Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid. I. Antigenicity of the glycolipids and the production of specific antibodies in rabbits.

Abstract

The antigenicity of lipids is still ill-defined, and there is very little evidence that any pure lipid by itself will act as an antigen . To produce an immune response they must be complexed with or coupled to carriers (1-6) . Only glycosphingolipids and phosphatides have been found to function as haptens when mixed with carriers such as serum albumin (7), methylated bovine serum albumin (8), or other auxillary lipids (9-11), or when the glycolipid was incorporated into liposomes (12) . Antibodies to glycolipids are usually directed primarily against the carbohydrate moiety (1, 7, 13, 14), and there is little evidence that antibodies can be formed to the free lipids even though free fatty acids were reported to cause inflammatory delayed-type allergic skin reactions (15, 16) .The purpose of this investigation was to synthesize and study the antigenicity and immunological properties of a series of glycolipid antigens prepared by coupling isomaltose oligosaccharides-varying in size from isomaltose to isomaltoheptaose-to a lipid carrier, stearylamine, and to compare the antigenicities of the different stearylisomaltosyl oligosaccharides thus obtained, either when injected alone or when incorporated into liposomes. Isomaltose oligosaccharides were chosen because they are the predominant structural units of a1--),6 dextran (17). Antibodies raised to them should cross-react with dextrans, and the heterogeneity of the response and the sizes of their antibody combining sites could be studied by quantitative precipitin and precipitin inhibition assays and could be compared with antidextran (18-22) .The stearyl-isomaltosyl oligosaccharides (stearyl-IM3 to IM7) when incorporated into liposomes were agglutinated in the presence of specific antibodies and were lysed ifcomplement was also present . When injected into rabbits they were antigenic, either emulsified directly with complete Freund's adjuvant (CFA) l or incorporated into * Aided by grants from the National Science Foundation (PCM 76-81029) and Cancer Center Support grant CA 13696 to the Cancer Center, Columbia University .f Portions of this work are from part I of a dissertation submitted by C . Wood in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences, Columbia University, New York .'Abbreviations used in this paper: BSA, bovine serum albumin ; CFA, complete Freund's adjuvant ; DNPCap-PE, DNP-e-aminocaproylphosphatidyl-ethanolamine ; yglo, gammaglobulin; IM2, isomaltose (DGlcal~6DGlc) ; IM3, isomaltotriose ; IM4, isomaltotetraose ; IM5, isomaltopentaose ; IM6, isomaltohexaose ; IM7, isomaltoheptaose ; PBS, phosphate-buffered saline (0 .01 M phosphate, 0 .8 percent sodium chloride, pH 7 .2) ; SDS, sodium dodecyl sulfate; SRBC, sheep erythrocytes ; TCA, trichloroacetic acid . 432J. Exp . MED .

Polyclonal human antibodies against glycans bearing red meat-derived non-human sialic acid N-glycolylneuraminic acid are stable, reproducible, complex and vary between individuals: Total antibody levels are associated with colorectal cancer risk

“…We reasoned that such antibodies against a single monosaccharide might not be as specific because the binding pockets of antibodies typically accommodate 3 to 5 monosaccharides [ 71 , 72 ]. Based on our prior work showing that antibodies against the more extended epitope Neu5Gcα2–6Lacβ–R were relatively common in the population [ 20 ], we next tried an ELISA assay against chemically synthesized Neu5Gcα2–6Lacβ-HSA.…”

Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides

“…Borch et al (1971) made use of this principle by including water-absorbing molecular sieves to accelerate conjugation reactions between highly hindered cyclic ketones, such as norbornanone, and ammonia or dimethylamine. Wiegandt & Ziegler (1974) reported that successful conjugation of sialyl-lactose to stearylamine (in 50 % yield) was achieved by refluxing in methanol for 2 h. For conjugating dextran oligomers to stearylamine in water-containing reaction mixtures [290 (v/v) water in tetrahydrofuran] an incubation time of 3 weeks was required (Wood & Kabat, 1981).…”

The 10th Anniversary of Carbohydrate Microarrays (2002–2012)

“…Thus, microarray-based assays are intrinsically optimized for binding equilibrium to take place, which is the basis for this class of the hyper-sensitive binding assays [44]. Carbohydrate microarrays have higher detection sensitivity than most conventional carbohydrate analytic tools, such as carbohydrate-specific ELISA and the glycolipid-based “Eastern blot” assays that were developed in 1980s by a number of early researchers in this field [45,46]. Historically, this situation is very similar to the relationship between conventional blotting methods for nucleic acids or proteins, such as Southern, Northern, and Western blots, and the nucleic acid–based or protein/peptide–based microarrays.…”