Dataset Information

Specificity and prevalence of natural bovine anti-alpha galactosyl (Galalpha1-6Glc or Galalpha1-6Gal) antibodies.

ABSTRACT: Immunity against the carbohydrate components of microorganisms mediated by antibodies is an important part of host defenses. Humans and closely related primates, but not other mammals, possess natural anti-Galalpha1-3Gal antibodies which also, although less avidly, react with melibiose (Galalpha1-6Glc). Using an enzyme-linked immunosorbent assay (ELISA) with melibiose-bovine serum albumin as an antigen, we analyzed bovine anti-alpha galactosyl antibodies with respect to specificity and distribution in individual animals. Inhibition assays showed that melibiose was the strongest inhibitor, followed equally by stachyose (Galalpha1-6Galalpha1-6Glcbeta1-2Fru) and raffinose (Galalpha1-6Glcbeta1-2Fru) and then by Galbeta1-6Gal, Gal, and Galalpha1-2Gal. Others, including Galalpha1-3Gal and Galalpha1-4Gal, only exhibited minor inhibition. Thus, these bovine anti-alpha galactosyl antibodies appeared to preferentially react with Galalpha1-6Glc or Galalpha1-6Gal. The distinction of this specificity from that (Galalpha1-3Gal) of human antibodies was further demonstrated by the poor reaction of bovine serum to the Galalpha1-3Gal antigen in comparison to human serum. All 27 healthy bovine serum samples of the three age groups (newborn, calf, and adult) tested contained such antibodies with titers increasing with age. The antibodies purified by affinity chromatography using a melibiose-agarose column were mainly of the immunoglobulin G (IgG) isotype with a concentration of >23 microg/ml in most samples. IgG1 was found to be the primary antimelibiose IgG isotype in all age groups by isotype-specific ELISA, but a significant increase in IgG2, an isotype more related to innate immunity, was observed in calves and adults, compared to newborns. The purified antibodies reacted with the type II bovine strain of Streptococcus agalactiae, a common pathogen of bovine mastitis. Thus, these anti-Galalpha1-6Glc or Galalpha1-6Gal antibodies in cattle might be involved in defense against microbes bearing this or the related epitopes.

SUBMITTER: Ni Y

PROVIDER: S-EPMC95899 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Specificity and prevalence of natural bovine anti-alpha galactosyl (Galalpha1-6Glc or Galalpha1-6Gal) antibodies.

Ni Y Y Powell R R Turner D D DD Tizard I I

Clinical and diagnostic laboratory immunology 20000501 3

Immunity against the carbohydrate components of microorganisms mediated by antibodies is an important part of host defenses. Humans and closely related primates, but not other mammals, possess natural anti-Galalpha1-3Gal antibodies which also, although less avidly, react with melibiose (Galalpha1-6Glc). Using an enzyme-linked immunosorbent assay (ELISA) with melibiose-bovine serum albumin as an antigen, we analyzed bovine anti-alpha galactosyl antibodies with respect to specificity and distribut ...[more]

PMID: 10799466

Similar Datasets

Project description:Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

Project description:The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.

Dataset Information

Specificity and prevalence of natural bovine anti-alpha galactosyl (Galalpha1-6Glc or Galalpha1-6Gal) antibodies.

Publications

Specificity and prevalence of natural bovine anti-alpha galactosyl (Galalpha1-6Glc or Galalpha1-6Gal) antibodies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets