Immobilization of antibodies (Ab) on hydrogels though long studied is still a challenge on account of the continuous development of new immune technologies. Enabling methodologies for antibody orientation, antibody stability and maximum recognition of their corresponding antigens is an object of intense study. Mini Ab such Fab fragments are less susceptible to conformational changes on surfaces, or unwanted reactivities compared to a whole Ab molecule. Herein, we have developed an immobilization protocol for a Fab anti epsilon toxin from Clostridium perfringens. Fine tuning of variables during immobilization showed a crucial role in the orientation and the spatial distribution of the Fab across the support surface. The final optimized immune-matrices demonstrated quantitative adsorption of antigen (1:1 molar ratio Fab to antigen) meaning that both the Fab biological activity was maintained after immobilization and an optimal orientation was achieved during the immobilization process. Immobilized Fab gained stability after immobilization as demonstrated by real time protein unfolding. (C) 2015 Elsevier Ltd. All rights reserved.