PBMC悬液中细菌内毒素检测方法的建立

Establishment of bacterial endotoxin detection method for PBMC suspension

目的:建立定量检测人外周血单个核细胞(PBMC)悬液中细菌内毒素的方法,并验证检测方法的可行性。方法:参考2020年版《中华人民共和国药典》(简称《中国药典》)三部<通则1143细菌内毒素检查法>及USP<1085细菌内毒素检查法应用指导原则>,从标准曲线可靠性验证、供试品干扰初筛试验2个方面,初步建立PBMC样本细菌内毒素含量的检测方法,并用此方法对3批PBMC悬液样品的细菌内毒素进行定量检测。结果:在标准曲线可靠性验证中,得到线性回归方程:lgt=2.9109-0.3001lgC,r=-0.9998,阴性对照的t>3600 s,平行管t的RSD均小于3%,表明标准曲线建立成功;干扰初筛试验结果显示,PBMC悬液在10倍稀释时对鲎试剂检测有干扰作用,在80倍稀释时回收率接近100%,表明80倍为最佳干扰稀释倍数;3批供试品的试验结果显示,在80倍稀释下,其内毒素含量均小于限值1.0 EU·mL^(-1),RSD均≤10%,阳性回收率在80.8%~100.3%,符合2020年版《中国药典》的规定。结论:本方法能有效定量检测PBMC悬液中细菌内毒素的含量。

Objective:To establish a method for the quantitative detection of bacterial endotoxins in human peripheral blood mononuclear cell(PBMC)suspensions and to validate the feasibility of the detection method.Methods:According to the standards for photometric determination of bacterial endotoxin in General Notice 1143 of Chinese Pharmacopoeia(ChP)partⅢ(2020 edition)and 1085 of USP.A preliminary detection method of bacterial endotoxin content in PBMC suspension was established from standard curve reliability verification and primary interferential screening test.Bacterial endotoxins in three batches of PBMC suspensions were quantitatively detected by this method.Results:In the establishment of standard curve and reliability verification test,the linear regression equation was lgt=2.9109-0.3001lgC,r=-0.9998,negative control t>3600 s,repeated reaction tube RSDs were less than 3%,the standard curve was established successfully.The results of the primary interferential screening test showed that the PBMC suspension had interference on limulus test at the dilution ratio of 10 and the recovery rate was close to 100%at the dilution ratio of 80,indicating that 80-fold was the best interference dilution ratio without interference.The results of the three batches of samples showed that the endotoxin contents were all less than 1.0 EU·mL^(-1)at the dilution ratio of 80,and the RSDs were less than 10%.The positive recoveries were 80.8%-100.3%,which complied with ChP 2020.Conclusion:This method can quickly and quantitatively detect the content of endotoxin in PBMC suspension.