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Total serum IgG level is a surrogate marker of hepatitis C (HC) severity. Antibodies (Abs) to microbial glycans could be markers of HC severity caused by the translocation of microbial products. The level of anti-glycan (AG) Abs was analysed in serum samples of patients ( n = 128) with chronic HC in ELISA using fourteen synthetic glycans present in microbes and adhesins to evaluate the association of Abs with clinical parameters and the efficacy of antiviral treatment. The anti-GlcNAc β IgG level was significantly higher in patients with fibrosis ( P = 0.021) and severe portal inflammation ( P < 0.001) regardless of other clinical parameters. The ROC curve analysis showed sensitivity of 0.59, specificity of 0.84, and AUC of 0.71 in discriminating F0 from F1-4 (HCV genotype-1b-infected patients). The level of anti-GA2 Abs before Peg-IFN/RBV treatment was significantly higher in nonsustained viral response (non-SVR) to treatment than in SVR ( P = 0.033). ROC analysis showed sensitivity of 0.62, specificity of 0.70, and AUC of 64. Correlations of AG Abs to clinical parameters were found. The quantification of anti-GlcNAc β Abs deserves attention in assessment of the hepatic damage while anti-GA2 Abs may be a sign of immune response related to the antiviral treatment.

Natural antibodies to the tumor-associated Thomsen-Friedenreich antigen (TF) are related to tumor immunosurveillance and cancer patients' survival. Hidden IgG antibodies (HAbs) to TF, their lectin reactivity, avidity, and clinical relevance were studied. HAbs were present in cancer patients and controls. A decreased level of IgG HAbs was detected in cancer. The HAbs level positively correlated with the sialospecific SNA lectin binding in purified total IgG (tIgG) in donors and cancer patients, indicating that HAbs are higher sialylated. The avidity of anti-TF IgG in tIgG samples was lower in cancer patients ( P = 0.025) while no difference in the avidity of free anti-TF IgG was established. A negative correlation between the avidity of anti-TF IgG in tIgG and SNA binding in both groups was observed ( P < 0.0001). The HAbs level negatively correlated with the anti-TF IgG avidity in tIgG only in donors ( P = 0.003). Changes in the level of HAbs and Abs avidity showed a rather good stage- and gender-dependent diagnostic accuracy. Cancer patients with a lower anti-TF IgG avidity in tIgG showed a benefit in survival. Thus the TF-specific HAbs represent a particular subset of anti-TF IgG that differ from free serum anti-TF IgG in SNA reactivity, avidity, diagnostic potential, and relation to survival., Competing Interests: The authors declare that they have no competing interests.

Unlabelled: The elevated anti-GalNAcβ IgG level of serum was shown to be associated with the significantly better survival of patients with gastrointestinal cancer., Aim: To characterize the specificity of IgG antibodies to GalNAcβ-terminated glycans of long-term gastric cancer survivors., Methods: Serum antibodies and affinity-isolated antibodies were analysed by the indirect and competitive ELISA using glycan-polyacrylamide (PAA) conjugates as well as by isoelectric focusing and Western blotting., Results: In the serum probes, a partial cross-reactivity of antibodies to GalNAcβ, GalNAcβ1-3Galβ (X2di), GalNAcβ1-3GalNAcβ (PFdi) and GlcNAcβ was observed. The isolated anti-GalNAcβ IgGs demonstrated the cross-reactivity to the X2di glycan mainly. The affinity of the X2di-PAA to anti-GalNAcβ IgGs was 11-21 times lower than that of the GalNAcβ-PAA. Anti-X2di and anti-PFdi IgGs demonstrated monoreactivity to their key glycans-PAA used in isolation. The IC50 values of key glycoconjugates ranged from 1 to 5 · 10(-7) M. No polyreactivity of antibodies to the unrelated antigens (ferritin, casein and DNA) was found. The polyclonal or oligoclonal distribution of IgG bands was established and the monoreactivity of antibodies was not associated with the clonal distribution of bands., Conclusion: The cross-reactivity of anti-GalNAcβ antibodies to X2di and related glycans deserves attention in the clarification of the role of antibodies in cancer progression and enhancement of the prognostic potential in the combined determination of antibody markers.

