论文部分内容阅读
目的:建立Biacore法测定重组人乳头瘤病毒双价(16/18型)疫苗原液的动力学常数。方法:分别将HPV 16·V5中和单抗与HPV 18·J4中和单抗偶联到CM5芯片上,然后根据Biacore X-100 Kinetics/Affinity控制软件将HPV 16L1和HPV18L1原液分别稀释至适当的不同浓度,采用50 mmol·L-1氢氧化钠溶液作为再生液进行动力学分析。根据Biacore X-100Evaluation分析软件进行拟合,得到样品的动力学常数K D值。结果:Biacore法测定重组人乳头瘤病毒16型、18型原液动力学常数K D分别为4.844×10-10mol·L-1和1.67×10-10mol·L-1,HPV 16L1和HPV 18L1原液在浓度为0.625~40μg·mL-1时线性关系良好(HPV16L1:Y=17.77X-0.0184,r=1.000;HPV 18L1:Y=18.73X+5.282,r=0.9957)。HPV 16L1和HPV18L1原液精密度和稳定性试验的RSD均<20%。并且Biacore法测定的结合常数k a值和体外相对效力测定(IVRP法)的EC50值之间具有一定的相关性。结论:该方法实时、简便、快速、准确、灵敏,适用于重组人乳头瘤病毒原液的动力学分析。
OBJECTIVE: To establish a kinetic constant of Biacore method for the determination of bivalent (type 16/18) vaccine of recombinant human papillomavirus. Methods: HPV 16 · V5 neutralizing McAb and HPV 18 · J4 neutralizing monoclonal antibody were respectively coupled to CM5 chip, and then the HPV 16 L1 and HPV18 L1 stock solutions were respectively diluted to appropriate levels according to Biacore X-100 Kinetics / Affinity control software Different concentrations, using 50 mmol · L-1 sodium hydroxide solution as a regenerant for kinetic analysis. Fitting according to the Biacore X-100 Evaluation software, the KD value of the kinetic constant of the sample was obtained. Results: The kinetic constants Kp of recombinant human papillomavirus type 16 and 18 were 4.844 × 10-10 mol·L-1 and 1.67 × 10-10 mol·L-1, respectively. The concentrations of HPV16L1 and HPV18L1 in the concentration The linearity was 0.625 ~ 40μg · mL-1 (HPV16L1: Y = 17.77X-0.0184, r = 1.000; HPV 18L1: Y = 18.73X + 5.282, r = 0.9957). The RSDs for both the HPV 16 L1 and HPV 18 L1 stock precision and stability assays were <20%. And the Biacore method to determine the binding constant k a value and in vitro relative potency determination (IVRP method) EC50 between a certain correlation. Conclusion: The method is real-time, simple, rapid, accurate and sensitive and suitable for the kinetic analysis of recombinant human papillomavirus.