Detection of plum pox potyvirus using monoclonal antibodies to structural and non‐structural proteins¹

Abstract

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non‐structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture‐PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA‐I and immunoprinting‐ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D‐specific monoclonal antibodies can be used in routine tests with high affinity.

Plum pox virus strains: Diversity and geographical distribution in Serbia

“…The production and use of specific monoclonal antibodies improved PPV diagnosis by ELISA technique. Monoclonal strain-specific antibodies (Mabs) specific to structural PPV CP have been developed for: PPV-D (Cambra et al, 1994), PPV-M (Boscia et al, 1997), PPV-EA (Myrta et al, 1998), PPV-C (Myrta et al, 2000) and PPV-W (Croft et al, 2008). A PPV-M specific antibody detects both PPV-M and the later described PPV-Rec isolates and this analysis needs to be supplemented with molecular tests for precise strain identification.…”

Genetic Diversity and Population Structure

“…Numerous assay kits for PPV diagnostics based on sandwich-ELISA with polyclonal antibodies and conjugated alkaline phosphatase are well represented on the market. Indirect sandwich-ELISA [20] with 5B monoclonal antibodies to 94 DRDVDAG 100 , a universal epitope found till now in the CPs of all studied isolates [21], seems to be the most reliable. In RT-PCR any PPV isolate can be found with universal primers specific for CP gene or 3´-NCR [22,23].…”

“…Variability of PPV isolates can be studied differently [25]. In particular, the strain can be identified in indirect sandwich ELISA-test with monoclonal antibodies to D, M, C and EA [20,[26][27][28], in RT-PCR with primers specific to D, M, C, CR, EA, Rec, and W [29][30][31][32][33][34], and by sequencing CP gene or its hyper variable N-terminal fragment followed by phylogenetic analysis. Sometimes the PPV strain can be identified by sequencing and phylogenetic analysis of 243 bp product along with its restriction analysis using RsaI and AluI endonucleases [22].…”

Epidemiología de Plum pox virus y Citrus tristeza virus en bloques de plantas de vivero. Métodos de control

“…El protocolo OEPP/EPPO (2004b) recomienda el anticuerpo monoclonal para la detección universal de PPV, 5B-IVIA (Cambra et al, 1994). Este anticuerpo es el único que puede detectar todos los aislados de PPV citados hasta la fecha (Cambra et al, 2006a).…”

“…Este anticuerpo es el único que puede detectar todos los aislados de PPV citados hasta la fecha (Cambra et al, 2006a). En cuanto a la caracterización de los aislados, el mismo protocolo indica que para la caracterización de los aislados PPV-D recomienda los anticuerpos monoclonales 4DG5 y 4DG11 (Cambra et al, 1994) y para la caracterización de aislados PPV-M recomienda el anticuerpo monoclonal AL 1. Introducción 15 (Boscia et al, 1997).…”