Aim: To study whether alterations in the glycosylation of immunoglobulin G (IgG) specific to the Thomsen-Friedenreich glycotope (TF) have diagnostic and prognostic potential in gastric cancer., Methods: Serum samples were obtained from patients with histologically verified gastric carcinoma (n = 89), healthy blood donors (n = 40), and patients with benign stomach diseases (n = 22). The lectin-enzyme-linked immunosorbent assay-based glycoprofiling of TF-specific IgG (anti-TF IgG) was performed using synthetic TF-polyacrylamide conjugate as antigen, total IgG purified by affinity chromatography on protein G sepharose, and lectins of various sugar specificities: mannose-specific concanavalin A (ConA), fucose-specific Aleuria aurantia lectin (AAL) and sialic acid-specific Sambucus nigra agglutinin (SNA). The sensitivity and specificity of the differences between cancer patients and controls were evaluated by receiver operator characteristic (ROC) curve analysis. Overall survival was analyzed by the Kaplan-Meier method. Time-dependent ROC curve statistics were applied to determine cut-off values for survival analysis. All calculations and comparisons were performed using the GraphPad Prism 5 and SPSS 15.0 software., Results: The level of TF-specific IgG was significantly increased in cancer patients compared with non-cancer controls (P < 0.001). This increase was pronounced mostly in stage 1 of the disease. Cancer patients showed a higher level of ConA binding to anti-TF-IgG (P < 0.05) and a very low level of SNA lectin binding (P = 0.0001). No appreciable stage-dependency of the binding of any lectin to anti-TF IgG was found. A strong positive correlation between the binding of AAL and SNA was found in all groups studied (r = 0.71-0.72; P < 0.0001). The changes in ConA reactivity were not related to those of the fucose- or sialic acid-specific lectin. Changes in the SNA binding index and the ConA/SNA binding ratio demonstrated good sensitivity and specificity for stomach cancer: sensitivity 78.79% (95%CI: 61.09-91.02) and 72.73% (95%CI: 57.21-85.04); specificity 79.17 (95%CI: 65.01-89.53) and 88.64% (95%CI: 71.8-96.6), for the SNA binding index and the ConA/SNA binding ratio, respectively. The other combinations of lectins did not improve the accuracy of the assay. The low level of ConA-positive anti-TF IgG was associated with a survival benefit in cancer patients (HR = 1.56; 95%CI: 0.78-3.09; P = 0.19), especially in stages 3-4 of the disease (HR = 2.17; 95%CI: 0.98-4.79; P = 0.048). A significantly better survival rate was found in all cancer patients with a low reactivity of anti-TF IgG to the fucose-specific AAL lectin (HR = 2.39; 95%CI: 1.0-5.7; P = 0.038)., Conclusion: The changes in the TF-specific IgG glycosylation pattern can be used as a biomarker for stomach cancer detection, and to predict patient survival.

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes-genetic markers of IgG heavy chains and kappa-type light chains, respectively-contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin gamma2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.

Glycan structures of IgG strongly influence the affinity for Fcgamma receptors and antibody effector functions. However, no particular attention has been paid yet to the glycosylation of tumor antigen-specific IgG. The objectives of this study were (i) to investigate the concanavalin A lectin (ConA) reactivity of human anti-Thomsen-Friedenreich (TF) and anti-alphaGal specific IgG in gastric cancer patients and healthy controls and (ii) to evaluate whether the ConA-reactivity of anti-TF and anti-alphaGal specific IgG is associated with the survival rate of patients with cancer. Total IgG was purified from the sera of patients with gastric cancer and healthy blood donors. The anti-TF and anti-alphaGal glycotope specific IgG were detected with ELISA using synthetic saccharide-polyacrylamide conjugates as antigen. In parallel plate, the ConA reactivity of the anti-TF or anti-alphaGal IgG was determined and the ConA index was calculated. Results show that serum anti-TF specific IgG antibodies of patients with cancer contain significantly higher content of ConA positive IgG glycoform compared to IgG of controls. No correlation between the ConA reactivity of anti-TF IgG and anti-alphaGal IgG was observed. High level of anti-TF IgG ConA reactivity was associated with a significantly lower survival rate of patients with gastric cancer.

