Repertoire of Abs primed by bacteria in gnotobiotic mice.

Innate immunity natural Abs (NAbs) execute a number of functions, including protection and surveillance. Despite active research, the stimuli that induce the formation of NAbs are still described only hypothetically. Here, we compared repertoires of anti-glycan Abs in the peripheral blood of mice that received per os various bacteria. The repertoires of Abs of mice primed in this way were compared using a microarray that included about 350 glycans, as well as 150 bacterial polysaccharides. Sterile mice did not possess anti-glycan Abs. Oral inoculation of a single strain or combination of two to four strains of bacteria, as well as putting the animals on short-term nutrition with non-sterile food, did not contribute significantly to the formation of Abs, whereas a single gavage of digested food of non-sterile mice induced the formation of a repertoire close to the natural ones. Interestingly, the priming with polysaccharide Ags (in a composition of the bacterial cell envelope), that is, dominant Ags of bacteria, led to the induction of Abs against typical glycans of mammalian glycoproteins and glycolipids (e.g. Abs of the ABH blood group system) that do not have a structural similarity to the polysaccharides. The results support the importance of early contact with a naïve immune system with microorganisms of the environment to form a normal NAbs repertoire.

Printed glycan array: antibodies as probed in undiluted serum and effects of dilution.

Using printed glycan array (PGA) we compared the results of antibody profiling in undiluted, moderately (1:15) and highly (1:100) diluted human blood serum. Undiluted serum is suitable for studying blood as a tissue in its native state, whereas to study the serum of newborns or small animals one usually has to dilute the starting material in order to have sufficient volume for PGA experimentation. The PGA used in this study allows for the use of whole serum without modifications to the protocol, and the background is surprisingly low. Antibodies profiles observed in undiluted serum versus 1:15 dilution were similar, with only a limited number of new signals identified in the undiluted serum. However, unexpected irregularities were found when IgG and IgM are measured separately, namely, at a 1:15 dilution more intensive IgG signals for many glycans are observed. We believe that in conditions of moderate dilution IgG and IgM antibodies can compete with each other for antigen and as a result, the higher affinity anti-glycan IgGs give rise to more intense signals. Therefore depending on the purpose, different dilutions of serum will be optimal: in competitive 1:15 conditions the observed IgG/IgM ratio corresponds to their titer, whereas at 1:100 dilution the measured ratio corresponds to real molar concentration of IgG and IgM.

Profiling of serum antibodies with printed glycan array: room for data misinterpretation.

Using an example of Galß1-3GlcNAc (Le(C)) related glycans, we here demonstrate a risk of data misinterpretation when polyclonal antibodies are probed for their glycan-binding specificities with help of a printed glycan array (PGA). Affinity isolation of antibodies from human serum using Le(C)-Sepharose or 3'-O-SuLe(C)-Sepharose in conditions of excess of the adsorbents generated identical material regardless of the affinity ligand, with the antibodies equally capable of binding to Le(C) and to 3'-O-SuLe(C) disaccharides, as well as to 3'-O-SiaLe(C) trisaccharide. More detailed profiling has shown that the isolated antibodies bind to the inner part of Galß1-3GlcNAc disaccharide. We therefore conclude that serum does not contain different subsets of antibodies specific either to Le(C) or to 3'-O-SuLe(C), despite their visibly different binding signals to these glycans on PGA.