in 96-well plates-has been introduced for influenza virus, mumps virus, several kinds of flavivirus and some other viruses [2,12,14,15]. Although the principle of the FRNT is the same as that of the PRNT, they differ in the surface area of the culture wells, the volumes of reagents added and the visualization steps.…”

“…It can be used to visualize viral foci by immuno-staining before plaque formation, resulting in a shorter test period than with the PRNT. The sensitivity of the FRNT is sufficiently high for detecting seroconversion and comparable to that of the PRNT [2,14,15]. In this study, to overcome the problems of conventional VN tests for EHV-1, we developed an FRNT for EHV-1 that can be used to test large numbers of samples in a short period of time and with sufficient sensitivity.…”