Some Human Anti-Glycan Antibodies Lack the Ability to Activate the Complement System.

Naturally occurring human antibodies against glycans recognize and quickly eliminate infectious bacteria, viruses and aberrantly glycosylated neoplastic malignant cells, and they often initiate processes that involve the complement system. Using a printed glycan array (PGA) containing 605 glycoligands (oligo- and polysaccharides, glycopeptides), we examined which of the glycan-binding antibodies are able to activate the complement system. Using this PGA, the specificities of antibodies of the IgM and IgG classes were determined in the blood serum of healthy donors (suggested as mostly natural), and, then, using the same array, it was determined which types of the bound immunoglobulins were also showing C3 deposition.

Localization of synthetic glycolipids in the cell and the dynamics of their insertion/loss.

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss.

Specificity profile of αGal antibodies in αGalT KO mice as probed with comprehensive printed glycan array: Comparison with human anti-Galili antibodies.

Background: The α1,3-galactosyltransferase gene-knockout (GalT KO) mice are able to produce natural anti-αGal antibodies apparently without any specific immunization. GalT KO mice are commonly used as a model immunological system for studying anti-αGal responses to Gal-positive xenografts in human. In this study, we compared the specificity of mouse and human αGal antibodies to realize the adequacy of the murine model.

Human Natural Antibodies Recognizing Glycan Galβ1-3GlcNAc (Le).

The level of human natural antibodies of immunoglobulin M isotype against Le in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors' blood using Le-Sepharose (Le is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with Le, which implies the impossibility of binding to regular glycoproteins of non-malignant cells.

Specificity of human natural antibodies referred to as anti-Tn.

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g.

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.

Improved spot morphology for printed glycan arrays.

Despite considerable success studying glycan-binding proteins using printed glycan arrays (PGAs), unambiguous quantitation of spot intensities by fluorescent readers remains a challenge. The main obstacles are the varying spot shape and size and in-spot fluorescence distribution caused by uneven drying of the printed drops. Two methods have been suggested for solving this problem: using polymeric glycoconjugates, which makes it possible to equalize the physicochemical properties (hydrophobicity, charge, and size) of different glycans, and applying a glycan solution on a slide coated with a thin oil mask, which hinders evaporation of the drop.

Repertoire of BALB/c Mice Natural Anti-Carbohydrate Antibodies: Mice vs. Humans Difference, and Otherness of Individual Animals.

One of the most common genetic backgrounds for mice used as a model to investigate human diseases is the inbred BALB/c strain. This work is aimed to characterize the pattern of natural anti-carbohydrate antibodies present in the serum of 20 BALB/c mice by printed glycan array technology and to compare their binding specificities with that of human natural anti-carbohydrate antibodies. Natural antibodies (NAbs) from the serum of BALB/c mice interacted with 71 glycans from a library of 419 different carbohydrate structures.

Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid-independent glycotope.

A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9.

Natural Antibodies Against Sialoglycans.

Natural antibodies, part of the innate immunity system, are produced at strictly regulated levels in normal sera without immunization and thus are part of the innate immune system. The best studied natural antibodies are those directed against blood group antigens A and B and xeno-antigens including glycolylneuraminic acid containing Hanganutziu-Deicher (HD) glycolipid. Abnormal levels of anti-glycan antibodies were found in a number of pathologies.

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.