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目的:建立大鼠血浆中重组人钙调磷酸酶B亚基(rhCNB)的测定方法,并研究其药动学特征。方法:采用酶联免疫吸附(ELISA)双抗体夹心法,将1μg/ml的rhCNB单克隆抗体mAb包板,加入待测样品,再加入rhCNB多克隆抗体pAb(稀释比例为1∶5 000)和辣根过氧化酶(HRP)标记抗免疫球蛋白G(IgG)(稀释比例为1∶10 000),以四甲基联苯胺显色,用酶标仪在450 nm波长处测定吸光度(OD值)。测定6只大鼠iv 2.5 mg/kg rhCNB后2、15、30、60、120、240、480、720 min的血药浓度,以BAPP 3.0软件计算药动学参数。结果:rhCNB检测质量浓度的线性范围为0.195~12.5 ng/ml(r~2=0.995 0),定量下限为0.195 ng/ml,准确度为97.300%~103.622%(RSD均小于7.5%,n=6);批内、批间和反复冻融3次的RSD均不大于8.5%(n=6、18、15)。该药在大鼠体内的药动学特征符合二室模型,AUC_(0-720 min)为173.038 mg·min/L、t_(1/2)为94.62 min。结论:建立的方法特异性强、灵敏度高、准确度及精密度好,可用于生物样品中rhCNB的定量检测和药动学研究。
Objective: To establish a method for the determination of recombinant human calcineurin B subunit (rhCNB) in rat plasma and to study its pharmacokinetic characteristics. Methods: The monoclonal antibody mAb of rhCNB (1μg / ml) was added to the test sample by enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method. Then the rhCNB polyclonal antibody pAb (diluted 1: 5000) Horseradish peroxidase (HRP) labeled anti-immunoglobulin G (IgG) (dilution 1:10 000), with tetramethylbenzidine color, with a microplate reader at 450 nm wavelength absorbance (OD value ). The plasma concentrations of 6 rats at 2.5, 15, 30, 60, 120, 240, 480 and 720 min after 2.5 mg / kg rhCNB were determined. The pharmacokinetic parameters were calculated by BAPP 3.0 software. Results: The linear range of rhCNB was 0.195 ~ 12.5 ng / ml (r ~ 2 = 0.995 0), the limit of quantification was 0.195 ng / ml, the accuracy was 97.300% ~ 103.622% (RSD less than 7.5%, n = 6). The RSDs of intra-lot, inter-lot and repeated freeze-thaw three times were no more than 8.5% (n = 6,18,15). The pharmacokinetics of the drug in rats was in accordance with the two-compartment model. The AUC_ (0-720 min) was 173.038 mg · min -1 and the t 1/2 was 94.62 min. Conclusion: The established method is specific, sensitive, accurate and precise, and can be used for the quantitative detection and pharmacokinetic study of rhCNB in biological samples.