PRODUCTION OF MONOCLONAL AND POLYCLONAL ANTIBODIES FOR THE DIAGNOSIS OF BANANA STREAK VIRUS, GENUS BADNAVIRUS

Abstract

BSV was isolated and purified from infected leaves of IITA plantain hybrids (TMPx) by Methods A & B. Method A involved the use of sucrose cushion and caesium gradient during ultracentrifugation, while method B involved the use of sucrose cushion only. Method B yielded 15-fold more BSV particles than method A. Mouse polyclonal antibodies raised separately against the BSV purified using these methods were BSV-specific and had high antibody titres. BSV-specific and high titre polyclonal (from rabbit and chicken) antibodies were also raised against purified BSV by method B. The rabbit antiserum, chicken antiserum and chicken egg immunoglobulin G produced had BSV antibody titres of 1:102,400, 1:32,000 and 1:2000 respectively. Two BSV-specific monoclonal antibodies (coded BSV 3F9/1 and BSV 3D4/2), were produced. The two monoclonal antibodies were of IgG 2a isotype. BSV 3F9/1 and BSV 3D4/2 culture fluids had antibody titres of 1:204,800 and 1:6400 respectively. BSV 3F9/1, BSV 3D4/2 and mouse polyclonal antibodies detected BSV in 22 out of 29 symptomatic Musa plants of different genotypes by the method of triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). These Muse samples were from the collections of the International institute of

Tropical Agriculture, Ibadan (IITA). The detection of BSV in IITA Musa samples and the monoclonal antibodies additivity index of 13% indicated that the monoclonal antibodies were detecting a common epitope of a BSV strain. The sap dilution end-point determination carried out on BSV-infected leaf sap showed that the sap dilution end-points were 1:885,735 by Immunocapture polymerase chain reaction (IC-PCR); 1:3,645 by Immunosorbent electronmicroscopy (ISEM); 1:1215 by TAS-ELISA and 1:1215 by antigen-coated dot-ELISA. These indicated that IC-PCR was the most sensitive technique for BSV detection at the lowest virus concentration/sap dilution. In another experiment, out of the 47 Musa leaf samples with BSV-Iike symptoms collected in Oyo, Ondo, Ogun and Ekiti states (in Nigeria), from Ghana and Germany, 34 tested positive to BSV by ISEM, 20 by IC-PCR and 13 by TAS-ELISA. The breakdown of the number of leaf samples that tested positive to BSV by ISEM, IC-PCR and TAS-ELISA respectively, over the total number of leaf samples that showed BSV-Iike symptoms in Oyo State was 15/18, 8/18 and 7/18: in Ogun State, it was 4/4, 1/4 and 0/4, in Ondo State, it was 5/5, 4/5 and 1/5; in Ekiti State, it was 1/1, 1/1 and 1/1; in Ghana, it was 4/13, 1/13 and 0/13; and in Germany, it was 5/8. 5/6 and 5/6. Therefore, overall, ISEM technique detected more BSV isolates than IC-PCR.

ISEM and ISEM-plus-decoration differentiated BSV isolates into serotypes using BSV rabbit polyclonal antibodies and the two monoclonal antibodies for trapping and decoration. In addition, longer (360 nm-600 nm) as against normal length (130 nm-150 nm) BSV particles were revealed in Musa leaf-sap by ISEM. Also detected in Musa leaf-sap by ISEM were bacilliforrn particles (60-67 nm wide and 267 nm-330 nm long), suspected to be a new virus belonging to the genus Rhabdovirus.