O-glycans as a source of cross-reactivity in determinations of human serumantibodies to Anisakis simplex antigens

Background Anisakis simplex is a seafood-borne parasite that may both infec t humans and cause allergy. Serodiagnosis of anisakiasis and allergy caused by this nematode is difficult since most Anisakis antigens show cross-reac tivity problems. Objective To analyse the possible role of sugar epitopes contained in Anisa kis simplex antigens as causes of false-positive results in serodiagnostic assays. Methods The antigens UA2R and UA3R recognized by two anti-Anisakis monoclon al antibodies were used in this study. Capture ELISA techniques were used t o compare the reactivities with native or O-deglycosylated antigens of sera from Anisakis-free children (most of them infected by several other parasi tes) and from Anisakis allergy patients. O-deglycosylation was done by mild alkali treatment with NaOH. SDS-PAGE and immunoblotting were used to chara cterize the effects of NaOH or N-glycanase F treatment on UA3R. Results Native UA2R was recognized by IgG1 and IgM antibodies in the sera o f both Anisakis-free subjects and allergy patients. Native UA3R was recogni zed by most sera from allergy patients (92% considering immunoglobulin (Ig) G1, 100% considering IgE), but also by a significant proportion of sera fr om Anisakis-free subjects (36% considering IgG1, 14% considering IgE). O-de glycosylation of UA3R greatly improved specificity: none of the sera from A nisakis-free patients showed either IgG1 or IgE reactivity with O-deglycosy lated UA3R, while the proportion of sera from allergy patients showing IgE reactivity with this antigen was practically unaffected. O-deglycosylation of UA2R did not improve the specificity of assays using this antigen. Our r esults also show that the protein core of glycoproteins may be altered by e ven very mild alkali treatment, depending on the nature of the protein. Conclusion Native glycoproteins of A. simplex should not be used for diagno stic purposes. O-deglycosylated UA3R seems to be an excellent candidate for use as target antigen in the serodiagnosis of anisakiasis and A. simplex a llergy.