ELISA detection for HLB using a pathogen-secreted protein as the biomarker

CLARK, K.; MULCHANDANI, A.; MA, W.; DE FRANCESCO, A.; TRAN, T.T; VIDALAKIS, G.; JIANFENG, L.; PAGLIACCIA, D.

Orlando

Conferencia; V International Research Conference on Huanglongbing (IRCHLB); 2017

Florida Citrus Mutual

We have identified detection markers from the HLB-associated bacterial pathogen, Candidatus Liberibacter asiaticus (CLas), and developed an enzyme-linked immunosorbent assay (ELISA) using CLas-specific antibodies. The antibody specifically recognizes epitopes from a Sec-secreted protein of CLas (called SDE1), which is presumably present in the phloem of infected trees. As such, this biomarker may have a greater distribution in the trees than the bacterial DNA, and there by allowing robust HLB detection. Using this antibody, we have developed a competitive indirect ELISA protocol, which is based on a competition for the HLB-specific antibody between the purified antigen coated on the ELISA plate and the biomarker present in citrus tissues that may have been infected with CLas. The protocol has been applied for HLB detection using freeze-dried field samples from Texas and Florida, which were simultaneously analyzed by qPCR (Li et al., 2006). Our data showed that the ELISA protocol gave comparable results with qPCR and in some cases could provide more definite diagnosis, e.g for samples gave positive qPCR results but they are higher than the cutoff point (Ct value >38). Our work demonstrates that the ELISA is a valuable tool for HLB diagnosis.Non-Technical summary: A critical measure of HLB management is to have an efficient disease detection system. This Project aims to develop serological detection methods, based on antibodies against a protein produced by CLas and secreted into the phloem during infection. Antibody-based detections are widely used for diagnosis due to its low cost, easy available equipment and reagents, and simple protocol, so they are ideal as a long scale diagnostic tool. This approach provides direct detection of the pathogen itself, instead of being focused in symptom, or physiological changes in the host, so that it should be very specific, even more allowing quantification of the pathogen-derived protein. The specificity is also given by the fact that the biomarker is only produced by CLas, avoiding unspecific results. From our current evaluation, we believe this protocol will enhance our capacity of HLB detection/diagnosis.