In the absence of quantitative real-time PCR as a means of detecting the oncoprotein BCR-Abl, can one reliably use Western blotting (WB) in the diagnosis of CML? Since BCR-abl is a 210kDa protein when the major break point cluster region is involved, and 190kD when the minor region is involved, it is therefore theoretically possible to detect these proteins using WB. My question is, is WB validated for such purpose? In practice, WB can as well be quantitative, and can therefore be used for both diagnosis and measurement of minimal residual disease for BCR-Abl and similar fusion proteins such as PML-RARA, etc. We are contemplating introducing an assay for BCR-abl in our hospital but do not have qPCR facility, so I was wondering if we can reliably use Western since it appears cheaper (at least for a resource-poor setting like ours), and less technically demanding than a qPCR. I would appreciate any reference, please. Thanks.
There are good antibodies to selectively detect BCR-ABL fusion protein in immunoblotting. The protein is detected easily. However, the immunodetection on the membrane is semi-quantitative. Therefore, it may not be suitable for diagnostic purpose. Further, post-transcriptional mechanisms that may alter protein's stability in cells from certain individuals could make this approach unsuitable and risky for diagnosis. In summary, the approach is good for research purpose, but may not be suitable for following the disease progression in patients due to limitations listed above.
There are good antibodies to selectively detect BCR-ABL fusion protein in immunoblotting. The protein is detected easily. However, the immunodetection on the membrane is semi-quantitative. Therefore, it may not be suitable for diagnostic purpose. Further, post-transcriptional mechanisms that may alter protein's stability in cells from certain individuals could make this approach unsuitable and risky for diagnosis. In summary, the approach is good for research purpose, but may not be suitable for following the disease progression in patients due to limitations listed above.
Do you have the equipment to do fluorescence in situ hybridization (FISH)? This would be an easy, cheap and fast option to detect the BCR-ABL1 fusion.
Or you could try the commercially available HemaVision(TM) from DNA Diagnostics in Denmark (http://dna-diagnostic.com/). They have two options: Either RT-PCR end point detection or a RT-qPCR kit. Both are extremely sensitive and robust, although they do require clean room facilties and experience in handling RNA to avoid contamination and breakdown, respectively.
We used c-ABL antibody from Santa Cruz and it was great to detect BCR-ABL (210 or 230 kDa) in different CML cell lines. You can also do qRT-PCR, and it should work :)
http://www.ncbi.nlm.nih.gov/pubmed/25557649
For diagnostic purpose Western blot is not appropriate because it does not tell the fusion protein is from BCR and ABL.
As you mentioned, breakpoint PCR is the prefered method for detecting BCR-ABL in clinical samples by RT-PCR. A fantastic reference for this is the BIOMED-1 study where they publish validated primer sequences. http://www.nature.com/leu/journal/v19/n7/full/2403761a.html
You will have a difficult time convincing anyone that a western blot is truly quantitative; additionally, this is very time consuming. An alternative to this could be flow-detection of the fusion protein in cell lysates. Check out this product: https://www.bd.com/resource.aspx?IDX=17739
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