PKA含量动力学检测方法的研究与建立

目的 建立激肽释放酶原激活剂(prekallikrein activator,PKA)含量的动力学检测方法,并对该方法进行验证.方法 对比不同样品稀释缓冲液的pH、离子强度,各步骤的孵育时间、孵育温度等因素,确定检测方法的最适条件.并对该方法的准确度、专属性、精密度、线性、稳定性和耐用性进行方法学验证.结果 确定以0....

Establish20ment of kinetic detection method for PKA content

Objective To develop and verify a kinetic method for the determination of prekallikrein activator(PKA) content. Methods The optimal reaction conditions were determined by comparing the factors of pH and ionic strength of different sample dilution buffers, incubation time of each procedure, and incubation temperature. The accuracy, specificity, precision, linearity, stability and durability of the method were validated. Results The sample was diluted with 0.05 mol/L Tris-HCl buffer(pH8.5, containing 0.15 mol/L NaCl) and incubated by prekallikrein(PK) at 37℃ for 20 min. After that, the substrate S-2302 was added. Within 10 min before the measurement, the absorbance change rate reached △A405/min. The validation results indicated that the linear range of the method was(0.5~4.0)IU/mL, while the recovery of calibration standard was 96.9%~103.7% with the R2 value more than 0.99. The specificity test showed that human serum albumin, excipients of intravenous human immunoglobulin(pH4), low pH and protein content had no significant effect on the detection of PKA, The recovery rates of standard sample solution in the specificity experiment were 98.0%(0.9% sodium chloride solution), 95.3%(0.46% sodium caprylate solution), 96.7%(10% maltose solution, pH4.0), 94.0%(20%BSA),and 94.0%(5%BSA, pH4.0),respectively. The accuracy and precision of the method can meet requirements in the range between 0.5 and 4.0 IU/mL. The inter-batch recovery rate of quality control samples were between 96.4%~109.5% with the coefficients of variation(CV) between 0.2%~6.9%, while the intra-batch recovery rate were between 101.5%~102.9% with the CV between 2.6%~5.9%. The linearity, accuracy and precision of the assay can meet the requirements when PK and S-2302 were placed at room temperature for less than 6 hours, with the recovery rate of quality control samples between 94.9%~109.9%. The end-point method and kinetic method were used to determine the PKA in 20 batches of human serum albumin, and the consistency showed that there was no significant difference between the two methods(P>0.05). Conclusion A kinetic method for determination of PKA content with good linearity, specificity, accuracy, precision, stability and durability has been established. Compared with the method in ChP, the new method is more convenient, accurate and rapid to determine the content of PKA in human albumin and human immunoglobulin(pH4) for intravenous injection.