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

Abstract

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.

A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Galβ1–4GlcNAc) and LacdiNAc (GalNAcβ1–4GlcNAc) Motifs Found on Vertebrate and Insect Cells

“…Glycan Array Scanning-Glycan arrays were fabricated as described previously (22,23) and contained 317 carbohydrate ligands (supplemental Table 1). The chips were first treated for 30 min with blocking buffer (50 mM ethanolamide in 50 mM borate buffer, pH 8.0) and subsequently for 30 min with 0.1 M phosphate-buffered saline (PBS) (10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 138 mM NaCl, and 2.7 mM KCl, pH 7.4) (Sigma) containing 0.1% Tween 20 (ICN, MP Biomedicals, Santa Ana, CA).…”

Biological roles of glycans

“…In addition to the blood groups already mentioned, intra-and interspecies variations in glycosylation can result in strongly antigenic epitopes. Indeed a significant fraction of circulating Ig found in normal humans may be directed against foreign glycan antigens (971)(972)(973). Certain types of modifications of N-glycans found on plant and invertebrate glycoproteins can trigger immune reactions in humans (974)(975)(976)(977), including therapeutic glycoproteins (835)(836).…”

High-Throughput Profiling of Anti-Glycan Humoral Responses to SIV Vaccination and Challenge

“…Our group [13], [33], [34], [35] and others [16], [17], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45] have used glycan array technology to profile repertoires of serum antibodies in humans, but much less is known about macaque antibody repertoires and the optimal methods for measuring their levels on a glycan array. In addition, the macaque samples available for this study were heat treated to inactivate SIV and the set included a mixture of serum and plasma.…”