If I could help you, in our paper [Medicine (Baltimore). 2015 Oct;94(40):e1699], for a determination of specific IgE, IgA1, and IgG1 antibodies against Anisakis simplex recombinant antigens by ELISA, we established the cutoff for each antibody with each antigen, by a randomly selection of 25 sera from the healthy control individuals and calculated the mean of the optical density values and the standard deviation. The cutoff was fixed as the mean plus 4 standard deviations, as previous described in other authors' paper (Allergy. 2000;55:627-633).