Comparison and in-silico modeling of monoclonal antibodies recognizing the immuno-dominant Ana o 3 cashew nut allergen

Abstract

Food allergies negatively affect quality of life and can be a significant medical burden. Allergic reactions to cashew nuts can often be severe and are rarely outgrown. The Ana o 3 protein is an immuno-dominant cashew allergen. Ana o 3 is a small 13kDa protein composed of 2 subunits held together by disulfide bonds that can be disrupted by treatment with reducing agents. The stability and solubility properties of Ana o 3 make it an excellent target for detection methods and tools. Several monoclonal antibodies targeted towards Ana o 3 were generated and 4 of them were characterized by immunoblot and ELISA. Selected Ana o 3 cognate antibody genes were sequenced, and in-silico modeling was used for epitope prediction. All 4 of the monoclonal antibodies recognized both recombinant and native Ana o 3. The calculated KD for each of the antibodies for native Ana o 3 is as follows, 2H5 - 21.7 nM, 5B7F8 - 9.2 pM, 6B9C1 - 0.465 nM, and 19C9A2 - 0.365 nM. The 2H5 antibody was specific for cashew nut extract, while the 6B9C1 and 19C9A2 antibodies also recognized pistachio extract, and the 5B7F8 antibody also recognized pecan extract. The 6B9C1 and 19C9A2 antibodies likely recognized conformational epitopes as binding by these antibodies was disrupted after treatment of Ana o 3 with a reducing agent. In contrast, recognition of Ana o 3 by the 2H5 and 5B7F8 antibodies was increased after reduction of Ana o 3, suggesting linear epitopes. The amino acids predicted to be important for interaction with Ana o 3 epitopes are unique for each of the 4 antibodies. The 5B7F8 antibody is likely the best candidate for continued study and assay development for the creation of cashew allergen detection or diagnostic devices. This collection of antibodies represents useful reagents for the study and characterization of the Ana o 3 cashew nut allergen using cell based and immunoassay methods.