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Activation of B cells through the B cell receptor or T cells through the CD3 complex follows a similar signaling pathway although, in many cases, different members of the same kinase families are used. ZAP-70 and Syk are members of the same family and have similar sequences and structures. Crosslinking of the CD3 receptor complex leads to the activation of Lck and Fyn which are Src family tyrosine kinases1,2. These kinases phosphorylate ITAM regions on the receptor which act as docking sites for ZAP-70. Phosphorylation of ZAP-70 by Lck leads to its activation. ZAP-70 then goes on to phosphorylate a number of adaptor proteins and helps activate other kinases as well as phospholipase Cγ. B cells are activated by crosslinking of the Ig receptor3. This activates Lyn, another member of the Src family. Through a similar sequence of events, Syk is activated. Syk is found in various hematopoietic cells such as B cells, platelets, macrophages, and mast cells. ZAP-70 is found primarily in T cells and is not expressed in normal B cells.
Chronic lymphocytic leukemia, a tumor of mature B cell origin, is one of the most common leukemias. The B cells of these donors express an unmutated variable region of the Ig heavy chain gene. More recent work has shown that these donors also express ZAP-70 and that this may be a better marker indicating the aggressive form of the disease4,5. Furthermore, it has been shown that signaling through the Ig receptor is better in ZAP-70 containing CLL cells as opposed to cells that only have Syk6. Responsiveness to anti-Ig is also increased in B cells transfected with ZAP-70.
Adding to our portfolio of ZAP-70 antibody reagents for flow cytometry, western blot, and IHC, BD Biosciences has developed a BD Cytometric Bead Array (CBA) Flex Set assay that measures total ZAP-70 from SDS denatured cell lysates. The BD CBA Flex Set assay utilizes an antibody-conjugated 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 cells were run and compared to a standard curve. As can be seen (Figure 1), some of the patients had very high levels (>1000 MFI, 400-800 units), others had intermediate levels (several hundred, 150-400 units), and others had less than 50 MFI (0-70 units). Our results correlated with Dr. Kipps results using other methods (western blots, flow cytometry) to determine which donor cells expressed ZAP-70.
Please visit the following websites for additional product information:
bdbiosciences/flexset.com
for BD CBA Flex Set product details
bdbiosciences/bdimag.com
for BD IMag cell separation reagent product details
References
1. Palacios, E.H. and Weiss, A. (2004) Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23:7990-8000.
2. Abraham, R.T. and Weiss, A. (2004) Jurkat T cells and development of the T-cell receptor signalling paradigm. Nature Reviews Immunol. 4:301-308.
3. DalPorto, J.M. et al. (2004) B cell antigen receptor signaling 101. Mol. Immunol. 41:599-613.
4. Wiestner, A. et al. (2003) ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 101:4944-4951.
5. Rassenti, L.Z. et al. (2004) ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. New Engl. J. Med. 351-893-901.
6. Chen, L. et al (2005) ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia, Blood 105:2036-2041.
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