All human immunoglobulins are glycosylated. The changes in IgG glycosylation are associated with autoimmune disorders and pregnancy. Little is known about IgG glycosylation in patients with cancer. A lectin enzyme-linked immunosorbent assay (LELISA) based method was developed for measuring the Concanavalin A - positive IgG in the serum. Its rationale is as follows: PtA was used as a capture agent for binding IgG via the Fc fragment. Then IgG and the ConA-positive glycans on the IgG were detected using an anti-human IgG-F(ab)2 alkaline phosphatase conjugate or biotinylated ConA, respectively. The index ConA binding/total IgG was calculated. Serum samples from patients with gastric carcinoma (n=53) and healthy blood transfusion donors (n=24) were analysed. The protein A-agarose and ConA-sepharose affinity chromatography was applied to the purification of IgG, ConA-positive IgG, and Fab fragments. The LELISA, SDS-PAGE and Western blot methods were used to analyse the purified IgG and Fab fragments. A significantly higher ConA binding to IgG was found in patients with cancer compared to that of blood donors (ConA index = 1.07+/-0.08 (95% CI) and 0.81+/-0.08, respectively; P=0.0002). In donors, a significant correlation between the level of IgG bound to PtA and the ConA binding (r=0.85; p<0.001) was observed. Patients with gastric cancer showed a less pronounced, though significant correlation (r=0.33; P=0.02). Only the Fd fragment of the Fabs derived from both total serum IgG and ConA-positive fraction of IgG contained the ConA-positive glycans. The comparison of the purified IgG and Fab fragments derived from healthy blood donors and patient with gastric cancer showed no difference in either SDS-PAGE, immunoblotting or LELISA pattern. The LELISA is simple, reproducible and suitable for the evaluation of IgG glycosylation changes. The level of ConA positive serum IgG was found to be increased in patients with cancer. No convincing evidence of the presence of asymmetrically glycosylated F(ab)2 fragments was found. A trend towards a better survival of patients with a lower level of the ConA-positive IgG was observed suggesting a possible blocking effect of the latter on tumor immunity.

Unlabelled: Earlier we found two unusual IgG-antibody specificities to GalNAc beta and GalNAc beta1-3GalNAc beta (para-Forssman disaccharide, PFdi) carbohydrate ligands in human serum. The aim of the study was to evaluate whether elevated antibody levels are related to the progression of gastrointestinal cancer and the histopathological grading., Methods: Specific IgG levels were tested in 159 patients with gastric cancer, 88 patients with colorectal cancer and 96 blood donors by the ELISA using synthetic polyacrylaamide (PAA) conjugates, GalNAc beta-PAA and PFdi-PAA. Biochemical and haematological analyses were performed using automatic equipment., Results: The anti-PFdi IgG levels were significantly higher in patients with gastric and colorectal cancer than in donors: in stages II-IV, P = 0.0002 - 0.04 (U-test). The elevated anti-PFdi IgG level was associated with the advanced gastric cancer: in stages II, III, IV vs stage I (P = 0.004 - 0.06) and in case of the tumor size T2 + T3 vs T1 (stages I, II; P = 0.03). Differences in anti-GalNAc beta IgG level were insignificant. No relation between antibody levels and the regional and distant metastases of gastric or colorectal cancer was found. The lower anti-GalNAc beta IgG level was associated with lower-differentiated carcinomas (P = 0.01 - 0.04). Prolonged postoperative changes in the levels of both antibodies during the follow-up were established. An elevation of both antibody levels in patients with gastrointestinal cancer was revealed after a surgical removal of G3-tumors (P = 0.003 - 0.01). The anti-PFdi IgG levels correlated with the levels of the C-reactive protein: r = 0.50, P = 0.003. The anti-GalNAc beta IgG levels correlated with the percentage of peripheral blood monocytes: r = 0.42, P = 0.002., Conclusion: The association of the anti-PFdi IgG level with cancer progression suggests the implication of antibodies in the pathogenesis of gastrointestinal cancer. Further studies are required to identify natural targets of antibodies, their relation to other diseases, prognostic significance in cancer.

