Development of Double-Antibody Sandwich ELISA for the Detection of Clostridium perfringens ε Toxin (ETX)

Clostridium perfringens Epsilon toxin (ETX), recognized as the world's third most potent known biological toxin, causes severe economic losses in the livestock industry due to ETX poisoning. Therefore, there is an urgent need to establish a diagnostic method for ETX that is highly specific, sensitive and easy to operate, and suitable for large-scale sample screening.

This study aimed to produce mouse anti-ETX monoclonal antibodies (mAbs) and establish a double-antibody sandwich ELISA (DAS-ELISA) for ETX detection, thereby providing a material foundation for early ETX diagnosis, epidemic surveillance, and the formulation of prevention and control strategies.

BALB/c mice were immunized with inactivated crude ETX (inactivated ETX, iETX) and non-toxic recombinant ETX (rETX_m1) as immunogens. An indirect ELISA method and an indirect immunofluorescence assay, established using rETX_m1 as the coating antigen, were employed for mAb screening. The obtained mAbs were tested for ETX neutralizing activity, and mouse monoclonal ascites were prepared. Purification of mouse ascites was performed using Protein A affinity chromatography, followed by identification via SDS-PAGE and Western-blot analysis. A DAS-ELISA for ETX detection was established using rabbit polyclonal anti-Clostridium perfringens ε toxin antibody (capture antibody) and neutralizing monoclonal antibody (detection antibody). Optimal reaction conditions were determined by optimizing capture and detection antibody concentrations via a checkerboard approach. The method's cutoff value, specificity, sensitivity, and reproducibility were validated prior to clinical application.

A total of three hybridoma cell lines capable of stable passage and producing ETX-specific mAbs were selected and designated ETX-CH, ETX-ZH, and ETX-NH, respectively. Toxin neutralization assays revealed that ETX-ZH exhibited the highest neutralizing titer at the cellular level, while ETX-NH showed no neutralizing activity. In vivo toxin neutralization experiments in mice further demonstrated that ETX-ZH retained neutralizing activity against ETX in vivo. The purified ascites contained 2.3 mg·mL^-1 of ETX-ZH protein. Using the checkerboard method, the optimal dilution for capture antibody was determined to be 1 : 100, and for detection antibody, 1 : 1600. The optimized conditions for this method were: capture antibody coating at 4 ℃ for 12 h, blocking with 5% skim milk at 37 ℃ for 2 h, antigen incubation at 37 ℃ for 90 min, detection antibody incubation at 37 ℃ for 30 min, incubation with enzyme-labeled secondary IgG antibody at 37 ℃ for 1 h, and TMB color development at 37 ℃ for 15 min. The positive cutoff value was 0.161, and the negative cutoff value was 0.143. The assay demonstrated high specificity with no cross-reactivity to other Clostridium perfringens toxins or purified proteins. It exhibited good reproducibility, with an intra-assay coefficient of variation ≤4.69% and an inter-assay coefficient of variation ≤5.32%. The lower limit of detection for rETX_m1 as was 31.25 ng·mL^-1, and for ETX it was 0.5 MLD. Testing of eighteen multi-valent dry powder vaccines containing ETX-like toxins yielded a 100% detection rate for ETX.

The DAS-ELISA method developed using the prepared ETX mAb exhibited high specificity and good reproducibility, making it suitable for ETX detection.