Nurmalasari (2017) Pengembangan Metode Imunodiagnosis Penyakit Mosaik Tebu Menggunakan Antibodi Untuk Rekombinan Protein Kapsid Sugarcane Mosaic Virus. Disertasi thesis, UNIVERSITAS AIRLANGGA,.

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Abstract

Serology-based detection method (e.g ELISA) utilizing the ability of antibodies, which are made in animals, recognized specific protein of virus targeted. ELISA (enzyme linked immunosorbent assay) is a widely used serological test for detection of plant disease because of easy preparation, can be done in a simple laboratory and no need expertise. Limiting factor of serological methods is providing specific antibody which has no cross reaction to plant protein. Improving accuracy and specifity of serological antibody could be acquired through using monoclonal antibodies and recombinant antibodies. This study aimed to obtain polyclonal antibodies specific for rapid detection test to sugarcane mosaic disease caused by Sugarcane Mosaic Virus (SCMV). The study begins with searching for capsid protein gene of SCMV (CP-SCMV) by conducting survey in five areas of sugar cane nursery in East Java (Magetan, Madiun, Lumajang, Jember and Bondowoso region). Sugarcane leaf samples that show visual symptoms of mosaic disease analyzed by molecular methods of RT-PCR (reverse transcriptase PCR) using degenerate primers CP-SCMV. From this observation SCMV Jember isolate had been obtained, which is different from existing SCMV isolates in Gene Bank. The next stage is to find the complete gene sequences of CP-SCMV by designing a complete primer for CP-SCMV gene. Then, further research is redesigned conserved region primers with restriction enzyme sequences for cutting side marker at the stage of molecular cloning. Molecular cloning begins with cloning in E.coli DH5α host for storage and reproduction of gene sources. Sub cloning was done on expression vector and E.coli BL21 as host to produce recombinant proteins. Recombinant proteins produced from E. coli BL21 by inducer IPTG (isopropyl thiogalactosidase) to produce recombinant proteins. After isolation and purification, recombinant CP-SCMV ready for use as antigens to induced antibodies in rabbit and chicken. Animals immunized with recombinant antigens produced polyclonal antibodies as raw material for serological testing. Crude antibodies were tested for specificity and titer using Agar Gel Precipitation Test (AGPT), direct agglutination and Westen blot. Development of immunodiagosis methods using crude antibodies were done succesfully through Indirect ELISA method.

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