Development of a novel rapid micro-neutralization ELISA for the detection of neutralizing antibodies against Chandipura virus

•Development of MN-ELISA using Indian strain of CHPV to detect Nabs to CHPV.•MN-ELISA gives read-out in the form of optical density values and has a shorter TAT.•The sensitivity, specificity, positive predictive value and negative predictive value of the new test was 100% when compared with the conventional MN-CPE method.•New assay detects very minute difference in Nab titer and also low-titer sero-conversion.•MN-ELISA; a better alternative to the conventional MN-CPE in sero-surveillance and in future vaccine studies.

BackgroundChandipura virus (CHPV) is a leading cause of acute encephalitis with high mortality in paediatric population in India. A micro-neutralization ELISA (MN-ELISA) assay was developed for the detection of neutralizing antibodies (Nab) against CHPV. This novel method gives read-out in the form of ELISA optical density (OD) values and has a shorter turn-around time (TAT) as compared to the conventional cytopathic effect (CPE)-based neutralization assay (MN-CPE). The assay was developed using an Indian strain of CHPV. During the development of the assay different parameters such as cell count, dilution of primary and secondary antibodies and time point for the test termination were optimized. The new and conventional assays were run in parallel where known positive and negative human serum samples were used as test controls. The conventional MN-CPE was terminated at 48 h post-infection (p.i.) and stained with Amido black, while in the new assay, MN-ELISA was terminated at pre-determined 18 h p.i. and the infected cells were fixed with acetone, followed by in-situ ELISA. Results of both the assays were compared.The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the new test was 100% when compared with the conventional MN-CPE method as a 'gold standard'. The MN-ELISA showed two-fold higher antibody titer in one sample and one sample was additionally positive than MN-CPE ELISA.ConclusionThe MN-ELISA is rapid, more sensitive and read-out of results is by measurement of OD, which could be more accurate than manual observation of reduction in CPE. This novel test could be used as an alternative to the conventional MN-CPE based assay in sero-surveillance and in future vaccine studies.