Collaborative study for the calibration of a replacement International Standard for diphtheria toxoid for use in flocculation test.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for diphtheria toxoid for use in flocculation test and its calibration in Lf units. Calibration was performed using Ramon flocculation method, standardized using the 2nd IS. The candidate standard was assigned a unitage of 1870 Lf/ampoule based on results from 25 laboratories in 15 different countries and was established as the 3rd IS for diphtheria toxoid for use in flocculation test by the WHO Expert Committee on Biological Standardization (ECBS) in October 2015. The study also assessed the use of alternative methods for measuring Lf. Participants were asked to determine the Lf value of the candidate standard using an Enzyme Linked Immunosorbent Assay (ELISA) established at NIBSC, or other suitable in-house method. 10 laboratories performed ELISA according to the NIBSC protocol, 1 laboratory performed flocculation using laser-light scattering according to an in-house protocol, and 1 laboratory performed another in-house ELISA. Results suggest these methods may provide suitable alternatives to the Ramon flocculation test, subject to validation, and that the new standard could act as a suitable reference preparation in these methods.