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Biomedical subjects

J M Woof

Publications and source records attributed to J M Woof.

9 recordsLinked to original sources
PubMed1989

Interaction of human IgG chimeric antibodies with the human FcRI and FcRII receptors: requirements for antibody-mediated host cell-target cell interaction.

Chimeric monoclonal antibodies (McAb), specific for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP), expressing human IgG1, IgG2, IgG3 and IgG4 subclass constant domains, have been examined for their ability to interact with the human FcRII receptor. Human red blood cells (RBC) sensitized by each of these McAbs have been assayed for their ability to form rosettes with the human histiocytic lymphoma U937 cell line, human B cell line Daudi and erythroblastoid K562 cell line. IgG1 and IgG3 sensitized RBC formed significant rosettes with the FcR- and FcRII+ Daudi and K562 cell lines, the percentage of cells forming rosettes being directly proportional to the degree of sensitization of the RBC. Bromelin treating Daudi cells did not alter this pattern of reactivity, whereas bromelin treated FcRI+ and FcRII+ U937 cells formed significant resettes with IgG1, IgG3 and IgG4 sensitized RBC, demonstrating a difference in the IgG subclass specificity between human FcRI and FcRII. Murine IgG2b anti-NIP sensitized RBC did not form rosettes with any cell line tested; however, RBC sensitized by some members of a panel of murine IgG1 McAb, specific for the glycophorin A molecule, were able to form rosettes with Daudi, U937 and K562 cells. This interaction was enhanced by bromelin treating the Daudi or U937 cells and can be correlated to the disposition of the epitopes recognized, relative to the target cell membrane, those McAbs recognizing epitopes furthest from the RBC surface being most effective in interacting with FcRII. The data are interpreted in terms of a simple model for antibody-mediated cell--cell interaction.

Antibodies, Monoclonal↗
PubMed1988

Microassay for measurement of binding of radiolabelled ligands to cell surface molecules.

An improved technique for measuring the binding of radiolabelled ligands to cell surface molecules has been developed by modification of a procedure using centrifugation through a water-immiscible oil to separate free and cell-bound ligand. It maximises the percentage of ligand bound since cell-bound and free ligand can be separated easily and reproducibly even when very small reaction volumes are used. This permits low levels of ligand radiolabelling and relatively low numbers of cells to be used.

Cell Line↗
PubMed1988

Localization of the binding site for the human high-affinity Fc receptor on IgG.

A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen.

Amino Acid Sequence↗
PubMed1988

Molecular recognition of antibody (IgG) by cellular Fc receptor (FcRI).

Earlier studies from this and other laboratories have provided indirect evidence for the involvement of the C gamma 2 domain of human IgG in the binding of IgG to the high affinity monocyte Fc receptor (FcRI). Two approaches have been used to extend these studies and to further localize the site of interaction on human IgG. Firstly, monoclonal antibodies (MAbs) directed against different epitopes on IgG were assayed for their capacity to inhibit the binding of radiolabelled IgG to human monocytes or U937 cells. The capacity of the MAbs to interact with their respective epitopes on FcR-bound IgG was also studied using indirect radiobinding and immunofluorescence assays. Secondly, a number of IgGs from several different species and fragments of human IgGs were assayed for their ability to inhibit the binding of radiolabelled IgG to human monocytes. The amino acid sequences of those IgGs exhibiting relatively tight, intermediate or weak binding to monocyte FcRs were compared. On the basis of these studies a possible monocyte FcR-binding site on human IgG is postulated, involving the lower hinge region of IgG (residues Leu 234-Ser 239) with possible involvement of the nearby N-proximal bend and two beta-strands (Gly 316-Lys 338).

Amino Acid Sequence↗
PubMed1988

Immunogenic and antigenic epitopes of immunoglobulins binding of human monoclonal anti-D antibodies to FcRI on the monocyte-like U937 cell line.

Seventeen human monoclonal IgG1- or IgG3 anti-D-secreting clones have been examined for their ability to sensitise O+ red cells for Fc-receptor-mediated rosette formation with U937 cells. IgG3 but not IgG1 anti-D antibodies were able to mediate stable rosette formation with unstimulated U937 cells via interaction with the FcRI receptor. Decreasing FcRI density by incubating U937 cells with di-butyryl cAMP almost completely abolished rosette formation, whilst increasing FcRI density by incubating U937 cells with interferon-gamma increased the percentage of cells forming rosettes with IgG3- and IgG1-sensitised red cells. These data suggest that rosette formation between IgG anti-D-sensitised red cells and FcRI-expressing cells is dependent upon the density of IgG3 on the red cell surface, the density of FcRI on the effector cell, multiple FcRI/IgG interactions are required for stable rosette formation and that more FcRI/IgG1 than FcRI/IgG3 interactions are required.

Binding Sites, Antibody↗
PubMed1986

The use of anti-IgG monoclonal antibodies in mapping the monocyte receptor site on IgG.

Monoclonal antibodies (MAbs) directed against epitopes on the C gamma 1, C gamma 2, C gamma 3 and C gamma 2-C gamma 3 interface regions of human IgG were used to attempt to localize the monocyte Fc receptor (FcR) binding site. The MAbs have been assayed for their capacity to inhibit the interaction between 125I-labelled IgG (125I-IgG) and human monocytes or human histiocytic lymphoma U937 cells. Two MAbs specific for epitopes on the N-terminal region of the C gamma 2 domain, and one MAb recognizing an epitope in the C gamma 2-C gamma 3 inter-domain region inhibited binding of 125I-IgG to monocyte FcRs. The remaining MAbs, against a C-terminal C gamma 3 domain epitope, another C gamma 2/C gamma 3 region epitope and the G1m(f) allotope on the C gamma 1 domain did not inhibit the interaction. The capacity of the MAbs to bind to their respective epitopes on cell surface FcR-bound IgG was also studied, using indirect radiobinding and immunofluorescence assays. All of the MAbs, except those with C gamma 2 domain specificities, were able to detect FcR-bound IgG under these conditions. The results confirm the role of the C gamma 2 domain in the interaction of IgG with monocytes and demonstrate that epitopes in the C gamma 3 and C gamma 2-C gamma 3 regions are not involved in the binding site.

Antibodies, Anti-Idiotypic↗