muramidase and 4-hydroxy-5-nitrophenyl-acetic-acid

H2-DM (DM, previously H2-M) facilitates the exchange of peptides bound to MHC class II molecules. In this study, we have used H2-DM-deficient (DM(-/-)) mice to analyze the influence of DM in the priming of B cell responses in vivo and for Ag presentation by B cells in vitro. After immunization, IgG Abs could be raised to a T-dependent Ag, 4-hydroxy-5-nitrophenylacetyl-OVA, in DM(-/-) mice, but closer analysis revealed the IgG response to be slower, diminished in titer, and composed of low-affinity Abs. The Ab response correlated with a vast reduction in the number of germinal centers in the spleen. The presentation of multiple epitopes by H2-A(b) from distinct Ags was found to be almost exclusively DM-dependent whether B cells internalized Ags via fluid phase uptake or using membrane Ig receptors. The poor B cell response in vivo could be largely, but not completely restored by expression of a H2-Ea(d) transgene, despite the fact that Ag presentation by H2-E(d/b) molecules was found to be highly DM dependent. Hence, while substantial Ab responses can be raised in the absence of DM, this molecule is a crucial factor both for Ag processing and for the normal maturation of T-dependent humoral immune responses in vivo.

Topics: Animals; Antibody Affinity; Antibody Formation; Antigen Presentation; B-Lymphocyte Subsets; Germinal Center; H-2 Antigens; Haptens; Hemocyanins; Histocompatibility Antigens Class II; HLA-D Antigens; Hybridomas; Immunoglobulin G; Injections, Intraperitoneal; Mice; Mice, Knockout; Mice, Transgenic; Muramidase; Nitrophenols; Ovalbumin; Peptide Fragments; Phenylacetates; Receptors, Fc; T-Lymphocytes; Transgenes

In order to obtain further information on the interaction between antigens (Ags) and B cell Ag receptors (BCR) for a better understanding of the relationship between signals resulting from Ag binding and B cell activation, effects of Ag valence and size on the apparent association constant, i.e. the avidity as well as the molecular stoichiometry of immune complexes in Ag-antibody (Ab) interactions were studied. Hapten conjugates using proteins of various molecular weights, such as hen egg lysozyme (HEL), ovalbumin (OVA), bovine serum albumin (BSA), and chicken gammaglobulin (CGG), were prepared for this purpose. Different ratios of the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP), to the protein were used for conjugation, and interactions between anti-NP monoclonal Abs (mAbs) and the NP conjugates were evaluated by surface plasmon resonance. It was founded that the two binding sites of an Ab were able to simultaneously accommodate two NP(1)-HEL, resulting in a tri-molecular complex, Ag(2)Ab(1). However, NP conjugates of the higher-molecular-weight proteins, OVA and BSA, formed only Ag(1)Ab(1), irrespective of hapten valence. This was thought to be due to steric hindrance caused by the binding of the first Ag. These results suggested that the stoichiometry depended largely on the size of the Ag involved and that mAbs with a low affinity are more efficient at raising the binding strength through divalent interaction since the avidity of two mAbs in interactions with highly haptenated BSA was not significantly different in spite of a 10-fold difference in affinity to the monovalent NP(1)-HEL.

Topics: Antibody Affinity; Antigen-Antibody Reactions; Antigens; gamma-Globulins; Haptens; Models, Immunological; Muramidase; Nitrophenols; Ovalbumin; Phenylacetates; Serum Albumin, Bovine; Surface Plasmon Resonance; Thermodynamics