Serological detection of Grapevine leafroll-associated virus 4 in grapevine growing areas of India using polyclonal antiserum raised against the recombinant coat protein

Antibodies specific for the recombinant coat protein (rCP) of the p25 gene of Citrus tristeza virus (CTV) were developed in goats and rabbits and further evaluated as a complete kit for the detection of the virus using healthy and CTV-infected tissue. The combination of goat T1 used as primary (coating) and rabbit C3 as intermediate (detecting) rCP antibodies reacted efficiently, with optical density at 405 nm (OD405) values between 0.250 and 2.000 with samples from an international collection of diverse CTV isolates. The CTV isolates tested cause a broad spectrum of disease syndromes in different citrus hosts. The OD405 values for healthy tissue were less than 0.100. Likewise, the combination of goat T1 and rabbit C3 rCP antibodies gave consistent results for CTV-positive and -negative sample discrimination when directly compared with the Central California Tristeza Eradication Agency (CCTEA) antibodies used for large-scale CTV detection and a commercially available CTV serological detection kit. The combination of goat T1 and rabbit C3 rCP antibodies showed its suitability for large-scale indexing with samples collected in commercial groves as part of the CCTEA's regular monitoring program. The evaluation included 41,195 samples from 301 commercial groves from districts 1, 2, and 3. In total, 26 trees (0.063%) were found to be CTV positive using the T1/C3 rCP antibody combination. Results of this research provide evidence that rCP antibodies can be efficiently used for both capturing and detecting CTV antigens in double-antibody sandwich indirect enzyme-linked immunosorbent assay.

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First Detection of Rupestris stem pitting associated virus Particles by Antibody to a Recombinant Coat Protein

Plant Disease ◽

10.1094/pdis.2003.87.5.510 ◽

2003 ◽

Vol 87 (5) ◽

pp. 510-514 ◽

Cited By ~ 18

Author(s):

Natasa Petrovic ◽

Baozhong Meng ◽

Maja Ravnikar ◽

Irena Mavric ◽

Dennis Gonsalves

Keyword(s):

Coat Protein ◽

Polyclonal Antiserum ◽

Enzyme Linked Immunosorbent Assay ◽

Grapevine Virus ◽

Virus Particles ◽

Grapevine Virus A ◽

Grapevine Virus B ◽

Rupestris Stem Pitting ◽

Recombinant Coat Protein ◽

Stem Pitting

Rupestris stem pitting associated virus (RSPaV), a member of the genus Foveavirus, is associated with the Rupestris stem pitting component of the Rugose wood (RW) disease complex of grapevines. Heretofore, particles of RSPaV have not been visualized. In this work, flexuous rod particles approximately 723 nm in length were detected in the sap of infected grapevines by immunosorbent electron microscopy (ISEM), using a polyclonal antiserum produced to a recombinant coat protein of RSPaV. Particles of RSPaV were detected in tissue culture-, greenhouse-, and field-grown grapevines infected with RSPaV, but not in healthy control plants. Detection of virus particles by ISEM corresponded with detection of RSPaV by Western blot, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. Virus particles were decorated with the antibodies specific to RSPaV but not with antibodies to Grapevine virus A or Grapevine virus B, two other viruses believed to be associated with RW. This definitive identification of RSPaV particles will help define the etiology of RW.

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