N-Hydroxysuccinimide (NHS)-functionalized GNPs were used as scaffolds to conjugate synthetic Galf and Rhap monosaccharides functionalized at their anomeric position with a short (C3) aliphatic amino ending spacer. GNPs with either monosaccharide or a combination of both thus generated Galf-GNPs, Rhap-GNPs, and Mix-GNPs, respectively (Fig. 1A). Control-GNPs were prepared by reacting the 5 nm NHS-gold nanoparticles with ethanolamine. The presence of Galf on the Galf- and Mix-GNPs was tested in an ELISA with the rat monoclonal antibody EB-A2 that is widely used to detect circulating Galf-containing galactomannan in the serum of aspergillosis patients and was demonstrated previously to also recognize Galf-GNPs. Here, EB-A2 bound to Galf-GNPs in a dose-dependent manner (25 to 3.125 μg/mL), while Rhap- or Control-GNPs did not show any binding up till the highest tested concentration of 50 μg/mL (Fig. 1B). Interestingly, it was previously thought that EB-A2 is only able to bind galactomannan having branches of a length of at least four β1-5 galactofuranosyl moieties. Although a recent glycoarray study has shown that two β1-5 galactofuranosyl moieties are sufficient to enable EB-A2 binding, our data suggest that other Galf conformations also enable this antibody to bind. To understand the interaction between EB-A2 and Galf moieties on GNPs in detail, studies, like done previously for the interaction between antibody CS35 and arabinofuranosides, could be conducted. Unexpectedly, EB-A2 did not recognize the Mix-GNPs, although they also contain multiple Galf moieties on their surface. Clearly, IgM and IgG antibodies in the human sera of this study were capable of binding to the Mix-GNPs, irrespective whether the sera contained antibodies that also recognized Rhap-GNPs or Galf-GNPs alone (Fig. 1C), thereby demonstrating that the monosaccharides coupled to the GNPs are presented in a way that enables antibody binding. Taken together, these results indicate that the Galf-moieties only enable binding of EB-A2 antibodies to Galf-GNPs if presented in a particular manner and that the Rhap-moieties present on the Mix-GNPs prevent this.
Next, we used these synthesized GNPs to test the presence of antibodies recognizing Galf and Rhap in 108 individual sera obtained from 4 different groups, i.e. patients infected with a drug-sensitive strain of Mycobacterium tuberculosis (MTB), those infected by a multi-drug resistant strain (MDR), patients suffering from chronic obstructive pulmonary disease (COPD) and healthy individuals (HC). Raw data can be found in the supplementary files S1, S2 and S3.
This experiment shows that many individuals, irrespective from their disease background, have serum IgM and IgG antibodies that can recognize Galf or Rhap (Fig. 1C-1I). Several other studies have shown human serum antibodies against Rhap, however, to our knowledge, anti-Galf antibodies have never been demonstrated before in human sera.
We decided to classify any serum sample that produced an optical density at 450 nm (OD450) value for a particular GNP above 0.5 and at least 1.5 times the value of reactivity against Control-GNPs as being positive against that particular GNP. So for example, samples A04 and F28 from the exemplar results shown in figure 1C were not classified as positive for either Galf-, Rhap- or Mix-GNPs (in total 37 samples were negative for IgM and 30 for IgG). Samples with a profile similar to A08 were classified as having antibodies against all three glycan-GNPs (20 for IgM and 16 for IgG); samples similar to A20 as having antibodies against Rhap-GNPs only (17 for IgM and 13 for IgG); samples like B30 as having antibodies against Galf only (8 for IgM and 3 for IgG); samples like D02 as having antibodies against Rhap- and Mix-GNPs (24 for IgM and 39 for IgG); and samples similar to F08 as having antibodies against Galf- and Mix-GNPs (2 for IgM and 4 for IgG). Just one sample (B15) was classified as only positive for having IgG against Mix-GNPs, as the signal against Rhap-GNPs did not pass the OD450 threshold of 0.5. Two samples (A04 and B29) were classified as positive only for IgG antibodies against Galf-GNPs and Rhap-GNPs, but not for Mix-GNPs. In both cases, this seems to be caused by marginal differences in measured OD450 values.
In total, 27 sera resulted in value above 0.5 when incubated with the Control-GNPs and secondary detection for IgM antibodies. 5 of those had a value above 1 like shown for A04 (Fig. 1C, grey bar). In the IgG detection experiment, these numbers were much lower (9 above 0.5 and none above 1), indicating the higher specificity of the IgG isotype.
All data for both IgM and IgG can be aggregated into the Venn diagram shown in figure 1D describing the overlap between Rhap and Galf recognition (for tables for IgM and IgG separately see Suppl. data file S4). Of the 108 individual sera tested in this study, 15 were not classified to contain either IgM or IgG antibodies reacting to any of the GNPs (14%), where 93 individuals (86%) seem to have at least one type of antibody capable of recognizing an epitope on the Galf-, Rhap-, or Mix-GNPs (Fig. 1D). Of those, 84 individuals had antibodies that could recognize Rhap (90%), while 43 possessed antibodies that reacted against Galf (46%).
When the levels of IgM antibodies against Galf are compared with those against Rhap (Fig. 1E) a significant positive correlation with a Spearman rank coefficient (rs) of 0.66 (p <0.001) is found, comparable as what has been described for correlations between anti-Rhap IgM and IgM antibodies recognizing other two other haptens (Jakobsche et al., 2013). There was also a positive correlation between anti-Galf IgG and anti-Rhap IgG in our study (Fig. 1F), albeit much weaker (rs = 0.27 and p = 0.053). As expected correlations for antibodies recognizing the single glycan-GNPs with the ones reacting with the Mix-GNPs are high (Suppl. data file S5).
IgM antibodies against Rhap (56%) were slightly less abundant than IgG antibodies against Rhap (65%), whereas, for anti-Galf antibodies, this is reversed, with 28% of the sera having IgM antibodies against this hapten and 23% having IgG antibodies. There was no correlation between reactivity against either Rhap or Galf based on IgM antibodies and reactivity based on IgG antibodies within individual samples (data not shown).
When the age of all tested individuals is plotted against the amount of IgM antibodies shows a negative correlation for anti-Galf-GNP IgM (rs = -0.009 and p = 0.0003), anti-Rhap-GNP (rs = -0.014 and p <0.0001) and anti-Mix-GNP (rs = -0.014 and p <0.0001) show a negative correlation (Fig. 1G), which is in line with previous studies. The negative correlation with age is much less pronounced for IgG antibodies (Fig. 1H), with only anti-Mix-GNPs showing a significant correlation (rs = -0.006 and p = 0.012). This is important, as the average age of the individuals of the four groups in this study differed significantly at time of sampling, with MDR (44.0 years ±13.7) and HC (40.0 years ±11.4) being relatively low and MTB (56.9 years ±11.5) and COPD (69.3 years ±8.0) being relatively high (Suppl. data file S6). When all data is clustered per patient group, it becomes immediately clear that more antibodies of both isotypes against Rhap and Mix were found across the 4 groups compared to anti-Galf antibodies (Fig. 1I). Interestingly, the highest number of individuals with IgG antibodies against Rhap-GNPs (74%) was found in the COPD group, despite the high average age in that group.