目的: 建立酶联免疫吸附（ELISA）方法分别检测高浓度静注人免疫球蛋白制品（10%IVIG）中人凝血因子Ⅻ（factor Ⅻ,FⅫ）和前激肽释放酶（prekallikrein,PK）的残留量,并对该方法进行验证。方法: FⅫ、PK特异性抗体预包被在微孔板上并封闭。10%IVIG中的FⅫ、PK分别与预包被抗体结合反应,经洗涤,分别加入FⅫ、PK特异性生物素化检测抗体与结合抗体反应,经第2次洗涤后,分别加入辣根过氧化物酶、链霉亲和素-过氧化物酶反应,再加入显色剂反应后,添加终止液终止反应,置于酶标仪450 nm波长处测定吸收度。参照2020年版《中华人民共和国药典》验证检测方法,并采用经验证的方法对10%IVIG工艺中间品、成品中FⅫ、PK残留量进行测定。结果: 验证结果如下：稀释线性显示FⅫ检测方法存在基质效应,PK检测方法存在钩状效应,可分别通过稀释5~20倍、80~120倍解决;加标实验测定结果的平均回收率分别为110.3%、99.1%,RSD分别为3.6%、2.7%,该方法准确度良好;重复性测定结果的RSD分别为8.3%、7.7%;中间精密度实验的RSD分别为7.0%、9.2%;方法精密度良好;定量限精密度验证符合要求,定量限分别为1.0、1.25 ng · mL-1;该方法在线性范围内线性关系良好（r=0.999 9）。耐用性验证了孵育时间、试剂盒开启有效期以及不同批号试剂盒,结果显示方法的耐用性良好。应用该方法检测10%IVIG中FⅫ、PK残留量均维持较低水平,说明辛酸沉淀结合2步阴离子层析工艺能够较好地去除10%IVIG中的FⅫ、PK。结论: 建立的方法适用性良好,能够满足实验室对10%IVIG静注人免疫球蛋白中FⅫ、PK残留量的测定。
余雨蓉
,
刘勇
,
杨龙
,
唐良玉
,
肖春桥
,
李丹
,
菅长永
. 酶联免疫吸附法测定高浓度静注人免疫球蛋白中人凝血因子Ⅻ与前激肽释放酶的残留量*[J]. 药物分析杂志, 2025
, 45(3)
: 514
-521
.
DOI: 10.16155/j.0254-1793.2024-0235
Objective: To establish, validate and apply an enzyme-linked immunosorbent assay (ELISA) method for determination of residual human factor Ⅻ(FⅫ) and prekallikrein (PK) in high concentration human intravenous immunoglobulin (10%IVIG). Methods: Microplates were pre-coated with specific antibodies for FⅫ and PK, respectively, and then blocked. The FⅫ and PK in 10%IVIG were allowed to bind with the pre-coated antibodies on the plates. After appropriate washing steps, biotinylated detection antibodies specific for FⅫ and PK were added. Following a second washing, HRP/Streptavidin-Peroxidase conjugates were added and unbound conjugates were washed away with another washing step. After the addition of a chromogenic substrate and a stop solution, the absorbance was measured at 450 nm using a microplate reader. The detection method was validated according to the 2020 edition of Chinese Pharmacopoeia. Then the validated method was used to determine the residual levels of FⅫ and PK in process intermediates and final products 10%IVIG. Results: The dilution linearity results showed a matrix effect and a hook effect in FⅫ and PK detection methods, respectively, which could be solved by diluting 5-20 times and 80-120 times, respectively. The average recoveries in the spiked experiments were 110.3% and 99.1% for FⅫ and PK respectively, with RSD of 3.6% and 2.7%. indicating good accuracy of the method. The RSDs of the repeatability validation were 8.3%、7.7% for both methods, and the RSD of intermediate precision validation were 7.0% and 9.2% for FⅫ and PK, respectively, indicating good precision of the two methods. The limits of quantitation of the two methods were 1.0 ng · mL-1 and 1.25 ng · mL-1 respectively. Both methods exhibited good linearity (r = 0.999 9). The robustness validation, which included incubation time, the effective period of the opened reagent kit, and different batches of reagent kits, showed good robustness of the method. The residues of high concentration human immunoglobulin FⅫ and PK maintained at low levels by using the method, indicating that octanoic acid precipitation combined with two-step anionic exchange chromatography process could effectively remove FⅫ and PK in 10%IVIG. Conclusion: The detection method shows good applicability and can be used for the determination of FⅫ and PK residues in 10%IVIG.
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