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Research Tools for Analysis of ZAP-70

bead as the capture surface. A phycoerythrin conjugated detector antibody is also employed. In the case of the BD CBA total ZAP-70 Flex Set, ZAP-70 present in the lysate binds to the capture bead. The amount of PE-anti-ZAP-70 detector antibody that binds to the captured antigen is a measure of the amount of that protein in the sample. A recombinant protein is used to generate a standard curve and to calibrate the relative amount of ZAP-70 in the sample. The specificity of the assay was tested using cell lines as well as purified B and T cells. Table 1 shows that Jurkat (human T cell line) and EL4 (mouse T cell line) cell lysates gave very good responses while there is no signal with A431 (human fibroblast cell line), Daudi (human B cell line), EB1 (human B cell line) or Ramos (human B cell line) cell lysates. Similar results were seen using BD IMag-purified human B and T cells. T cells gave a response of over 5000 MFI while an equal number of B cells gave a background level signal. These experiments not only show how specific the assay is, but also reaffirm that normal B cells do not contain any ZAP-70.

The percentage of B cells in the PBMC fraction from CLL donors ranges from 70-95%. Because of the presence of contaminating T cells which naturally express ZAP-70, it was necessary to determine how many contaminating T cells would result in a positive signal from normal or ZAP-70 CLL cells. By setting a threshold of 100 units/ml, it was shown that between 3,000 and 4,000 T cells will produce this signal (Table 2). If 50,000 cells are read, then a contamination of 6-8% is permissible.

B cells from CLL donors, which were kindly provided by Dr. Tom Kipps at University of California, San Diego, were purified with BD IMag cell separation reagents using negative selection to remove contaminating T cells. This resulted in greater than 98% purity of B cells in all cases. In the assay that was used, 50,000 CLL B
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