“…BAT was performed with heparinized whole blood stimulated by allergens for 30 minutes at 37°C as described by Mukai et al 25 One hundred microlitres of whole blood was mixed with 100 μL of RPMI medium or with the flowing stimulants: purified AK, SCP, TIM, and Mimotopes obtained from phage display experiments were aligned using the multiple sequence alignment program ClustalW, to find a consensus pattern of amino acids. 27 For conformational epitope mapping, the parameter of amino acids in mimotopes was calculated, analysed using LocaPep (http://atenea.montes.upm.es), 28 and mapped onto the three-dimensional structure of allergens by PyMOL (https://pymol.org/2/). Analysis of the frequency of single amino acids was performed by ExPASy (https://web.expasy.org/ protparam).…”