No coin nor oath required. For personal study only.
Despite the comprehensive range of techniques available for the detection of specific IgE, there is now a vast literature that shows poor correlation between the observed allergic clinical symptoms and specific IgE levels. By adding a biological readout, current developments on basophil activation tests are showing a much improved clinic relevance (DBPFCF) than specific IgE alone. Noteworthy, some advances in the area have demonstrated that basophil activation of cell lines without full degranulation is a good predictor of clinical outcome. Hence in order to develop a highthroughput diagnostic device which combines the advantages of basophil activation tests with the numerical power of protein microarrays, we have demonstrated as proof-of-principle that purified human peripheral blood basophils stripped and resensitised with an allergic donor's serum can bind to a protein array containing appropriate allergens, and that activation can be detected using an antibody to CD63. In order to avoid the costs, logistics and intrinsic difficulties associated with the use of peripheral blood basophils, we sought to replace the human cells with a human or humanised basophil or mast cell line. Binding and incubation parameters (buffer, incubation time, temperature, cell density, washing steps, non-specific binding blocking reagents, addition of extracellular matrix components) were experimentally optimised for 3 cell lines. Further, as a support for the array we developed a comprehensive 4 immunoglobulin profiling tool for the simultaneous detection of IgM, IgA, IgG and IgE. The current developments on the array profiling technique and alternative ways for detection of basophil cell activation in an array format involving Annexin V binding, Calcium influx and fluorescent inducible reporter systems (NFAT-EGFP and RnIL-4p-DsRed) will be discussed.
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