Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis

Abstract

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1-4(Fucα1-3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.

Figure 1

Glycan array analysis of anti-glycan antibodies in the sera of parasite infected individuals. Sera from individuals seropositive for trichinellosis (T, n=5), leishmaniasis (L, n=5) or echinococcosus (E, n=5), as well as from healthy blood donors (HC, n=2) were 1:100 diluted and analyzed for the presence of anti-glycan IgG antibodies using printed array Version 2.1 of the Consortium for Functional Glycomics. (A) Examples are shown of an array from an individual seropositive for trichinellosis and from a healthy blood donor. RFU=Relative Fluorescence Units. The RFU for the LDNF glycan (#91) is indicated in both arrays. (B) Scatter plot of the RFU values for 5 selected glycan antigens, of all 17 sera analyzed. Three glycan antigens were selected that showed differential recognition by sera from parasite-infected individuals and healthy controls (#91, LDNF antigen, GalNAcβ1-4(Fucα1-3)GlcNAc-R; #189, Manα1-2Manα1-2Manα1-3Man-R; #191, Manα1-2Manα1-3Man-R). In addition, one glycan antigen was selected that showed a very low binding, and one that showed high binding, respectively (#135, Le^X antigen, Galβ1-4(Fucα1-3)GlcNAc-R; #156, GlcNAcα1-3Galβ1-4GlcNAc-R). CFG monosaccharide symbols used are: green circle: Man; black square, GlcNAc; yellow square, GalNAc; yellow circle, Gal; red triangle, Fuc.

Figure 2

Recognition of glycan antigens in Trichinella by specific monoclonal antibodies in ELISA. Anti-Le^x (IgM, Calbiochem), anti-LDN (mAb SMLDN1.1) (Nyame et al., 1999) and anti-LDNF (mAb SMLDNF) (Nyame et al., 2000) antibodies were used for glycan recognition on crude antigen from T. spiralis muscle larvae (ML) and excretory/secretory (ES) antigen.

Figure 3

Analysis of the binding of anti-glycan Ig from serum of individuals seropositive for trichinellosis to different neoglycoconjugates in ELISA. BSA and neoglycoconjugates [BSA-chitotriose (Chi), BSA-LDN (LDN) and BSA-LDNF (LDNF2 and LDNF5)] were coated at concentrations of 5 (white bars) and 10μg/ml (grey bars). Bars represent the mean binding of five Trichinella-positive sera tested. Ratio values equal or higher than 1 (line) are considered positive.

Figure 4

Plot of the ratio distribution of the Trichinella-positive serum samples tested by the ES-ELISA and LDNF5-ELISA for the detection of total Ig. Ratio values equal or higher than 1 (line) are considered positive