Oriented immobilisation of anti-pneumolysin Fab through a histidine tag for electrochemical immunosensors

• Published in: Biosensors & bioelectronics Vol. 23; no. 2; pp. 210 - 217
• Main Authors: Vallina-García, Romina, del Mar García-Suárez, María, Fernández-Abedul, M. Teresa, Méndez, Francisco Javier, Costa-García, Agustín
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 30.09.2007; Elsevier Science
• Subjects: Bacterial Proteins - analysis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Binding Sites; Biological and medical sciences; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Biosensors; Biotechnology; Coated Materials, Biocompatible - chemistry; Electrochemical immunosensor; Electrochemistry - instrumentation; Electrochemistry - methods; Equipment Design; Equipment Failure Analysis; Expressed Sequence Tags; Fab fragment; Fundamental and applied biological sciences. Psychology; Gold film; Histidine - chemistry; Histidine - genetics; Histidine - immunology; Histidine tag; Immunoassay - instrumentation; Immunoassay - methods; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - immunology; Methods. Procedures. Technologies; Pneumolysin; Protein Binding; Reproducibility of Results; Sensitivity and Specificity; Streptolysins - analysis; Streptolysins - chemistry; Streptolysins - genetics; Streptolysins - immunology; Various methods and equipments; Fab fragment; Pneumolysin; Gold film; Electrochemical immunosensor; Histidine tag; Antibody; Immobilization; Fab-Fragment; Orientation; Immunosensor; Streptococcus pneumoniae; Streptococcaceae; Toxin; Histidine; Biosensor; Electrochemistry; Tagging; Bacteria; Micrococcales
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2007.04.001

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 His 6 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 (His 6-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:His 6-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.