Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia.
BACKGROUND
The goal of this study was to optimize a cell staining procedure for flow cytometric detection of zeta-chain associated protein-70 (ZAP-70). Our specific objectives were to improve antibody selection criteria, identify a cell permeabilization procedure better tailored to ZAP-70 analysis, as well as to establish objective criteria to control antigen stability.


METHODS
Sequentially titrated 2F3.2-FITC, 1E7.2-FITC, and 1E7.2-Alexa Fluor 488 anti-ZAP-70 antibodies were used to stain normal B and T cells and Scatchard analysis was applied to calculate K(d) and B(max) values from saturation curves of specific binding. ZAP-70 staining was compared in cells permeabilized with two commercially available kits, Triton X-100, and a custom saponin procedure.


RESULTS
Normal B-cells were found to provide an excellent measure of nonspecific staining while varying ZAP-70 antibodies and concentrations. Comparing Scatchard analyses of specific T-cell binding revealed that 1E7.2-Alexa Fluor 488 had the highest binding affinity of the tested anti-ZAP-70 antibodies and was the best choice. The highest levels of ZAP-70 fluorescence occurred when cells were permeabilized using a noncommercial saponin procedure. Decrease of chronic lymphocytic leukemia cell viability correlated with diminished ZAP-70 expression; when viability was lower than 95% the percentage of bright positive samples was significantly decreased, indicating a possibility of false-negative results.


CONCLUSIONS
The efficiency and reliability of flow cytometric detection of ZAP-70 can be optimized by using Scatchard analysis to help select the most effective antibodies and antibody concentrations that maximize specific to nonspecific binding, by using a "custom" ZAP-70 permeabilization procedure, and by better controlling antigen stability by measuring cell viability.
Preobrazhensky
Sergey N
SN
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA. preobrs@aruplab.com
Bahler
David W
DW
D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D002470:Cell Survival / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006834:Hydrazines; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood* / Q000175:diagnosis* / Q000276:immunology; D018655:Lymphocyte Count / Q000295:instrumentation / Q000379:methods; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000097:blood* / Q000276:immunology
C417561:Alexa 488 hydrazide; D000906:Antibodies; D015415:Biomarkers; D006834:Hydrazines; D051746:ZAP-70 Protein-Tyrosine Kinase