Arik Arubil Fatinah, Estri Laras Arumingtyas, Sri Widyarti, Muhaimin Rifa'I
Early detection techniques, including symptom identification, microscopy, and molecular methods, are crucial for preventing diseases caused by phytoplasmas. Currently, antibodies are under development for early detection, but their low titers, specificities, and sensitivities make them unsuitable for phytoplasma as an antigen. This study suggests utilizing phytoplasma data from the gene bank to create antigens to generate high-Titer antibodies. Data for the target protein was obtained from NCBI using the SecA protein target. A follow-up in silico confirmation test is conducted to evaluate the epitope target, antigenicity, toxicity, allergenicity, and potential cross-reactivity through homology testing using online services. The target epitope SecA(395-470) was utilized in antigen production through recombinant protein techniques. The analysis through SDS-PAGE and western blotting indicates that the recombinant protein produced demonstrates a purity greater than 90%, with a molecular weight of 12.5 kDa. The SecA(375-490) protein is subsequently administered to male New Zealand White rabbits to produce polyclonal antibodies. The polyclonal antibodies underwent ELISA to ascertain the antibody titer. The measured antibody titer exceeds 1:512,000. Developing antigens through a combination of in silico and in vitro techniques can yield high antibody titers, representing a significant challenge in advancing antibody-based phytoplasma detection methods. © Published under licence by IOP Publishing Ltd.
Animal, Fish and Plant Quarantine Authority Region Central Kalimantan, Indonesian Quarantine Authority, Central Kalimantan, Palangka Raya, 73112, Indonesia; Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, East Java, 65145, Indonesia
