Characterization of the influence of heat- and acid-treatment on the FVIII-antibody interaction in order to improve inhibitor diagnostics

Introduction We have previously reported, that acid-treatment of FVIII-containing plasma samples is advantageous over heat-treatment to detect FVIII-specific antibodies in treated patients, as heat-treatment often leads to false-positive results. However, the number of samples in which we could detect FVIII-specific antibodies using acid-treatment was low. Therefore, here we aimed to further characterize the impact of heat- and acid-treatment on the binding properties of FVIII-specific antibodies.

Method A total of 23 murine FVIII-specific monoclonal antibodies were analysed in two different ELISAs: first, binding of antibodies to immobilized FVIII in the presence of soluble FVIII with or without acid-treatment was analysed, to identify if binding to immobilized FVIII could be competed with FVIII and if the competition could be reversed by acid treatment of the soluble FVIII-antibody mixture. Second, we tested binding of heat- and acid-treated antibodies to native, as well as heat- and acid-treated immobilized FVIII, to further quantify the susceptibility of FVIII and antibodies to these treatments.

Results Analysis of the influence of heat- and acid-treatment on antibodies and FVIII, revealed that antibody-binding to FVIII was more influenced by heat- than acid-treatmtent. Interestingly, if FVIII was also treated with heat or acid, only heat-treatment led to a further decrease of binding, pointing out that a high proportion of antibodies might still bind to acid-treated FVIII. However, in the competition ELISA for 17 of 23 antibodies, binding to immobilized FVIII could be competed by soluble FVIII and restored by acid-treatment. Competition resulted in a mean of 66,18 % (± 13,29 %) reduction of the ELISA signal and acid-treatment resulted in a mean restoration of 88,26 % (± 18,01 %) of the initial ELISA signal. Remaining 6 antibodies either could not be competed by soluble FVIII (2) or could be competed with soluble FVIII but binding was not restored by acid-treatment (4), like previously shown for the human antibody BO2C11.

Conclusion Our data indicate, that FVIII as well as antibodies are more affected by heat-treatment than by acid-treatment. Treatment of FVIII in complex with antibodies should ideally ensue the denaturation of FVIII while still being mild for the antibodies. When comparing binding of acid-treated antibodies to native and acid-treated FVIII, binding was not significantly reduced by additional acid-treatment of FVIII, indicating, that antibodies might still bind to acid-denatured FVIII in the plasma sample. However, analysis of 23 monoclonal murine antibodies revealed, that most antibodies bound again to FVIII after acid-treatment of the antibody and competing FVIII and binding was only reduced by a mean of 12 % compared to binding without competition and acid treatment. Thus acid-treatment of FVIII-containing plasma samples appears to be a beneficial approach to detect masked immune responses in treated haemophilia patients.

Conflict of Interest

The authors declare no relevant conflict of interest

Publication History

Article published online:
20 February 2023