An Indirect ELISA Using a Recombinant Envelope Protein for Detecting Antibody Against Duck Tembusu Virus Infection

Abstract

Duck Tembusu virus (DTMUV) belonging to Flavivirus group, causes severe central nervous system disorders and decreased egg production as found in Thailand. To detect antibody against the DTMUV infection, we developed an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant envelop (E) protein for coating antigen. The E protein coding gene of KPS54A61DTMUV was cloned into pPICzαA vector and expressed by using 2% (v/v) of absolute methanol as inducer in Pichia pastoris expression. The recombinant E protein was detected in the culture medium at a concentration of 4.55 mg/ml and analyzed a protein expected size by SDS-PAGE at 65 kDa. Western blotting analysis showed specific reactivity of convalescent duck antibodies against DTMUV with the recombinant E protein. Mouse and duck antibody against the recombinant E protein were tested its specificity by immunoperoxidase monolayer assay and immunohistochemistry. The indirect ELISA coated with the recombinant E protein at concentration of 22.75 µg/ml demonstrated a coefficient of variance (%CV) range of 2.8-14.7% and 2.9-14.28% for intra assay and inter assay, respectively. The specificity and sensitivity were 88.24% and 93.22%, respectively with a kappa coefficient measured at 0.81. The indirect ELISA based on the recombinant E protein are routinely useful for DTMUV antibody detection. A retrospective study of serology was done on duck sera collected in different areas and years indicating that DTMUV infection has occurred in Thailand since the first outbreak of HPAI H5N1.