title: Oriented immobilisation of anti-pneumolysin Fab through a histidine tag for electrochemical immunosensors.
abstract: Orientation of reagents is a key step in the construction of immunosensors. When the immunoreagent is a recombinant protein, this can be achieved by employing hexahistidine tags. The orientation of recombinant histidine-tagged Fab fragments of monoclonal anti-pneumolysin antibodies on gold films is evaluated. Using histidine as a chelator of Ni or employing an anti-polyhistidine antibody for capturing the His6 residue is considered. Measurements are based in the signal of indigo, which comes from the hydrolysis of 3-indoxylphosphate by alkaline phosphatase (AP). The attachment of the enzyme occurs through the interaction of biotin with AP-labelled streptavidin or employing AP-conjugated immunoreagents. In the case of the interaction Ni-histidine, for the study of the self-assembling process a His-tagged and biotinylated protein (His6-GST-B) was employed. General conditions were studied and non-specific adsorption was avoided with the use of 1-hexanethiol. Improvements of the signal compared with the direct adsorption were only achieved by the use of histidine capturing antibodies. With an optimised ratio anti-polyhis:His6-Fab the signal increases approximately a 100%. Precision is adequate and the response is linear with the concentration of pneumolysin between 0.1 and 10 ng/mL.
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, Asturias, Spain. r_vallina@yahoo.es
authors.forename: Romina
authors.initials: R
authors.lastname: Vallina-García
authors.forename: María
authors.initials: M
authors.lastname: del Mar García-Suárez
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Fernández-Abedul
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Méndez
authors.forename: Agustín
authors.initials: A
authors.lastname: Costa-García
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020224:Expressed Sequence Tags; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013301:Streptolysins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D020099:Coated Materials, Biocompatible; D007140:Immunoglobulin Fab Fragments; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D006639:Histidine