中文摘要：

目的建立动物组织内P53蛋白的免疫检测分析方法,为P53生物药物的代谢分布研究提供检测手段。方法辣根过氧化物酶（HRP）标记抗P53蛋白单克隆抗体3P40,应用标记后的抗体及重组P53蛋白的检测,建立竞争性ELISA检测方法;应用此方法建立血清样品中P53蛋白标准曲线,测定腹腔注射P53后不同时间小鼠血清中P53浓度。结果利用亲和层析纯化的3P40,进行HRP标记,ELISA检测3P40HRP滴度可达1∶200 000;建立的竞争性ELISA方法可以检测PBST中P53蛋白,灵敏度达30 ng/ml,日内精密度较高,相对标准偏差（RSD）小于5%,体现较好的重复性;分别应用正常小鼠血清、PBST稀释的重组P53蛋白样品进行竞争性ELISA检测,结果显示小鼠血清成分对P53蛋白检测有一定的影响;应用血清制备P53蛋白的标准曲线,检测了腹腔注射P53蛋白后不同时间点小鼠血清样品中的P53蛋白浓度。结论进行了抗P53单克隆抗体3P40的HRP标记,建立了竞争性ELISA方法,可用于P53蛋白药物血药浓度检测。

英文摘要：

Objective To establish a method of competitive ELISA to measure P53 or P53-associated bio-drug level in animal tissues.It could be effective in the research of the metabolism and distribution of bio-reagents based on P53 protein.Methods Horseradish peroxidase,HRP,was used to label monoclonal antibody 3P40 against P53.Based on the specific recognition of P53 and 3P40HRP,a competitive ELISA method was established and its within-day precision was evaluated.The influence of mouse serum on the binding of P53 and 3P40HRP was determined by comparing the result from PBST control.Results HRP labeled 3P40,3P40HRP could specifically bind with P53 by ELISA with the titer of 1∶ 200,000.The competitive ELISA method had a good within-day precision and the relative standard deviation（RSD） was lower than 5%.In order to assay the influence of tissue content on the binding of 3P40HRP with P53,mouse serum was added to the binding solution.Compared with PBST,mouse serum could affect the binding signal in this assay.Using a standard curve obtained with P53 protein in serum sample,the P53 concentration in the serum of mouse ip injected with P53 protein was measured.Conclusion Monoclonal antibody against P53,3P40,was labeled with HRP.Based on P53 protein binding with 3P40HRP and then decreased the amount of 3P40HRP binding with P53 coated on 96 well plate,a competive ELISA method was established.This method could be well used to measure the P53 concentration in mouse serum.