ELISA法测定微生物谷氨酰胺转氨酶残留量

目的：建立微生物谷氨酰胺转氨酶（microbial transglutaminase，MTGase）残留量检测的酶联免疫吸附法（ELISA法），并进行方法学验证，用于MTGase催化偶联的生物制品的质量控制。方法：利用MTGase单抗和MTGase兔多抗，建立了MTGase的双抗体夹心ELISA方法，并验证了该方法的定量限、精密度和准确度、稀释可靠性、稳定性和专属性等，对3批PEG-IFNα2a原液进行重复测定。结果：该方法的定量限为0.165~10 ng·mL^-1，在0.05~40 ng·mL^-1范围内拟合度高，R²>0.99，板内和板间精密度和准确度均在（±）15%以内；样品经不同倍数稀释后，回归计算样品的准确度在±15%以内，精密度小于15%；样品室温放置2 h或冻融1次和3次，结果显示质控样品采用该方法检测的精密度均小于15%，回收率为80%~120%；应用该方法在不同稀释倍数的PEG-IFNα2a原液中等量添加5 ng·mL^-1质控样品，结果显示加标样品回收率为97.7%~101.1%。3批PEG-IFNα2a原液MTGase残留量重复测定的板内和板间精密度均在15%以内，MTGase的残留量均低于总蛋白含量的0.01%。结论：ELISA法可用于生物制品中MTGase残留量的检定。

Objective:To establish an enzyme-linked immunosorbent assay(ELISA)for the determination of residual microbial transglutaminase(MTGase)for the quality control of the biologics which are catalyzed conjugation by MTGase.Methods:A double antibody sandwich-ELISA detection method using MTGase monoclonal antibody and MTGase rabbit polyclonal antibody was established for quantitative detection of MTGase.We had verified the limit of quantitation(LOQ),precision and accuracy,dilution reliability,stability,and specificity of the method. The method was used for repeated determination of residual MTGase in three batches of PEG-IFNα2a bulk. Results:The detection LOQ of the method was 0.165-10 ng·mL^-1,the ELISA method for residual MTGase had good linearity(R²>0.99) in the concentration range of 0.05-40.0 ng·mL^-1,intra-and inter-assay precision was less than 15% and the accuracy varied between -15% and 15%;Using the method to analyze different times dilution QC samples,the precision and accuracy were both less than(±)15%;Leaving the samples at room temperature for 2 hours or freeze-thaw 1 and 3 times,the results showed that the detection precision of reference samples was less than 15% and recoveries were between 80% and 120%. The PEG-IFNα2a solution with differernt dilution times was added to the 5 ng·mL^-1 reference solution,the average recoveries of standard addition were between 97.7% and 101.1%. The method was used for the determination of residual MTGase in three batches of PEG-IFNα2a bulk,the results indicated that the intra-and inter-assay precisions were less than 15%,and the residual MTGase content was lower than 0.01% of the total protein content. Conclusion:The ELISA method can be used for the determination of residual MTGase in biologics.