Production of Polyclonal Antibody Against Tomato Leaf Curl New Delhi Virus Exploiting Synthesized Peptide as An Antigen

Abstract

The main objective of this research was to produce an antibody specific to Tomato leaf curl New Delhi virus (ToLCNDV), which is one of begomoviruses infecting cucurbits in Thailand. A peptide sequence, derived from the target epitope on the viral capsid protein based on African cassava mosaic virus (ACMV) and ToLCNDV (reported in Thailand) coat protein amino acid sequences and structures, was used as an antigen. The synthesized peptide contained 20 amino acid residues that were as follows: NIKTKNHTRRPTGTPQDFGE. It was then conjugated with bovine serum albumin (BSA) and used to immunize New Zealand White rabbits in the 1^st and 5^th weeks by subcutaneous and intramuscular injection, respectively. The antisera were collected weekly from the rabbits, over the period of the 6^th-15^th weeks. Titers of the antisera ranged from 25,600-51,200, of which the highest titer obtained was from the 9^th week (51,200). The titers at the 6^th-8^th and 10^th-15^th weeks were both 25,600. Determination of
the antiserum sensitivity and specificity were performed using an indirect plate-trapped antigen enzyme-linked immunosorbent assay (indirect PTA-ELISA) with the antiserum at 1:200 dilution. The results showed sensitivity up to 1:320 dilution of plant sap. This is the first report of the production of an antiserum that is specific to ToLCNDV only, and which does not cross react with the other 21 plant viruses brought into examination.