title: An experimental design approach to optimize an amperometric immunoassay on a screen printed electrode for Clostridium tetani antibody determination.
abstract: An immunoassay for the determination of anti-tetani antibodies has been developed using a screen printed electrode (SPE) as solid support for toxoid (antigen) immobilization. The assay was performed in guinea pig serum. The immunoreaction and the subsequent amperometric detection occurred directly onto the SPE surface. The assay consisted of spiking the anti-tetani sample directly onto the toxoid modified SPE, and then a second antibody, i.e. a HRP-labeled anti-immunoglobulin G, was deposited onto the biosensor. Subsequent amperometric detection was realized by spiking 10 µL of a hydroquinone (HQ) solution into 40 µL of buffer solution containing hydrogen peroxide. An experimental design approach was implemented for the optimization of the immunoassay. The variables of interest, such as bovine serum albumin (BSA) concentration, incubation times and labeled antibody dilution, were optimized with the aid of the response surface methodology using a circumscribed central composite design (CCCD). It was observed that two factors exhibited the greatest impact on the response, i.e. the anti-tetani incubation time and the dilution factor of the labeled antibody. It was discovered that in order to maximize the response, the dilution factor should be small, while the anti-tetani antibody incubation time should be long. The BSA concentration and the HRP-anti-IgG incubation had very limited influence. Under the optimized conditions, the immunoassay had a limit of detection of 0.011 IU/mL and a limit of quantification of 0.012 IU/mL. These values were below the protective human antibody limit of 0.06 IU/mL.
authors.affiliation: Université libre de Bruxelles (ULB), Faculty of Pharmacy, Campus Plaine, CP 205/6, 1050 Brussels, Belgium. Electronic address: spatris@ulb.ac.be.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Patris
authors.affiliation: Université libre de Bruxelles (ULB), Faculty of Pharmacy, Campus Plaine, CP 205/6, 1050 Brussels, Belgium.
authors.forename: Marie
authors.initials: M
authors.lastname: Vandeput
authors.affiliation: Université libre de Bruxelles (ULB), Faculty of Pharmacy, Campus Plaine, CP 205/6, 1050 Brussels, Belgium.
authors.forename: Gersonie Momo
authors.initials: GM
authors.lastname: Kenfack
authors.affiliation: Université libre de Bruxelles (ULB), Faculty of Pharmacy, Campus Plaine, CP 205/6, 1050 Brussels, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mertens
authors.affiliation: Université libre de Bruxelles (ULB), Faculty of Pharmacy, Campus Plaine, CP 205/6, 1050 Brussels, Belgium; Vrije Universiteit Brussel (VUB), Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Laarbeeklaan 103, B-1090 Brussels, Belgium.
authors.forename: Bieke
authors.initials: B
authors.lastname: Dejaegher
authors.affiliation: Université libre de Bruxelles (ULB), Faculty of Pharmacy, Campus Plaine, CP 205/6, 1050 Brussels, Belgium. Electronic address: jmkauf@ulb.ac.be.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Kauffmann
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology*; D003017:Clostridium tetani / Q000276:immunology*; D003217:Conductometry / Q000295:instrumentation*; D004566:Electrodes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006168:Guinea Pigs; D007118:Immunoassay / Q000295:instrumentation*; D010781:Photography / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies
keywords: Central composite design; Clostridium tetani; Immunoassay; Immunosensor; Screen printed electrode