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Biotinylation of human interleukin-2 for flow cytometry analysis of interleukin-2 receptors
1Central Research Laboratory, Ajinomoto Co., Inc., Kawasaki, Japan.
We designed a method to analyze receptors for interleukin-2 (IL-2R) using biotinylated IL-2 (b-IL-2). To optimize the condition of biotinylation of IL-2 for flow cytometry, the degree of biotinylation was controlled by monitoring the relative biotin contents in b-IL-2 with a newly developed ELISA. The b-IL-2 prepared by incubating 150 micrograms IL-2 in 150-300 micrograms/ml N-hydroxysuccinimidyl biotin retained biological activity and was appropriate for flow cytometry analysis. Positive fluorescence appeared in the IL-2R-bearing cell lines but not in those without IL-2R. This binding was inhibited by preincubation of the cells with unlabelled IL-2. The b-IL-2 bound to both low and high affinity IL-2Rs, but the binding to the latter was more intense. The advantage of this method is that expression of IL-2Rs of these two categories of affinity can be separately monitored.
We designed a method to analyze receptors for interleukin-2 (IL-2R) using biotinylated IL-2 (b-IL-2). To optimize the condition of biotinylation of IL-2 for flow cytometry, the degree of biotinylation was controlled by monitoring the relative biotin contents in b-IL-2 with a newly developed ELISA. The b-IL-2 prepared by incubating 150 micrograms IL-2 in 150-300 micrograms/ml N-hydroxysuccinimidyl biotin retained biological activity and was appropriate for flow cytometry analysis. Positive fluorescence appeared in the IL-2R-bearing cell lines but not in those without IL-2R. This binding was inhibited by preincubation of the cells with unlabelled IL-2. The b-IL-2 bound to both low and high affinity IL-2Rs, but the binding to the latter was more intense. The advantage of this method is that expression of IL-2Rs of these two categories of affinity can be separately monitored.