SPR analysis of the interaction between a recombinant protein of unknown function in Leishmania infantum immobilised on dendrimers and antibodies of the visceral leishmaniasis: A potential use in immunodiagnosis

Publication year: 2015
Authors: Souto D.E. 1, Fonseca A.M. 2, Barragan J.T. 1, Luz Rde C. 3, Andrade H.M. 2, Damos F.S. 3, Kubota L.T. 4
Affiliations:

1 - National Institute of Science & Technology in Bioanalytics, Institute of Chemistry/State University of Campinas - Unicamp, SP, Brazil
2- Federal University of Minas Gerais - UFMG, MG, Brazil
3- Federal University of Maranhão - UFMA, MA, Brazil.
4- National Institute of Science & Technology in Bioanalytics, Institute of Chemistry/State University of Campinas - Unicamp, SP, Brazil

Published in: Biosensors and Bioelectronics, 2015, Vol. 70, p. 275-81
doi: 10.1016/j.bios.2015.03.034

In this work, an SPR immunosensor was developed to elucidate the reaction kinetics between a protein of unknown function in Leishmania infantum (hypothetical C1 protein) and specific antibodies of the visceral leishmaniasis (VL). A platform, which is based on layer-by-layer assembly was formed by cysteamine in combination with a fourth-generation poly(amidoamine) dendrimer (PAMAM(G4)) on gold surface for the immobilisation of the protein. This film resulted in amplification of the signal of SPR. Then, a kinetic model based on a bivalent ligation suggested that the reaction between the C1 protein and the anti-C1 antibody occurs in two steps. The value of the equilibrium dissociation constant (KD1×KD2=1.64×10(-7)molL(-1)) demonstrated high binding affinity between the biomolecules. Furthermore, low limits of detection (LOD=7.37nmolL(-1)) and quantification (LOQ=7.83nmolL(-1)) were presented with the proposed SPR immunosensor. Afterwards, the addition of real samples consisting of positive and negative canine sera for VL was accompanied by high sensitivity and selectivity by SPR immunosensor. Therefore, this study quantitatively demonstrated the strong antigenic character of a hypothetical protein and consequently its potential use in the immunodiagnosis of the VL.


MP-SPR keywords: antibody (analyte) interaction, biosensor development, immunosensor, layer-by-layer assembly, parasite detection, protein/ antigen (ligand)