Development of microfluidic ELISA for measuring humoral responses to clostridial antigens in vaccinated cattle.

Clostridial diseases significantly threaten livestock health, particularly in cattle, underscoring the need for effective vaccination strategies. This study develops and optimizes a microfluidic-based enzyme-linked immunosorbent assay (ELISA) for the rapid (< 1 h) and cost-effective measurement of IgG antibody levels against various clostridial toxins in cattle vaccinated with a multivalent clostridial vaccine. This assay requires only 5 μL of sample and reagent volume, demonstrating high repeatability and reproducibility with coefficient of variation (CV) values ranging from 0.1 % to 8.5 % across all tested clostridial antigens. The Limit of Detection (LOD) of the assay ranged from 1:150 to 1:800, allowing for sensitive detection of antibody levels. For Clostridium perfringens ε toxin, antibody titers were measured using a commercial kit, while microfluidic ELISA was applied to assess titers against tetanus toxoid, Clostridium septicum α toxin, Clostridium novyi type B toxins, and Clostridium sordellii toxins. Significant increases in IgG antibody levels were observed for C. perfringens ε toxin and tetanus toxoid following both primary and booster vaccine doses, peaking by day 42. Antibody titers against C. septicum α toxins and C. novyi type B toxins increased after the primary dose, peaking at day 42, while no booster effect was seen for C. sordellii. These findings highlight the utility of microfluidic ELISAs as a practical and efficient tool for assessing humoral immune responses to clostridial toxins in vaccinated cattle, with potential application for the herd level disease surveillance and vaccine efficacy assessment.