Many investigators have demonstrated alteration of gastric mucins in H. pylori infected individuals. The inflammatory environment induced by H. pylori leading to aberrant glycosylation of MUC1 and demasking of core peptide MUC1 epitope could enhance immune responses to MUC1. IgG and IgM immune response to MUC1 in patients with gastric cancer (n = 214) chronic gastroduodenal diseases (n = 160) and healthy blood donors (n = 91) was studied with ELISA using bovine serum albumin-MUC1 60-mer peptide as antigen. H. pylori serologic status was evaluated with ELISA and CagA status by immunoblotting. Gastric mucosa histology was scored according to the Sydney system. Compared to H. pylori seronegative individuals, higher levels of IgG antibody to MUC1 were found in H. pylori seropositive patients with benign gastric diseases (p < 0.01) and blood donors (p < 0.03). Higher MUC1 IgG antibody levels were associated with a higher degree of gastric corpus mucosa inflammation in patients with chronic gastroduodenal diseases (p < 0.0025). There was a positive correlation between the levels of anti-H. pylori IgG and MUC1 IgG antibody levels in blood donors (p = 0.03), and in patients with benign diseases (p < 0.0001). In patients with gastric cancer (n = 214) a significantly higher level of anti-MUC1 IgG than in blood donors was observed (p < 0.001) irrespective of H. pylori status or stage of cancer. MUC1 IgM antibody levels were not related to the H. pylori serology. IgG immune response to tumor-associated MUC1 is up regulated in H. pylori infected individuals. This increase is associated with a higher IgG immune response to H. pylori and with a higher degree of gastric mucosa inflammation. High levels of MUC1 IgG antibody irrespective of H. pylori serologic status characterized patients with gastric cancer. The findings suggest that, in some individuals, the H. pylori infection may stimulate immune response to tumor-associated MUC1 peptide antigen thus modulating tumor immunity.

The Galalpha1-3Galbeta (alphaGal) hapten is xenogeneic for humans; natural anti-alphaGal antibodies are present in human serum. To study the possible abnormal expression of the alphaGal in humans and the pathophysiological role of antibodies, the method of affinity purification of human anti-alphaGal IgG was developed. The specificity of antibodies was evaluated using polyacrylamide (PAA)-based glycoconjugates in direct and competitive enzyme-linked immunosorbent assays (ELISA). The purified antibodies exhibited alphaGal-restricted specificity. The IC50 value for alphaGal-PAA was equal to 4 x 10(-8) M. In a competitive assay, the Galalpha1-3(Fucalpha1-2)Galbeta-PAA (trisaccharide of blood group B) was found to be one hundred times less active inhibitor than alphaGal-PAA. The multivalent alphaGal-PAA was 1100 times more potent an inhibitor than the monovalent spacered alphaGal-saccharide. The antibodies did not show any reactivity to the negatively charged antigens (DNA, human tumor-derived mucins). At a concentration of 2 microg/mL, the antibodies agglutinated rabbit erythrocytes but not hare erythrocytes. The high reactivity of antibodies to the alphaGal-glycosphingolipids of rabbit erythrocytes and the pig kidney was shown by a modified sensitive method of thin-layer chromatography with immunodetection.

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.

A possible association of serum anti-T IgM and IgG antibody levels with Lewis blood-group phenotype was investigated in 168 blood donors and 132 gastric cancer patients using ELISA with synthetic T-disaccharide-polyacrylamide conjugate as antigen. The donors of Le(a-b+) phenotype showed the highest anti-T IgM level irrespective of ABO(H) blood group. A significant decrease in anti-T IgM in serum was observed among cancer patients of Le(a-b+) phenotype: 95% of weak responders versus 17.5% for related groups of donors (p < 10(-6)). In contrast, no significant difference between patients and donors was found for Le(b-) individuals. Thus, a level of natural anti-T antibodies in serum of blood donors and its decrease in patients with gastric cancer are related to Le(a,b) phenotype. This should be taken into account where anti-T antibody level in the serum is used as a tumour marker or for monitoring patients during cancer immunotherapy with mucin-type vaccines.