Development of the method for detection of antigen-specific lymphocytes and antigen receptors using cell-microarray

淋巴细胞表达抗原特异性受体，抗原与受体结合后启动淋巴细胞活化和分化。淋巴细胞活化通常通过96孔板中大量培养淋巴细胞的增殖或蛋白分泌来监测。在克隆水平上，每个淋巴细胞表达具有不同特异性的抗原受体，并对抗原分子中的不同表位作出反应。当能够在克隆水平上监测淋巴细胞活化时，将获得更精确的淋巴细胞活化信息。为此，我们一直在开发在克隆水平上检测抗原特异性淋巴细胞的系统。淋巴细胞装载钙指示剂荧光探针fluo-4，并添加到含有约2 × 10^5微孔阵列的硅芯片（2 cm × 2 cm）上，其大小正好适合单个淋巴细胞。除去未进入微孔的残余细胞后，70 - 90%的微孔中含有单个淋巴细胞。激活前淋巴细胞的荧光由扫描仪监测。然后用抗原等刺激物激活淋巴细胞，扫描仪监测淋巴细胞的荧光。通过比较激活前后的荧光，利用微操作仪检测并回收芯片上的活化淋巴细胞，从单个淋巴细胞中回收抗原受体cDNA。利用该系统检测抗原应答的人b淋巴细胞，获得抗原特异性抗体。该系统操作简单，可用于抗体药物的研制。它还将用于检测抗原特异性t淋巴细胞，并在基础研究和临床应用中研究淋巴细胞活化

Lymphocytes express antigen-specific receptors and binding of antigen to the receptors initiates lymphocyte-activation as well as differentiation. Lymphocyte-activation is usually monitored by the proliferation or the protein-secretion of bulk culture of lymphocytes in 96 well plates. At the clonal level, each lymphocyte expresses antigen-receptors with distinct specificity and responds to distinct epitopes in an antigenic molecule. When lymphocyte-activation could be monitored at the clonal level, more precise information on lymphocyte-activation will be obtained. To that end, we have been developing the system for detecting antigen-specific lymphocytes at the clonal level. Lymphocytes are loaded with fluorescent probe of calcium indicator, fluo-4, and added to a silicon chip (2 cm x 2 cm) containing array of about 2 x 10^5 microwells of which size is just fit for a single lymphocyte. After removing the residual cells that do not enter microwells, 70 to 90% of wells contain single lymphocytes. Fluorescence of lymphocytes before activation is monitored by a scanner. Then lymphocytes are activated with stimulants such as antigens and fluorescence of lymphocytes is monitored by the scanner. By comparing the fluorescence before and after activation, activated lymphocytes are detected and recovered from the chip using micromanipulator, and cDNA of antigen-receptors is recovered from a single lymphocyte. By using this system, we detected antigen-responding human B-lymphocytes and obtained antigen-specific antibodies. This system is simple and useful to develop antibody medicine. It will be also used to detect antigen-specific T-lymphocytes and useful to study lymphocyte-activation in basic research as well as clinical application