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

• Published in: Biosensors & bioelectronics Vol. 70; pp. 275 - 281
• Main Authors: Souto, Dênio E.P., Fonseca, Aliani M., Barragan, José T.C., Luz, Rita de C.S., Andrade, Hélida M., Damos, Flávio S., Kubota, Lauro T.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-08-2015
• Subjects: Antibodies; Autoantibodies - immunology; Biomolecules; Dendrimer; Dendrimers; Dendrimers - chemistry; Equipment Design; Equipment Failure Analysis; Humans; Hypothetical C1 protein; Immunoassay - instrumentation; Immunologic Tests - instrumentation; Immunosensor; Immunosensors; Kinetic evaluation; Leishmania infantum; Leishmaniasis, Visceral - diagnosis; Leishmaniasis, Visceral - immunology; Mathematical models; Protein Interaction Mapping - instrumentation; Proteins; Reaction kinetics; Recombinant; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Reproducibility of Results; Sensitivity and Specificity; SPR; Surface Plasmon Resonance - instrumentation; Visceral leishmaniasis; Dendrimer; Kinetic evaluation; Hypothetical C1 protein; SPR; Visceral leishmaniasis; Immunosensor
• ISSN: 0956-5663; 1873-4235
• 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=1.64×10−7molL−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.
•By the SPR technique, the antigenic character of a hypothetical protein in L. infantum protozoan was kinetically demonstrated.•The dendrimer provided a greater accessible area for interaction with the recombinant C1 antigen.•A proposed kinetic model suggested that the reaction between the C1 antigen and the IgG anti-C1 occurs in two steps.•The SPR immunosensor presented high sensitivity and specificity towards real samples.