Development of immunoassay methods for ricin and application in the OPCW international proficiency test.

The Organisation for the Prohibition of Chemical Weapons (OPCW) organizes the annual Trial Biotoxins Proficiency Test (ToxPT) to implement standardized and transparent evaluation on the technical proficiency of laboratories applying for or retaining official designation for biotoxin analysis. Laboratories are required to pass all relevant ToxPT events to prove sustained detection capability. Launched in August 2025, the Second Trial ToxPT centered on ricin detection and marked the first inclusion of high-molecular-weight protein toxins within an OPCW proficiency test. This study aimed to construct an immunological detection platform for ricin using conventional analytical methods, including WB and ELISA, and to evaluate their application performance in the Second Trial ToxPT. By optimizing the experimental parameters of immunoassays-including antibody coating concentration, blocking buffer selection, and working concentration of enzyme-labeled secondary antibody-we successfully established WB and sandwich ELISA methods for ricindetection. As a quantitative method, the ELISA assay employed four-parameter nonlinear regression to fit the standard curve and derive the standard equation, with a correlation coefficient (R2) of 0.9974, a limit of detection (LOD) of 1 ng/mL, a limit of quantitation (LOQ) of 7 ng/mL, and a broad quantitative range of 7-400 ng/mL. When applied to sample analysis in the second trial ToxPT, the obtained results were accurate and consistent with official reference data. These validated methods exhibit high reliability for routine ricin detection and lay a solid technical foundation for laboratories to sustain their biotoxin analysis accreditation.