Biomedical subjects
Publications and source records attributed to E Muratti.
The chemical complexity of peptides limits the choice of coupling procedures which can be used in the preparation of gel-peptide conjugates. Several immunoadsorbents have been investigated, in order to select those that permit the isolation of antipeptide antibodies in the highest yield and with optimal specific activity.
Sepharose-peptide immunoadsorbents, employed for the isolation of specific antibodies from the sera of rabbits immunized with carrier protein-peptide conjugates, were digested with suitable proteolytic enzymes, in order to obtain the splitting of a part of the peptide bound to the gel. This new modified immunoadsorbent can be advantageously used for the isolation of antibody subsets, that do not cross-react with related peptides exhibiting high sequence homology with the immunogens.
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A rabbit was immunized with B lymphoblastoid cells, and subsets of the antibodies produced were isolated on affinity columns made from synthetic peptides corresponding to known amino acid sequences from the human class II antigens DQ and DP. Those peptides for which specific antibodies were isolated could be assumed to contribute to the antigenic properties of the intact antigen. The antibody subsets were tested for binding to synthetic peptides, to glycoprotein fractions isolated from cells with different DR and DQ specificities, and to the cells used for immunization of the rabbit. The isolation of those antibodies directed against well-defined amino acid stretches of the histocompatibility antigens is proof of the role of those regions in determining the antigenic properties of these molecules.
Repeated immunizations of rabbits with chemically synthesized peptides from selected regions of HLA-DP histocompatibility antigens resulted in the production of specific antibodies that were then isolated from the immune sera by chromatography on Sepharose-peptide immunoadsorbents. The purified antibodies, when tested with an enzyme-linked immunosorbent assay, specifically bound to the inciting fragments; moreover, two of them recognized glycoproteins extracted by nonionic detergents from human chronic lymphocytic leukemia cells, as revealed by binding assays. The results suggest that amino acid stretches 51-61 of the alpha chain and 80-90 of the beta chain of HLA-DP histocompatibility antigens are likely exposed on the surface of the protein molecule. The specific recognition of DP regions is strongly suggested by the difference in the binding of those antibodies to soluble membrane proteins, as compared to the binding of monomorphic anti-Class II monoclonal antibodies to the same antigens.
A group of 19 anti-class II monoclonal antibodies produced in different laboratories were tested in ELISA for their ability to bind to a panel of synthetic peptides selected from HLA-DQ alpha and beta chains. No one of the antibodies tested was found to react with the synthetic fragments, thus confirming the common finding that MoAbs generally fail to recognize fragments of the native antigen. The possibility that this result might be partly due to the procedure used for screening hybridoma supernatants is discussed.