Antibody

About: Antibody is a research topic. Over the lifetime, 113941 publications have been published within this topic receiving 4130181 citations. The topic is also known as: Ab & antibodies.

Profiling the humoral immune response to infection by using proteome microarrays: High-throughput vaccine and diagnostic antigen discovery

Abstract: Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.

Immunofluorescent studies of the development of pre-B cells, B lymphocytes and immunoglobulin isotype diversity in humans

Abstract: Immunofluorescent techniques were used to examine several aspects of B cell ontogeny in humans. Large lymphoid cells containing intracytoplasmic IgM (pre-B cells) were present in fetal liver as early as 7 weeks of gestation, approximately 2 weeks prior to the appearance of surface IgM positive (sIgM+) B lymphocytes. Pre-B cells outnumbered sIgM+ B lymphocytes in fetal liver up until the 13th week of gestation. In fetuses older than 13 weeks, pre-B cells and sIgM+ B lymphocytes were present in approximately equal proportions in liver and bone marrow. Pre-B cells in fetal liver, and fetal and adult marrow, were large and small (indicating a heterogeneous population of cytoplasmic IgM+. SIg- cells in these sites), while only the small pre-B cells were present in fetal spleen, blood and lymph node. Lymphocytes bearing sIgG were detected earlier than those bearing sIgD or sIgA, which were present by the 12th gestational week. Using double-staining techniques, we determined that during fetal life, (a) the proportion of B lymphocytes bearing only sIgM, as opposed to those bearing both sIgM and sIgD, was much higher in liver and bone marrow than in spleen, blood and lymph node, and (b) sIgG, sIgA and sIgD appear independently on lymphocytes bearing sIgM. Studies of the frequency of double-stained cells for each combination of the four sIg isotypes indicated that B lymphocytes from neonatal humans may simultaneously bear three or more sIg isotypes, whereas sIgG+ and sIgA+ B lymphocytes in adult blood usually express only the single isotype. Lower concentrations of anti-y antibodies were required for modulation of sIgM from B lymphocytes of fetal liver and adult bone marrow than for equivalent removal of sIgM from circulating B cells of mature individuals. In conjunction with data obtained in mice, our observations indicate that (a) the presence of large and small pre-B cells, (b) a high ratio of sIgM single to sIgM.sIgD double B lymphocytes, and (c) increased sensitivity to modulation of B cell sIgM by divalent anti-μ antibodies define the fetal liver and adult bone marrow as bursa-equivalent sites in humans. Our results are consistent with a model of isotype diversification in which immature sIgM+ cells give rise to B cell sublines devoted to synthesis of each of the Ig classes, and sIgD is secondarily expressed on unstimulated B cells of all of these sublines.

Production of antigen-specific human monoclonal antibodies from in vitro-primed human splenocytes.

Abstract: We have developed culture conditions for human lymphocytes that support primary in vitro immune responses to protein Ag of either human or nonhuman origin We now show that these primed B cells can be efficiently immortalized by fusion with a heterohybrid fusion partner to generate human, Ag-specific IgM or IgG antibody-producing heterohybridomas at a rate of 17 to 50 hybrids/10(6) lymphocytes fused Approximately 50% of the Ig-secreting clones were stable with respect to Ig secretion Levels of secretion attained with terminal cultures ranged from less than 1 to 100 micrograms/ml Fusions of cells between 2 and 5 days after initiation of in vitro exposure to Ag produced more Ag-reactive and Ag-specific antibodies than fusions at 1 day or fusions performed after 5 days Ag-reactive hybrids could be isolated at frequencies of 3 to 10%, depending on antigenicity of the immunogen Foreign proteins, horse spleen ferritin, and a murine monoclonal Ig, induced higher percentages of Ag-reactive mAb than immunization with the human-derived ferritin Ag-reactive IgG mAb were produced at relatively high frequency, depending on immunization conditions and the nature of the Ag The strategy for identification of the best hybrids included early elimination of unstable hybridomas and of hybridomas producing broadly cross-reactive antibody, followed by evaluation of units of Ag reactivity/micrograms Ig Ferritin-specific mAb selected according to these criteria showed immunocytochemical reactivity with ferritin-containing tissues and apparent affinities in the range of 10(7) to 10(8)/mol

Type I and type II Fc receptors regulate innate and adaptive immunity

Abstract: Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibody's Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.

CD4+ effector cells default to the Th2 pathway in interferon gamma-deficient mice infected with Leishmania major

Abstract: Mice with homologous disruption of the interferon gamma (IFN-gamma) gene on the C57BL/6 background were infected with Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice, deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6-8 wk. Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-gamma and lymphotoxin, typical of T helper type 1 (Th1) cells, the knockout mice developed CD4+ cells that contained transcripts for interleukin 4 (IL-4), IL-5, and IL-13, typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-gamma or IL-4 production by T cells in similar frequencies in the respective groups of mice, and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-gamma.