PRODUCTION OF POLYCLONAL ANTIBODY TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS IN CHICKEN EGGS

Citrus tristeza virus (CTV) is one of the most destructive diseases in many citrus growing areas of Indonesia. Effective strategies for controlling CTV depend on diagnostic procedure namely enzyme-linked immunosorbent assay (ELISA). Study aimed to purify the CTV antigen and produced its polyclonal antibody. Virion of the severe CTV isolate designated UPM/ T-002 was concentrated by polyethylene glycol (PEG) precipitation combined with low speed centrifugation. Semipurified antigen was further purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). The specific coat protein (CP) band of CTV with molecular weight of 25 kD was excised and eluted using elution buffer containing 0.25 M Tris-HCl pH 6.8 + 0.1% SDS, then used as antigen for injection into 6-month-old female of White Leghorn chicken. Results, showed than the specific polyclonal antibody raised against the 25-kDa CP had a titer of approximately 104, gave low background reaction with healthy plant sap and reacted specifically with CTV isolates. The reaction was equally strong for a severe, a moderate, a mild, and a symptomless isolate, suggesting a broad reaction range of this antibody toward different CTV isolates. Optimal virus titer can be obtained since virus loss during purification could be minimized and the highly purified antigen as an immunogen could be obtained by cutting out the CP band from SDS-PAGE gels. Large amount of highly titer of CTV antibody can be produced in chicken egg. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antibody can be produced in basic laboratory.

Download Full-text

Serological Detection of Citrus tristeza virus with Antibodies Developed to the Recombinant Coat Protein

Plant Disease ◽

10.1094/pdis-93-1-0011 ◽

2009 ◽

Vol 93 (1) ◽

pp. 11-16 ◽

Cited By ~ 5

Author(s):

M. M. Iracheta-Cárdenas ◽

P. Metheney ◽

M. L. Polek ◽

K. L. Manjunath ◽

R. F. Lee ◽

...

Keyword(s):

Coat Protein ◽

Large Scale ◽

Citrus Tristeza Virus ◽

Monitoring Program ◽

Enzyme Linked Immunosorbent Assay ◽

Serological Detection ◽

Central California ◽

Tristeza Virus ◽

Recombinant Coat Protein ◽

Citrus Tristeza

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.

Download Full-text

Comparison of Detection Methods for Citrus Tristeza Virus in Field Trees During Months of Nonoptimal Titer

Plant Disease ◽

10.1094/pdis.1997.81.5.525 ◽

1997 ◽

Vol 81 (5) ◽

pp. 525-529 ◽

Cited By ~ 31

Author(s):

D. M. Mathews ◽

K. Riley ◽

J. A. Dodds

Keyword(s):

Positive Result ◽

Citrus Tristeza Virus ◽

Virus Titer ◽

Significant Loss ◽

San Joaquin Valley ◽

Detection Methods ◽

Enzyme Linked Immunosorbent Assay ◽

Nucleic Acid Extract ◽

Tristeza Virus ◽

Citrus Tristeza

Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.

Download Full-text

Comparison of the Coat Protein Gene Sequences of Citrus Tristeza Virus Isolatesin New Zealand

International Organization of Citrus Virologists Conference Proceedings (1957-2010) ◽

10.5070/c50z51r3jp ◽

2000 ◽

Vol 14 (14) ◽

Author(s):

P. A. Mooney ◽

T. E. Dawson ◽

C. L. Niblett

Keyword(s):

New Zealand ◽

Coat Protein ◽

Coat Protein Gene ◽

Citrus Tristeza Virus ◽

Protein Gene ◽

Gene Sequences ◽

Tristeza Virus ◽

Citrus Tristeza

Download Full-text

Production of polyclonal antibodies against the recombinant citrus tristeza virus coat protein expressed in bacteria

International Journal of Virology ◽

10.3923/ijv.2005.7.7 ◽

2004 ◽

Vol 1 (1) ◽

pp. 7-7

Author(s):

Hala A. Amin . ◽

A. Barakat . ◽

A.A. Abou Zeid .

Keyword(s):

Coat Protein ◽

Citrus Tristeza Virus ◽

Polyclonal Antibodies ◽

Virus Coat Protein ◽

Virus Coat ◽

Tristeza Virus ◽

Citrus Tristeza

Download Full-text

Molecular detection and coat protein gene based characterization of Citrus tristeza virus prevalent in Sikkim state of India

Indian Phytopathology ◽

10.1007/s42360-019-00180-3 ◽

2019 ◽

Vol 73 (1) ◽

pp. 135-143 ◽

Cited By ~ 2

Author(s):

Ashish Warghane ◽

Amol Kokane ◽

Sunil Kokane ◽

Manali Motghare ◽

Datta Surwase ◽

...

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Molecular Detection ◽

Citrus Tristeza Virus ◽

Protein Gene ◽

Tristeza Virus ◽

Citrus Tristeza

Download Full-text

Breakdown of Cross-Protection of Grapefruit from Decline-Inducing Isolates of Citrus tristeza virus Following Introduction of the Brown Citrus Aphid

Plant Disease ◽

10.1094/pdis.2003.87.9.1116 ◽

2003 ◽

Vol 87 (9) ◽

pp. 1116-1118 ◽

Cited By ~ 8

Author(s):

C. A. Powell ◽

R. R. Pelosi ◽

P. A. Rundell ◽

M. Cohen

Keyword(s):

Immunosorbent Assay ◽

Citrus Tristeza Virus ◽

Cross Protection ◽

Enzyme Linked Immunosorbent Assay ◽

Experimental Area ◽

Toxoptera Citricida ◽

The Cross ◽

Tristeza Virus ◽

Citrus Tristeza

A 21-year-old replicated field planting of 84 ‘Ruby Red’ grapefruit trees cross-protected with three mild isolates of Citrus tristeza virus (CTV) was assessed for decline-inducing and non-decline-inducing isolates of the virus 5 years after the brown citrus aphid (BrCA) (Toxoptera citricida Kirkaldy) first was established in the experimental area. Prior to the introduction of the BrCA, the cross-protecting mild isolates had significantly reduced detectable infection with decline-inducing isolates of CTV for 16 years (average infection of 13% in cross-protected trees compared with 67% in unprotected trees). After the introduction of the BrCA, infections with decline-inducing CTV (measured by enzyme-linked immunosorbent assay) were 57, 81, and 71% for trees protected with three mild isolates, respectively, compared with 95% in unprotected trees. These results suggest that the introduction of BrCA accelerated the breakdown of cross-protection against decline-inducing isolates of CTV in grapefruit.

Download Full-text