人外周血单个核细胞热原检测法的研究

目的: 针对现有热原检测法存在的问题,研究建立人外周血单个核细胞热原(PBMC)检测法。 方法: 新方法依据人体发热机理设计,用人外周血单个核细胞模拟人体,将其分别与热原标准品(细菌内毒素)、受试品溶液进行孵育,通过酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测并比较其孵育体系中内源性热原白细胞介素(IL-6)的含量从而反映受试品的致热活性及热原污染情况。 结果: ①本方法孵育时间可选择17~24 h;②PBMC—IL-6法可检测多种热原 ,其对上述热原的反应总体较家兔法灵敏;③PBMC—IL-6法对细菌内毒素最低检测限为0.0125 EU·mL^-1,IL-6在LPS(0.0125~0.4 EU·mL^-1)范围内与LPS浓度间有较好的量效关系;④本试验中,PBMC—IL-6法可应用于甲型H1N1流感疫苗、双黄莲注射液、氯化钠注射液、葡萄糖注射液、甘露醇注射液、氟康唑注射液、棕榈酸帕利哌酮注射液的热原检测中;⑤本方法在4个实验室内重复性分别为100%,86.7%,91.7%,91%,在实验室间重复性分别为95.7%,90.5%,84%,86.7%,78.5%,80%;⑥PBMC—IL-6法灵敏度(即真阳性率)为90.1%(100/111),特异度(即真阴性率)为92.3%(72/78)。 结论: PBMC—IL-6法具有对热原的检测谱宽、灵敏度高、应用范围广、可靠性强等特点,作为现有热原检测方法的补充该方法应得到进一步应用。

Objective:To explore a novel pyrogen test using human peripheral blood monocytic cells (PBMC) from Chinese individuals,which improves conventional pyrogen tests.Methods:This novel pyrogen test based on the mechanism of human fever reaction uses human peripheral blood monocytic cells (PBMC) as model of body,which are incubated with standard (lipopolysaccharide,LPS),samples respectively,then compares the quantity of endogenous pyrogen interlukin 6 (IL-6) in culture of them by ELISA to reflect the pyrogen activity and contaminant of samples.Results:① Using IL-6 as indicator,17-24 h was recommended to the incubation time of PBMC test.② PBMC—IL-6 test had a wide detection spectrum to pyrogens [e.g.LPS from Gram-negative bacteria,lipoteichoic acid (LTA) from Gram-positive bacteria,zymosan from fungi],which was also more sensitive than rabbits to those pyrogens generally.③ The detection limit of PBMC—IL-6 test to LPS was 0.0125 EU·mL^-1,LPS dose dependently increased IL-6 secretion in the range of 0.0125-0.4 EU·mL^-1.④ The novel pyrogen testing could be widely applicable [e.g.biological products (H1N1 influenza vaccine),traditional Chinese medicine (shuanghuanglian injection),chemic drug (sodium chloride injection,glucose injection,mannitol injection,fluconazole injection,paliperidone palmitate injection)].⑤ The within-laboratory reproducibility of this method was 100%,86.7%,91.7%,91% in four laboratories respectively,the inter-laboratory reproducibility was 95.7%,90.5%,84%,86.7%,78.5%,80% in six pairs-wise laboratory.⑥ The sensitivity of PBMC—IL-6 test,which described the probability of correctly classifying positive samples,was 90.1% (100/111) and specificity,which described the probability of correctly classifying negative samples,was 92.3% (72/78).Conclusion:The PBMC—IL-6 test,with a wide detection spectrum to pyrogens,high sensitivity and wide application,should get further application as a supplement to conventional pyrogen tests.