title: Using QCM and SPR for the Kinetic Evaluation of the Binding Between A New Recombinant Chimeric Protein and Specific Antibodies of the Visceral Leishmaniasis.
abstract: In the present study, the surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) techniques were employed to kinetically evaluate the binding affinity of a new recombinant chimeric protein (CP10) toward anti-Leishmania infantum antibodies for the immunodiagnostics of the visceral leishmaniasis (VL). This chimeric protein was formed by the union in a same artificial coding DNA of ten different peptides, which showed themselves reactive toward positive canine serum for VL. Using the CP10 in enzyme-linked immunosorbent assays (ELISA), it was possible to detect 80% of the asymptomatic infected dogs. After this, SPR and QCM immunosensors were constructed by the covalent immobilization of the CP10 on a self-assembled monolayer (SAM) formed by adsorption of alkanethiol on gold substrates. The thickness (6.80 nm) and the refractive index (1.475) of the protein on the SAM were simultaneously determined through SPR curves measured in different wavelengths (670 and 785 nm). Interactions between the CP10 and its specific IgGs (anti-CP10 antibodies) were characterized by the electrochemical impedance spectroscopy, SPR and QCM techniques. The equilibrium dissociation constant obtained by SPR (K(D) = 8.27 x 10(-10) mol.L(-1)) and QCM (K(D) = 2.42 x 10(- 10) mol.L(-1)) demonstrated high binding affinity of the CP10 toward anti-CP10 antibodies. In this sense, this work quantitatively proves the strong antigenic character of a new recombinant chimeric protein, giving evidence to potential contribution for the use of this protein in programs of control of the VL.
authors.forename: Denio E P
authors.initials: DE
authors.lastname: Souto
authors.forename: Angelica R
authors.initials: AR
authors.lastname: Faria
authors.forename: Helida M
authors.initials: HM
authors.lastname: de Andrade
authors.affiliation: National Institute of Science & Technology of Bioanalytics, Institute of Chemistry / State University of Campinas -Unicamp, SP - Brazil. Kubota@iqm.unicamp.br.
authors.forename: Lauro T
authors.initials: LT
authors.lastname: Kubota
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000953:Antigens, Protozoan / Q000378:metabolism; D058266:Dielectric Spectroscopy; D004285:Dogs; D055767:Immobilized Proteins / Q000378:metabolism; D007118:Immunoassay; D007700:Kinetics; D007898:Leishmaniasis, Visceral / Q000276:immunology*; D011485:Protein Binding; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012031:Refractometry; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D000906:Antibodies; D000953:Antigens, Protozoan; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins