Establishment and Comparison of High‐Throughput Immunoassays for Quantifying Clostridioides difficile Infection Biomarkers

Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated diarrhea, most often occurring after antibiotic exposure. In this study, we established three immunoassays: indirect enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, and fluorescence-linked immunosorbent assay (FLISA), using antibodies specific to the major virulence factors, toxin A (TcdA) and toxin B (TcdB). Indirect ELISA demonstrated high specificity with an overall assay time within 21 h, achieving sensitive detection of both toxins. Although more time- and resource-intensive (22 h), sandwich ELISA is advantageous for applications where pre-coated plates or ready-to-use membranes can reduce assay time. FLISA, using optimized fluorescent dye-conjugated antibodies, offered the most rapid workflow and simplest preparation, with potential for multiplex detection, though its sensitivity was lower than that of ELISA. All three assays were implemented in a 96-well plate format, which supported parallel sample processing, reduced turnaround times, and offered scalability for high-throughput applications. Depending on assay requirements, such as analytical sensitivity, turnaround time, and cost-effectiveness, these methods provide complementary approaches for the detection of TcdA and TcdB.