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

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HPV 18L1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-100 Evaluation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^-10 mol·L^-1和1.67×10^-10 mol·L^-1，HPV 16L1和HPV 18L1原液在浓度为0.625~40 μg·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HPV 18L1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 surface plasmon resonance biosensor (Biacore X-100) to detect the two K_D values of recombinant human papillomavirus types 16 and 18 bulks. Methods: HPV 16·V5 and HPV 18·J4 neutralizing monoclonal antibodies were coupled to sensor chip CM5,respectively.With the help of the control software of Biacore X-100 Kinetics/Affinity,HPV 16 L1 and HPV 18L1 bulks were diluted to several suitable concentrations respectively and 50 mmol·L^-1 NaOH was used as a regeneration solution for kinetic analysis.The data was analyzed to calculate the K_D values with fitting process by Biacore X-100 evaluation software. Results: The K_Dvalues of HPV 16 and HPV 18 bulks by Biacore were 4.844×10^-10 mol·L^-1 and 1.67×10^-10 mol·L^-1,respectively.The linear concentration of original HPV 16L1 and HPV 18L1 solutions ranged from 0.325 to 160 μg·mL^-1(for HPV16L1,Y =17.77X-0.0184,r=1.000;for HPV 18L1,Y =18.73X+5.282,r=0.9957).The RSD of precision and stability study was below 20%.The k_a value detected by Biacore had certain relevance to the EC₅₀ values of IVRP. Conclusion: The method is real-time,simple,rapid,accurate and sensitive.It can be used to detect the kinetic constants of HPV bulks.