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目的:研究猪全血法体外检测热原的可行性。方法:采用猪全血,与不同浓度的各类热原[细菌内毒素(LPS)、脂磷壁酸(LTA)、酵母多糖]共同孵育20 h(37℃、5%CO2),以细胞因子(IL-1β、IL-6)为检测指标,用酶联免疫法(ELISA)检测孵育体系中相应细胞因子的含量,进行猪全血-IL-1β、IL-6法体外热原检测的方法研究。结果:猪全血经一定比例稀释后(猪全血:RPMI1640为1∶3),猪全血-IL-1β、IL-6法对LPS可检测到0.25 EU獉mL-1,对LTA可检测到0.5μg獉mL-1,对酵母多糖可检测到16.0μg獉mL-1;且在该孵育体系下,猪全血-IL-1β法对LPS的最低检测限为0.125 EU獉mL-1,线性范围为0.125~0.5 EU獉mL-1,r=0.9515;猪全血-IL-6法对LPS的最低检测为0.0625 EU獉mL-1,线性范围为0.0625~0.5 EU獉mL-1,r=0.9926。结论:初步研究认为,猪全血-IL-1β、IL-6法适用于检测热原。
Objective: To study the feasibility of porcine whole blood assay for detecting pyrogen in vitro. Methods: The porcine whole blood was used to incubate with various concentrations of pyrogen [LPS, LTA, Zymosan] for 20 h (37 ℃, 5% CO2) (IL-1β, IL-6) as the detection index, the corresponding cytokines in the incubation system were detected by enzyme-linked immunosorbent assay (ELISA) the study. Results: After a certain proportion of porcine whole blood was diluted (whole blood of pigs: 1: 3 of RPMI1640), whole blood-IL-1β and IL-6 could detect 0.25 EU 獉 mL-1 for LPS, The detection limit of LPS was 0.125 EU 獉 mL-1 for porcine whole blood-IL-1β under the incubation system of 0.5 μg 獉 mL-1, and that of zymosan was 16.0 μg 獉 mL-1. The linear range was 0.125 ~ 0.5 EU 獉 mL-1, r = 0.9515. The minimum detection limit of LPS was 0.0625 EU 獉 mL-1 in porcine whole blood-IL-6 method with a linear range of 0.0625-0.5 EU 獉 mL-1 = 0.9926. Conclusion: Preliminary studies suggest that porcine whole blood-IL-1β and IL-6 are suitable for the detection of pyrogen.