title: QCM immunoassay for recombinant cysteine peptidase: a potential protein biomarker for diagnosis of citrus canker.
abstract: Citrus canker is one of the most important agricultural citrus diseases worldwide. It is caused by Xanthomonas citri subsp. citri (Xcc) bacterium that infects leaves and the fruits produce a cysteine peptidase (CPXaC), which makes it a potential target for the development of effective and rapid detection methods for citrus canker. We report here the studies on the development of piezoelectric immunoassay for CPXaC using a polyclonal antibody against CPXaC (anti-CPXaC). Three different strategies for covalent immobilization of anti-CPXaC on gold surfaces were evaluated by monitoring the frequency (Δf) and energy dissipation (ΔD) variation in real time when 64.5×10(-8) mol L(-1) CPXaC was added. Anti-CPXaC immobilized with 11-mercaptoundecanoic acid (MUA) showed the best relation between the frequency and dissipation factor variation, and strong values for the kinetic and equilibrium binding constant were obtained. The immunosensor showed a detection limit of 13.0 nmol L(-1) with excellent specificity, showing no response for different proteins that include another cysteine peptidase that is used as a target to detect Xylella fastidiosa bacterium, responsible for another important citrus disease. These results provide good perspectives for the use of CPXaC as a new biomarker for citrus canker.
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905, São Carlos, SP, Brazil.
authors.forename: André S
authors.initials: AS
authors.lastname: Afonso
authors.forename: Bianca F
authors.initials: BF
authors.lastname: Zanetti
authors.forename: Adelita C
authors.initials: AC
authors.lastname: Santiago
authors.forename: Flavio
authors.initials: F
authors.lastname: Henrique-Silva
authors.forename: Luiz H C
authors.initials: LH
authors.lastname: Mattoso
authors.forename: Ronaldo C
authors.initials: RC
authors.lastname: Faria
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D015415:Biomarkers; D002957:Citrus; D057056:Cysteine Proteases / Q000032:analysis* / Q000276:immunology; D006046:Gold / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology; D007118:Immunoassay; D010935:Plant Diseases; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D014974:Xanthomonas / Q000201:enzymology*
chemical_list: D000906:Antibodies; D015415:Biomarkers; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D006046:Gold; D057056:Cysteine Proteases