Engineered Antibody Fragments for Immunodiagnosis of Papaya ringspot virus.

Abstract

The present study was undertaken to clone and express the genes encoding antibody to the recombinant coat protein (rCP) of Papaya ringspot virus (PRSV) and to assess the engineered antibody for the detection of PRSV. A 33-kDa rCP of PRSV, which was produced in Escherichia coli, generated PRSV specific antibody in immunized mouse. The heavy and light chain variable domain genes (VH and VL) of 351 and 360 nucleotides, respectively, were cloned from the mRNA isolated from the spleen of the immunized mouse with rCP of PRSV. The VH and VL belong to the family IgG1 and kappa chain, respectively, and contained the framework regions and complementarity determining regions. The VH and VL genes were individually used to develop the expression constructs in pET28a (+) vector and 14-kDa proteins were obtained in E. coli. The amount of purified VH and VL proteins was 3-4 mg/l of bacterial culture. Both the antibody fragments recognized PRSV in the crude sap; however, the VL antibody fragment showed higher affinity to PRSV. The mixture of VH and VL detected PRSV as effectively as polyclonal antibody. The recombinant antibody fragments mixture detected PRSV in the field samples with 100 % accuracy in dot immunobinding assay (DIBA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of the detection of PRSV using antibody fragments was 1.0 and 10.0 ng in DIBA and ELISA, respectively. The results showed successful isolation of functional single-domain antibody encoding genes to PRSV directly from the immunized spleen cells of mouse. This study for the first time demonstrates application of bacterial expressed recombinant antibody fragments in immunodiagnosis of PRSV.