Abstract
Microarrayed antigens are used for identifying serum antibodies with given specificities and for generating binding profiles. Antibodies bind to these arrayed antigens forming immune complexes and are conventionally identified by secondary labelled antibodies.In the body immune complexes are identified by bone marrow derived phagocytic cells, such as monocytes. In our work we were looking into the possibility of replacing secondary antibodies with monocytoid cells for the generation of antibody profiles. Using the human monocytoid cell line U937, which expresses cell surface receptors for immune complex components, we show that cell adhesion is completely dependent on the interaction of IgG heavy chains and Fcγ receptors, and this recognition is susceptible to differences between heavy chain structures and their glycosylation. We also report data on a possible application of this system in autoimmune diagnostics.Compared to secondary antibodies, fluorescent monocytesas biosensors are superior in reflecting biological functions of microarray-bound antibodies and represent an easy and robust alternative for profiling interactions between serum proteins and antigens.
Highlights
If blood comes into contact with antigens the binding of antigen specific circulating antibodies to their target willresults in the generation immune complexes
To identify the receptors that potentially mediate adhesion of U937 cells to immune complexes, IgG and complement receptor distribution on U937 cells was determined by flow cytometrywith receptor specific antibodies recognizing CD16(FccRIII), CD32(FccRII), CD64(FccRI), CD35(CR1), CD11b(CR3) and CD11c(CR4)
Binding of U937 cells to materials printed as microarrays For analyzing U937 cell binding preferences, IgG subclasses were printed onto nitrocellulose microarrays
Summary
If blood comes into contact with antigens the binding of antigen specific circulating antibodies to their target willresults in the generation immune complexes. A binding pattern called antibody profile, characteristic of the tested serum, is generated [1,2]. This pattern is usually revealed by secondary antibodies labeled suitably to allow qualitative and quantitative detection of the bound serum antibodies. Using secondary antibodies for the generation of antibody binding profiles we can obtain only a partial picture on the biological function of the detected immune complexes. T lymphocytes bind to MHCpeptide complexes printed as arrays [4], leukemia cells are captured by surface marker specific capture antibodies [5], mesenchymal cells adhere to peptides of extracellular matrix components [6], hepatocytes to glycans [7], just to mention a few types of binding. Cells on the array can be detected by fluorescent labeling and laser scanning [4] or without labeling, by microscopy [5]
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