Optimization of lateral flow assay for Canine morbillivirus detection and the application of the strip as sample substitute

Abstract

Canine distemper is an emerging disease, caused by the Canine morbillivirus (CDV) of the Paramyxoviridae family. The virus has evolved as a multi-host pathogen as it affects many wildlife animal species. The development of specific and sensitive diagnostic tests is the need for a control program. Several diagnostic tests are available for the detection of CDV antigen and antibody. Lateral flow assay (LFA) is the most promising point of care diagnostic test because of its specificity, easy use, and instant result. This study was designed to develop a lateral flow assay using the in-house developed monoclonal antibody (mAb) against the nucleocapsid protein (N) of the ‘CDV/dog/bly/Ind/2018’ isolate, which represents the circulating strains of India. The two mAbs included in the study showed high binding affinity in indirect ELISA and dot blot assay. Out of two, one mAb was selected due to its comparatively higher binding affinity in LFA format, and less non-specific binding to the biological matrix and buffer components. The limit of detection was found to be 106.5 TCID50/ml with the assay run time of 5 min. The fresh clinical samples collected on the spot were distinctly detected by the LFA, whereas the stored samples with a reduced titre of the virus showed inconsistent results. Moreover, the blood samples showed a clear distinction of positive and negative than the swab and tissue homogenates. The RNA extraction from the strip was successful with the some modifications in the Trizol RNA extraction method and the N and H gene fragments were amplified. Therefore, the study concludes that the LFA is suitable for CDV antigen detection in the field conditions and the strips can be used as the sample substitute for molecular study.