O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens.
BACKGROUND
Anisakis simplex is a seafood-borne parasite that may both infect humans and cause allergy. Serodiagnosis of anisakiasis and allergy caused by this nematode is difficult since most Anisakis antigens show cross-reactivity problems.


OBJECTIVE
To analyse the possible role of sugar epitopes contained in Anisakis simplex antigens as causes of false-positive results in serodiagnostic assays.


METHODS
The antigens UA2R and UA3R recognized by two anti-Anisakis monoclonal antibodies were used in this study. Capture ELISA techniques were used to compare the reactivities with native or O-deglycosylated antigens of sera from Anisakis-free children (most of them infected by several other parasites) and from Anisakis allergy patients. O-deglycosylation was done by mild alkali treatment with NaOH. SDS-PAGE and immunoblotting were used to characterize the effects of NaOH or N-glycanase F treatment on UA3R.


RESULTS
Native UA2R was recognized by IgG1 and IgM antibodies in the sera of both Anisakis-free subjects and allergy patients. Native UA3R was recognized by most sera from allergy patients (92% considering immunoglobulin (Ig) G1, 100% considering IgE), but also by a significant proportion of sera from Anisakis-free subjects (36% considering IgG1, 14% considering IgE). O-deglycosylation of UA3R greatly improved specificity: none of the sera from Anisakis-free patients showed either IgG1 or IgE reactivity with O-deglycosylated 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 results also show that the protein core of glycoproteins may be altered by even very mild alkali treatment, depending on the nature of the protein.


CONCLUSIONS
Native glycoproteins of A. simplex should not be used for diagnostic purposes. O-deglycosylated UA3R seems to be an excellent candidate for use as target antigen in the serodiagnosis of anisakiasis and A. simplex allergy.
Lorenzo
S
S
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Santiago de Compostela, Spain.
Romarís
F
F
Iglesias
R
R
Audícana
M T
MT
Alonso
J M
JM
Leiro
J
J
Ubeira
F M
FM
D000818:Animals; D017163:Anisakis / Q000276:immunology*; D000909:Antibodies, Helminth / Q000097:blood / Q000276:immunology*; D000918:Antibody Specificity; D000947:Antigens, Helminth / Q000276:immunology*; D003429:Cross Reactions; D006801:Humans; D016056:Immunodominant Epitopes; D011134:Polysaccharides / Q000276:immunology
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D016056:Immunodominant Epitopes; D011134:Polysaccharides