Establishment of a peptide-based electrochemical immunosensor for detecting antibodies against the GP5 protein from the porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases that affect the swine industry worldwide. Although enzyme-linked immunosorbent assays (ELISAs) are the gold standard for analyzing antibodies against viruses, they have some limitations, such as sample transport and analysis times and cost. Rapid serological surveillance of PRRSV remains a critical challenge for swine health management. Immunosensors are feasible diagnostic alternatives, where antigen–antibody interactions can be immediately quantitatively determined. Electrochemical sensor operation is based on changes in interfacial electron transfer at the working electrode surface, which are monitored using electrochemical impedance spectroscopy (EIS). In this approach, a redox probe ([Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻) is used to evaluate the charge-transfer resistance (R_ct) at the electrode interface. Variations in analyte concentration lead to changes in the interfacial properties of the electrode, particularly after biomolecular interactions such as antibody binding. These interactions hinder electron transfer, resulting in an increase in R_ct. The analytical signal is therefore based on the correlation between the change in R_ct (ΔR_ct) and the concentration of the target analyte, enabling quantitative analysis through calibration plots. The goal was the establishment of a peptide based electrochemical immunosensor for quantifying antibodies against PRRSV. Gold electrode functionalization was characterized using EIS, the GP5-B peptide, and casein protein. Balb/c mice were immunized according to control, BSA-immunized, and GP5-B peptide-immunized groups. Control pigs were free from the porcine respiratory disease complex (PRDC) and nonvaccinated. Vaccinated pigs were injected using commercial Ingelvac vaccine according to the manufacturer's instructions, and naturally infected pigs were also included in the study. Antibodies were measured using ELISA assays and an immunosensor. The immunosensor recognized antigen–antibody interactions, with a limit of detection (LOD) of 6 ng mL⁻¹, a repeatability of intra-assay CV of 5.05%, and a reproducibility inter-assay CV of 4.08% through batch to batch. Additionally, it achieved a sensitivity of 100%, and a specificity of at least 93%. Considering all the data, compared to immunoglobulin G detection via ELISAs, the proposed peptide-based electrochemical immunosensor is valuable for detecting antibodies against PRRSV. These results suggest the feasibility of biosensors for serological diagnosis in pigs.