Quantitative detection of zeta-chain-associated protein 70 expression in chronic lymphocytic leukemia

Abstract

Overexpression of zeta-chain-associated protein 70 (ZAP-70) was recently recognized as an independent prognostic marker for the aggressive form of chronic lymphocytic leukemia (CLL). The objective of this study was to demonstrate the feasibility and implementation of quantitative detection of ZAP-70 protein in B cells to clearly distinguish patients with CLL with the aggressive form of the disease. B cells were isolated from patient blood and lysed. Released ZAP-70 protein was detected using an immunomagnetic fluorescence assay. The assay protocol was developed using Jurkat cells and recombinant ZAP-70 (rZAP-70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP-70 protein. The signal response was linear over a wide dynamic range, from 125 to 40 000 Jurkat cells per test (R² = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R² = 0.9928). The results from 20 patients with CLL correlated strongly with flow cytometry analysis. Concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the two methods was 95%. The assay reported here is a simple, reliable and reproducible method for quantitative detection of ZAP-70 in patient leukemic cells, without the need for cell fixation or permeabilization. The ZAP-70 signal was linear over a wide dynamic range, which we believe enables quantitative assessment of small changes in ZAP-70 expression over the course of the disease and in response to therapeutic intervention.

Keywords::

• Chronic lymphocytic leukemia (CLL)
• ZAP-70
• flow cytometry
• immunomagnetic fluorescence assay
• Signalyte-II spectrofluorometer

Acknowledgements

The authors thank Susan Soto in Dr. Wiestner's laboratory for assistance in the collection of CLL blood samples and Yong-Qiang Wang for assistance in preparation of PBMCs from the samples. This research was supported in part by the intramural research program of NHLBI, NIH.

Potential conflict of interest

Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.

This work was supported by a grant R44 CA094430-02 from the National Institutes of Health.