Electrochemical enzyme-linked immunosorbent assay (e-ELISA) for parasitic nematode Ostertagia ostertagi (brown stomach worm) infections in dairy cattle

Abstract

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3′,5,5′-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H₂SO₄ based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N₇₀ control ratio 1.6 times higher than that of optical ELISA (TMB/H₂SO₄ combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N₂₅ = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N₂₅ = 3.8). The proposed e-ELISA is specific (selective Ab–Ag interactions) and highly sensitive – capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle.