SenGupta S, Petsche D, Gelfand E W, Chechik B E
J Immunol Methods. 1985 Jun 25;80(2):155-62. doi: 10.1016/0022-1759(85)90017-1.
The purpose of this study was to develop a flow cytometric method for the detection of adenosine deaminase (ADA) in a single cell suspension of mononuclear cells. Anti-human ADA antibody was purified by affinity chromatography on a column of Sepharose 4B to which calf ADA was covalently linked. This antibody was used for indirect immunofluorescent staining of cells fixed in 4% paraformaldehyde. The specificity of staining was proved by substitution of anti-human ADA with normal rabbit IgG and by absorption experiments. The fluorescence profile of the cells was then analyzed by flow cytometry. Two groups of cells were studied: (a) thymocytes, tonsil cells and peripheral blood mononuclear cells (PBMC), (b) ADA-positive and ADA-deficient cell lines. In each of these populations of cells a peak of specific immunofluorescence staining for the enzyme could be easily distinguished from weak background staining of control preparations. Within each group, the cell population with higher ADA activity also displayed a greater intensity of cell fluorescence. Flow cytometry provides a means for quantitation of ADA in individual mononuclear cells.
本研究的目的是开发一种流式细胞术方法，用于检测单核细胞单细胞悬液中的腺苷脱氨酶（ADA）。抗人ADA抗体通过在共价连接小牛ADA的Sepharose 4B柱上进行亲和层析纯化。该抗体用于对固定在4%多聚甲醛中的细胞进行间接免疫荧光染色。通过用正常兔IgG替代抗人ADA以及吸收实验证明了染色的特异性。然后通过流式细胞术分析细胞的荧光图谱。研究了两组细胞：（a）胸腺细胞、扁桃体细胞和外周血单核细胞（PBMC），（b）ADA阳性和ADA缺陷细胞系。在这些细胞群体中的每一个中，酶的特异性免疫荧光染色峰都可以很容易地与对照制剂的弱背景染色区分开来。在每组中，具有较高ADA活性的细胞群体也显示出更强的细胞荧光强度。流式细胞术为定量单个单核细胞中的ADA提供了一种方法。
