抗PR、抗ER单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies to estrogen and progesterone receptors

目的自行研制抗人孕激素受体（ＰＲ）和抗人雌激素受体（ＥＲ）单克隆抗体（ＭｃＡｂ），以期达到可替代国外ＭｃＡｂ之目的。方法以ＰＲ氨基端基因表达蛋白产物及ＥＲＤ合成肽为抗原，制备小鼠抗ＰＲ、抗ＥＲＭｃＡｂ，采用Ｗｅｓｔｅｒｎｂｌｏｔ，免疫组化ＳＰ法，免疫荧光技术和流式细胞仪鉴定自制ＭｃＡｂ的性质。结果（１）抗ＰＲＭｃＡｂ与ＰＲ阳性乳腺癌细胞Ｔ４７Ｄ的提取物，抗ＥＲＭｃＡｂ与ＥＲ阳性之ＭＣＦ７细胞的提取物转移于硝酸纤维膜上的蛋白显示特异反应带；（２）抗ＰＲ、抗ＥＲＭｃＡｂ分别检测４０和３５例乳腺癌组织石蜡切片，染色效果与Ｚｙｍｅｄ公司生产的ＰＲ、ＥＲＭｃＡｂ相当，阴、阳性符合率均为１００％。免疫组化与ＤＣＣ法一一对应比较，符合率分别为８５０％、９１４％。（３）荧光显微镜和激光共聚焦扫描显微镜观察荧光标记阳性反应物主要位于胞核。（４）流式细胞仪检测，荧光指数（Ｆ１）＞１．０，阴性对照Ｆ１＜１．０。结论自制抗ＰＲ、抗ＥＲＭｃＡｂ特异性强，灵敏度高，结果与Ｚｙｍｅｄ公司产品相同，操作简便。

Objective Preparation of anti human progesterone receptor (PR) and estrogen receptor (ER) monoclonal antibodies (McAb). Methods Characterization of the two McAbs were detected by Western blot, immunohistochemical, immunofluorescence and fluocytometer techniques. Results (1) A specific protein belt was obtained between anti PR McAb and the extract of cell line T47D and the same result was noticed between anti ER McAb and extract from MCF 7 cell line by Western blot. (2) The PR and ER status of 40 and 35 human breast cancer were studied by immunohistochemistry (IHC) using the anti PR and ER McAb. The efficacy of the McAbs prepared was just the same as that of the McAbs purchased from Zymed company. The corresponding rate of determination for ER and PR status between IHC and DCC were 85% and 91.4%, respectively. (3) The fluorescent markers were mainly localized in the cell nuclei when detected through indirect immunofluorescence techniques. (4)FCM showed that fluorescent index was larger than 1.0, and negative control F1 was less than 1.0. Conclusion The anti PR and ER McAbs prepared in this program are specific and sensative and have the similar efficacy and quality as the Zymed PR and ER McAbs.