可溶性PD-1酶联检测试剂盒的研制及其应用

Preparation of sPD-1 ELISA kit and its application

目的:建立人的可溶性PD-1(sPD-1)酶标检测试剂盒并探讨其检测的临床意义。方法:在已成功获得两株识别位点不同的鼠抗人PD-1分子单克隆抗体(1F2和5F10)的基础上,采用该室制备的鼠抗人PD-1分子单克隆抗体(1F2)作为包被抗体,应用本室制备的单抗5F10经生物素(biotin)标记后作为检测抗体,建立双单抗夹心的人sPD-1酶标检测方法,并对健康供血员、甲亢、血液病患者的血清中sPD-1的含量进行了检测。结果:成功研制了人sPD-1酶联检测试剂盒,其灵敏度为200.00pg/ml。该试剂盒4℃放置1个月,离散度(CV%)<±5.17,回收率为95%～115%,提示该检测试剂盒具有良好的灵敏度、稳定性和准确性。用该试剂盒测得血清中sPD-1的正常值为1.103±0.240ng/ml,再生障碍性贫血患者血清中sPD-1的含量明显高于正常对照组,而甲亢和器官移植病人血清中sPD-1的含量和正常值相比没有显著差异(P<0.05)。结论:成功研制了检测人sPD-1酶标检测试剂盒,并首次发现再生障碍性贫血患者血清中sPD-1的含量较高,该试剂盒在临床病人sPD-1的检测中具有潜在的应有价值。

Objective： To establish the sPD-1 ELISA kit and to detect the amounts of sPD-1 in different diseases. Methods： Two mAbs of mouse anfi-htrman PD-1 （ 1F2 and 5F10） established by our lab were used.The 1F2 mAb was used as coating antibody and the 5F10 which recognized a different epitope was selected to be labeled by biotin. The levels of sPD-1 from serum in normal volunteers and haematology,hyperthyroidism and transplantation patients were detected by the ELISA kit. Results： sPD-1 ELISA kit was established successhtUy with the measuring threshold of 200.00 pg/ml. The sensitivity did not dechne after the kit was stored at 4 ℃ for 1 month. The level of sPD-1 in normal volunteers was 1.103± 0.204 ng/ml. The levels of sPD-1 in aplastic anemia （AA） patients were showed highly increasing compared with those of normal control. The levels of sPD-1 in other patients such as hyperthyroidism and transplantation had no obviously increasing compared with normal control. Conelusion：A specific and sensitive ELISA kit to detect hunman sPD-1 is established. The application of the kit has been performed. It is firstly found that the level of sPD-1 in AA patients is higher than in normal control.h is suggested that the ELISA kit has a potential application in detecting chnic patients of sPD-1.