2.2.4. Cutoffs and Interpolation

WHO international standards TE-3 and 10/262 were used to convert MFI-BG for tetanus and diphtheria, respectively, to IU/mL. Dilutions of each standard were run on separate plates over the course of the study at approximately one-month intervals, with a total of seven individual runs averaged to create a single curve per antigen for all samples. Curve fitting and interpolation were performed in GraphPad Prism v.9 using 5PL non-linear regression. Conversion of values to IU/mL was conducted in SAS.

Tetanus and diphtheria antibody seroprotection were defined using the standard cut-off of 0.01 IU/mL, which corresponds to the minimum level of antibody required for protection against tetanus and diphtheria [20,21,24]. Tetanus IgG testing on MBA has been previously validated against the reference standard double antigen ELISA IgG with a sensitivity of 99% and specificity of 92% at the ≥0.01 IU/mL cutoff [5]. Diphtheria IgG testing on MBA was validated against the Vero cell neutralization assay IgG with a sensitivity of 95% and specificity of 83% at the ≥0.01 IU/mL cutoff for minimal protection [23]. Increasing antibody concentrations against tetanus are associated with decreased risk of infection and increased duration of protection [25], and tetanus antibody concentrations ≥1 IU/mL are typically associated with long-term protection [6,25]. Tetanus and diphtheria

antibody seroprotection were further categorized as IgG < 0.01 IU/mL (lack of protection), 0.01–<0.1 IU/mL (minimal protection), 0.10–<1 IU/mL (full protection), and ≥1 IU/mL (long-term protection) [5,6,23–26].
