Immunoassay based on peroxidase silver conjugate for the determination of human immunoglobulins against tick‐borne borreliosis (lyme disease) by voltammetry and spectrophotometry

Abstract

AbstractIn this work two strategies for the synthesis of peroxidase silver conjugates for the qualitative and quantitative determination of immunoglobulins (IgG) to ixodid tick‐borne borreliosis (ITBB) (Lyme disease) in human serum were proposed. The first approach for Ab‐HRP@AgNP conjugate synthesis involved silver nanoparticles (Ag NPs) capped with a commercial peroxidase conjugate (Ab‐HRP) by passive adsorption. The second strategy was based on the initial coupling of Ag NPs with human anti‐species antibodies (Ab) by passive adsorption followed by the introduction of horseradish peroxidase (HRP) enzyme into the reaction mixture as a blocking reagent for Ab@AgNP@HRP conjugate synthesis. The formation of peroxidase silver conjugates was proved by UV/Vis spectroscopy and Transmission Electron Microscopy (TEM). The catalytic activity of Ab‐HRP@AgNP and Ab@AgNP@HRP conjugates was evaluated by Michaelis‐Menten kinetics. A commercially available 96‐well microtiter plate with recombinant antigens to ITBB was used as a platform for immobilization of analyzed IgG. The HRP in Ab‐HRP@AgNP conjugate was found to retain a sufficient level of activity for interaction with the H2O2 substrate to form an intensely colored reaction product. Therefore Ab‐HRP@AgNP conjugate can be used in enzyme‐linked immunosorbent assay (ELISA) with spectrophotometric detection of 3,3’,5,5’‐Tetramethylbenzidine (TMB Ox) for quantitative determination of IgG to ITBB in human serum in the concentration range 12.5–800 ng ml−1 with LOD 2 ng ml−1. Ab@AgNP@HRP conjugate is recommended for the electrochemical determination of IgG to ITBB in human serum at LOD 3 ng ml−1 with registration of silver oxidation by linear sweep anodic stripping voltammetry (LSASV). Ag NPs in Ab‐HRP@AgNP and Ab@AgNP@HRP conjugates do not change electrochemical activity during storage and can be used as an electrochemical label in LSASV method in case of HRP inactivation. The immunoassay based on peroxidase silver conjugates expands the analytical potential for the determination of IgG to ITBB especially during the period of increasing incidence.