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=2.910 9-0.300 1lgC,r=-0.999 8,阴性对照的t＞3 600 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) part Ⅲ (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= 2.910 9-0.300 1lgC, r=-0.999 8, negative control t＞3 600 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.
[1] LI B, YANG C, JIA G, et al. Comprehensive evaluation of the effects of long-term cryopreservation on peripheral blood mononuclear cells using flow cytometry[J]. BMC Immunol, 2022, 23(1): 30
[2] DUCAR C, SMITH D, PINZON C, et al. Benefits of a comprehensive quality program for cryopreserved PBMC covering 28 clinical trials sites utilizing an integrated, analytical web-based portal[J]. J Immunol Methods, 2014, 409: 9
[3] MELLMAN I, COUKOS G, DRANOFF G. Cancer immunotherapy comes of age[J]. Nature, 2011, 480(7378):480
[4] SUN J, GAO M, LI K, et al. Quality of cryopreserved peripheral blood mononuclear cells recovered from the hepatitis/AIDS biobank[J]. Bioperserv Biobank, 2018, 16(6):397
[5] TYAGARAJAN S, SCHMITT D, ACKER C, et al. Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor-T cell manufacture[J]. Cytotherapy, 2019, 21(12): 1198
[6] ANDERSON WB, SLAWSON RM, MAYFIELD CI. A review of drinking-water-associated endotoxin, including potential routes of human exposure[J]. Can J Microbiol, 2002, 48(7): 567
[7] EIN RJ, WOLFF SM, MCADAM KP, et al. Properties of reference Escherichia coli endotoxin and its phthalylated derivative in humans[J]. J Infect Dis, 1981, 144(4): 329
[8] CHEN H, SCHURCH CM, NOBLE K, et al. Functional comparison of PBMCs isolated by cell preparation tubes (CPT) vs. lymphoprep tubes[J]. BMC Immunol, 2020, 21(1):15
[9] BRENTIGENS RJ, DAVILA ML, RIVIERE I, et al. CD19-Targeted T cells rapidly induce molecular remissions in adults with chemotherapy refractory acute lymphoblastic leukemia[J]. Sci Transl Med, 2013, 5(177):177
[10] NALEPKA JL, GREENFIELD EM. Detection of bacterial endotoxin in human tissues[J]. Biotechniques, 2004, 37(3): 413
[11] 李树馀,梁进.微量动态显色法定量检测3种大输液的细菌内毒素[J]. 中国药师,2014,17(6):1052
LI SY, LIANG J. Application of micro-kinetic chromogenic method in endotoxin quantitative detection of three kinds of infusions[J]. China Pharm, 2014, 17(6): 1052
[12] 袁超璐,张思师,陈绎如,等.动态显色法检测脐带间充质干细胞悬液中细菌内毒素含量[J]. 药物分析杂志,2022, 42(12): 2110
YUAN CL, ZHANG SS, CHEN YR, et al. Dynamic chromogenic method for detection of bacterial endotoxin content in umbilical cord mesenchymal stem cell suspension[J]. Chin J Pharm Anal, 2022, 42(12): 2110
[13] SARZOTTI-KELSOE M, NEEDHAM LK, ROUNTREE W, et al. The Center for HIV/AIDS Vaccine Immunology (CHAVI) multi-site quality assurance program for cryopreserved human peripheral blood mononuclear cells[J]. J Immunol Methods, 2014,409: 21
[14] PETSCH D, DECKWER WD, ANSPACH FB. Proteinase K digestion of proteins improves detection of bacterial endotoxins by the limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins[J]. Anal Biochem, 1998, 259(1): 42
[15] 陈星,贺利民,刘戎.细菌内毒素及分析检测方法研究进展[J]. 分析实验室, 2023,42(9): 1242
CHEN X, HE LM, LIU R. Progress on detection of bacterial endotoxin[J]. Anal Lab, 2023,42(9): 1242
[16] SHIMIZU T, OBATA T, SONODA H, et al. The ability of endotoxin adsorption during a longer duration of direct hemoperfusion with a polymyxin B-immobilized fiber column in patients with septic shock[J]. Transfus Apher Sci, 2013, 49(3):499
[17] ANGEL S, VON BRIESEN H, OH YJ, et al. Toward optimal cryopreservation and storage for achievement of high cell recovery and maintenance of cell viability and T cell functionality[J]. Biopreserv Biobank, 2016, 14(6):539
[18] SCHNEIER M, RAZDAN S, MILLER AM, et al. Current technologies to endotoxin detection and removal for biopharmaceutical purification[J]. Biotechnol Bioeng, 2020, 117(8): 2588
[19] SU W, DING X. Methods of endotoxin detection[J]. J Lab Autom, 2015, 20(4): 354
