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

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 (R(2) = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R(2) = 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.