Development of specific polyclonal antibodies and a fluorescence-based immunoassay for detecting tomato brown rugose fruit virus- Iranian isolate

Tomato brown rugose fruit virus (ToBRFV; Tobamovirus fructirugosum) is an emerging virus species within the Virgaviridae family. It poses a significant threat to tomato and pepper production in Iran and worldwide. Managing ToBRFV is particularly challenging due to its stability and the ease of mechanical transmission. An effective strategy for addressing this virus involves employing quick and sensitive diagnostic method to detect its presence before symptoms appear in plants, followed by the eradication of contaminated sources. Additional strategies include serological detection methods and fluorescence-based immunoassays, which require raising antibodies for an immunochemical reaction. The aim of this study was to develop specific polyclonal antibodies against the coat protein (CP) of the ToBRFV-Iranian isolate and to create fluorescence-based immunoassays utilizing green CdTe quantum dots (QDs) to enhance the sensitivity of conventional immunoassays. To achieve this, the coding region of the ToBRFV-CP was optimized for codon usage, then cloned and expressed in the Escherichia coli strain BL21 (DE3) using recombinant DNA technology. The resulting recombinant coat protein was purified and used to immunize two female New Zealand White rabbits with five injections at two-week intervals. Polyclonal antibodies obtained from the rabbit antiserum were conjugated to horseradish peroxidase (HRP) using the periodate method and to QDs using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) as chemical linkers. Their characteristics and sensitivity were evaluated through various serological assays. The results indicated that both ToBRFV-CP-IgG^HRP and ToBRFV-CP-IgG^QDs exhibited high sensitivity in detecting ToBRFV. The concentrations of the antigens (ToBRFV-CP) and the dilutions of the infected extracts identified by ToBRFV-CP-IgG^HRP were 250 ng/mL and 1:16, respectively. In contrast, for ToBRFV-CP-IgG^QDs, they were 50 ng/mL and 1:32. Actually, compared to traditional immunoassays, the fluorescence-based immunoassays demonstrated two to five times more sensitive. Another finding from the study is the antibodies' ability to distinguish between samples infected with the ToBRFV-Iranian isolate and the closely related tobamoviruses. Additionally, this research represents the first successful report of a fluorescence-based immunoassay for ToBRFV. Also, alignment results of the present isolate with other isolates available in NCBI showed that this isolate has 100 % identity in antigenic regions with isolates from the USA, Jordan, Italy, Lebanon, and Albania. The antibodies could likely detect the mentioned isolates.