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Updated: Jun 26, 2026
Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function
Published on: February 16, 2017
Optimization of methods for the detection of BCR-ABL activity in Philadelphia-positive cells
Ashley Hamilton1, Fatma Alhashimi, Svetlana Myssina
1Section of Experimental Haematology, Cancer Division, Faculty of Medicine, University of Glasgow, Paul O'Gorman Leukaemia Research Centre, Gartnavel General Hospital, Glasgow, UK.
Objective:
The recent success in treating chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has created a demand for reproducible methods to accurately assess inhibition of BCR-ABL activity within CML cells, including rare stem and progenitor cells, either in vitro or in vivo. The purpose of this study was to develop an enzyme-linked immunosorbent (ELISA) method to measure total tyrosine phosphorylation (P-Tyr) in small samples of cells that express BCR-ABL and to compare to more established methods.
Materials And Methods:
The assay was first validated in BCR-ABL wild-type and mutant vs BCR-ABL-negative cell lines. P-Tyr levels were then measured by ELISA in primary CD34(+) CML cells treated with IM.
Results:
In vitro exposure to TKI resulted in decreases in the level of P-Tyr, in both BCR-ABL-positive cell lines and primary CD34(+) CML samples, which were comparable to the reduction in P-Tyr by flow cytometry and phosphorylation of CrkL by either Western blot or flow cytometry.
Conclusion:
We have developed an accurate ELISA method to measure BCR-ABL activity within Ph(+) cells, which is comparable to other in vitro BCR-ABL assessment techniques in terms of sensitivity and could be adapted for high throughput.
Insights
A new ELISA method accurately measures BCR-ABL activity in chronic myeloid leukemia (CML) cells, comparable to existing techniques. This assay is sensitive and suitable for high-throughput screening of tyrosine kinase inhibitor (TKI) effectiveness.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Tyrosine kinase inhibitors (TKIs) like imatinib mesylate (IM) have advanced chronic myeloid leukemia (CML) treatment.
- Accurate assessment of BCR-ABL activity in CML cells, including stem cells, is crucial for monitoring TKI efficacy.
- Existing methods for measuring BCR-ABL activity may require large cell samples or are not high-throughput.
Purpose of the Study:
- To develop and validate an enzyme-linked immunosorbent assay (ELISA) for measuring total tyrosine phosphorylation (P-Tyr) in BCR-ABL-expressing cells.
- To assess the sensitivity and comparability of the developed ELISA method against established techniques.
- To enable accurate measurement of BCR-ABL activity in small cell samples, including primary CML stem and progenitor cells.
Main Methods:
- Validated an ELISA assay using BCR-ABL wild-type, mutant, and negative cell lines.
- Measured P-Tyr levels in primary CD34(+) CML cells treated with IM using the developed ELISA.
- Compared ELISA results with flow cytometry and Western blot analyses of CrkL phosphorylation.
Main Results:
- The ELISA method demonstrated accurate measurement of P-Tyr levels in BCR-ABL-positive cell lines and primary CML samples.
- In vitro TKI treatment led to significant decreases in P-Tyr levels, as detected by ELISA.
- ELISA results showed comparable sensitivity to flow cytometry and Western blot for assessing BCR-ABL activity.
Conclusions:
- An accurate and sensitive ELISA method for measuring BCR-ABL activity in Philadelphia chromosome-positive (Ph(+)) cells has been developed.
- The ELISA assay is comparable in sensitivity to existing in vitro BCR-ABL assessment techniques.
- The developed ELISA method has the potential for adaptation to high-throughput screening applications.
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