一种基于人体发热机理设计的新热原检测方法研究

目的: 针对现有热原检测法存在的问题,研究建立体外混合新鲜人全血热原检测法。 方法: 本试验用来源于5个不同个体的混合新鲜人全血模拟人体,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相关内热原(主要为细胞因子,如IL-1β、IL-6、TNF-α)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本试验热原刺激全血分泌的细胞因子中,IL-1β、IL-6较TNF-α更灵敏,更适用于检测热原;②新鲜人全血—IL-1β、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;③新鲜人全血—IL-1β、IL-6法的最佳孵育时间为17~24 h,所用全血应37 ℃保存,2 h内使用;④新鲜人全血—IL-1β、IL-6法对细菌内毒素最低检测限分别为0.05 EU·mL^-1和0.1 EU·mL^-1,两检测指标分别在LPS(0.05~1.6,0.1~1.6 EU·mL^-1)范围内与LPS浓度间均有较好的量效关系;⑤初步结果表明全血法可应用于生物制品 、中药注射液(如双黄莲注射液)及化药注射剂(如注射用甲氨蝶呤)的热原检测中。 结论: 体外混合新鲜人全血热原检测法具有对热原的检测谱宽、灵敏度高、应用范围较广等特点,可将该方法作为现有热原检测法(家兔法、细菌内毒素法)的补充方法对其做进一步论证研究。

Objective: To study a novel pyrogen test using pooled fresh human whole blood to compensate shortcomings of conventional pyrogen tests. Method: In this novel pyrogen test,pooled fresh human whole blood (from five donors) was used to establish a model of human body,and was incubated with standard pyrogen sample (LPS) and test solution respectively,then the contents of proinflammatory cytokines (e.g.IL-1β,IL-6,TNF-α) in incubation were determined and compared to evaluate the pyrogen activity and the contaminant of samples by ELISA. Results: ① This assay indicated that IL-1β and IL-6 were more sensitive than TNF-α in detecting pyrogens.②The human whole blood tests (WBT)—IL-1β and WBT—IL-6 could detect pyrogens from gram-negative bacterial (LPS),gram-positive bacterial (LTA),and fungi (zymosan),which were more sensitive than rabbit method.③ Taking IL-1β and IL-6 as indicators,a time period of 17-24 h was recommended for incubation;the blood should be stored at 37 ℃ and used in 2 h.④ The detection limit of IL-1β and IL-6 in fresh human whole blood to LPS was 0.05 EU·mL^-1 (IL-1β) and 0.1 EU·mL^-1 (IL-6),respectively;and the IL-1β and IL-6 secretions had good dose-dependent relation with LPS in the range of 0.05-1.6 EU·mL^-1 and 0.1-1.6 EU·mL^-1,respectively.⑤ According to primary results,the novel pyrogen tests could be widely applied to biological products,e.g.Recombinant Human Interferon α1b for Injection,Recombinant Human Granulocyte Colony-stimulating Factor Injection,Human Immunoglobulin (pH 4) for Intravenous Injection,Rabies Vaccine (Vero cell) for Human Use,Freeze-dried,Chinese traditional medicine (Shuanghuanglian Injection) and chemical drugs (methotrexate for injection). Conclusion: The whole blood test,which had a wide detection spectrum and was sensitive and widely applicable,should get further validation as the supplement of conventional pyrogen tests.