Protocol for simultaneous isolation of three important banana allergens

•Mus a 2, Mus a 4, and Mus a 5 allergens from banana were purified simultaneously.•Identity of purified proteins was confirmed by microsequencing and mass fingerprint.•Purified allergens preserved their IgG and IgE immunoreactivity.

Banana fruit (Musa acuminata) has become an important food allergen source in recent years. So far, 5 IgE reactive banana proteins have been identified, and the major allergens are: Mus a 2 (a class I chitinase, 31 kDa), Mus a 4 (thaumatin-like protein, 21 kDa), and Mus a 5 (β-1,3-glucanase, 33 kDa). Due to variations in allergen expression levels, diagnostic reagents for food allergy can be improved by using individual allergen components instead of banana allergen extracts. The purpose of this study was to optimize the purification protocol of the three major allergens present in banana fruit: Mus a 2, Mus a 4 and Mus a 5. By employing a three-step purification protocol (a combination of anion-exchange, cation-exchange and reversed-phase chromatography) three important banana allergens were obtained in sufficient yield and high purity. Characterization of the purified proteins was performed by both biochemical (2-D PAGE, mass fingerprint and N-terminal sequencing) and immunochemical (immunoblot) methods. IgE reactivity to the purified allergens was tested by employing sera of five allergic patients. The purified allergens displayed higher sensitivity in IgE detection than the routinely used extracts. The three purified allergens are good candidates for reagents in component-based diagnosis of banana allergy.