Antithrombin III(Grifols SA)

Interferon (IFN) is a type of crucial cytokine, capable of directly participating in the antiviral immune response of epithelial cells, mediating the natural immune response to induce the expression of Interferon Stimulated Genes (ISG), hence, swiftly establishing the body's natural immune defense line against viral infections.  Based on the structural characteristics, receptor binding, and biological activity of interferon, human interferons can be divided into three types: Type I, II, and III interferons. Their genes are located on human chromosomes 9p21-22, 12q24.1, and 19q13.13, respectively. Among the three types of human interferons, Type I interferons mainly include IFN-α, IFN-β, and other 20 subtypes, Type II interferons only consist of IFN-γ, and Type III interferons comprise four subtypes including IL-29 (IFN-λ1). Most cells infected by viruses can produce Type I interferon, the secreted IFN-α/β can bind to a common receptor on neighboring uninfected cells, and initiate a paracrine signaling cascade response. Type I and Type III interferons are considered classical antiviral interferons, with alpha interferons being the main subtype used in clinical antiviral treatment. These mainly include subtypes like alpha-1b, alpha-2b, alpha-2a, etc. Recent studies indicate that Type III interferons have a significant relationship with mucosal immunity, while IFN-gamma also exhibits strong virus inhibitory characteristics. Regulatory Mechanisms of Interferons● Classic JAK-STAT signaling pathway The signaling pathway triggered within the cell after type I and II receptors bind to their respective IFNs is very similar, which is the JAK-STAT signaling pathway. Interferon receptors and Type I & II interferons activate the classic JAK-STAT pathwayType I IFN binding to the IFN-α/β receptor can phosphorylate and activate two members of the JAK family: TYK2 (Tyrosine Kinase 2) and JAK1 (Janus Kinase 1). These kinases further phosphorylate STAT1α (STAT91), STAT1β (STAT84), and STAT2 (STAT113). These three activated STATs dissociate from the receptor and bind to the intracellular protein p48, and then this complex enters the cell nucleus and directly binds to the upstream regulatory region (ISREs) of IFN-sensitive genes, thereby regulating the expression of specific genes. Typically, type I and III interferon signals form STAT1-2 heterodimers and ISGF3 complexes by activating TYK2 and JAK1, while type II interferon signals induce the formation of STAT1 homodimers by activating JAK1 and JAK2.  ● PI3K/AKT/mTOR signaling pathway In addition to activating the JAK–STAT signaling pathway, interferons also have an activating effect on the PI3K/AKT/mTOR signaling pathway. The activating effect of Type I Interferon on the PI3K/AKT/mTOR pathwayThe activation of the PI3K/AKT/mTOR pathway depends on JAK1/TYK2, which phosphorylates IRS1 (insulin receptor substrate 1) and IRS2 (insulin receptor substrate 2). The phosphorylation of IRS1 and IRS2 leads to the activation of PI3K, which subsequently activates the target of mammalian mTORC1 (rapamycin complex 1). MTORC1 activates RPS6kB (ribosomal protein S6 kinase, also known as p70s6 kinase), then phosphorylates RPS6 (ribosomal protein S6), leading to the initiation of mRNA translation. Furthermore, mTORC1 also regulates the phosphorylation of EIF4EBP1 (eukaryotic initiation factor 4E binding protein 1), the phosphorylation of EIF4EBP1 leads to the inactivation of EIF4EBP1, causing the dissociation of EIF4E (eukaryotic translation initiation factor E), thereby initiating the translation of dependent mRNA. Type I interferons also activate mTORC2 (rapamycin-insensitive complex 2), which is necessary for the phosphorylation of RPS6KB, RPS6, and EIF4BP1. Biological Activities of Type III Interferons1) Antiviral Function Type I interferon is the key antiviral defense and modulation factor in the immune system. On the one hand, it directly activates immune cells; on the other hand, it can indirectly inhibit the replication process of viruses. It can also activate natural killer cells and macrophages, thereby promoting the activation of dendritic cells, while at the same time inducing CD4+ subtype T lymphocytes and T helper cells to produce a large amount of type I interferon on distinct effect cells, protecting CD8+ cells and preventing induced antibody cell death.  2) Antibacterial Function The antibacterial function of interferons is mainly manifested in IFN-γ. IFN-γ can decrease bacterial iron supply by down-regulating transferrin receptor, or inhibit intracellular bacterial propagation directly by inducing production of endogenous NO. It can also increase the phagocytic body of monocytes and macrophages, lysosome, to dissolve bacteria. By these means, it achieves the function of eliminating bacteria. 3) Anti-parasitic Activity The anti-parasitic activity of interferon is primarily manifested in IFN-γ. IFN-γ can activate macrophages (Mφ), and the activated Mφ can express high levels of inducible nitric oxide synthase (iNOS) which catalyzes the production of NO from L-arginine. NO has inhibitory and killing effects on inoculated pathogens. 4) Involvement in Immune Regulation IFN-γ can affect the expression of the Fc receptor for IgG, which facilitates macrophages' phagocytosis of antigens, NK cells' lysis of target cells and the activation of T and B lymphocytes, enhancing the body's immune response. IFN-γ can increase the expression of MHC class II molecules on the surface of macrophages, enhancing their antigen presentation capabilities. 5) Antitumor Activity IFN-γ is produced by T lymphocytes stimulated by specific antigens. It is the main macrophage-stimulating factor in the body, it resists acid, can activate effector cells, enhances the activity of natural killer cells, macrophages, and tumor-infiltrating lymphocytes, promotes monocyte circulation, increases the expression of antigens and antibodies on the surface of immune cells, stimulates the production of cellular factors such as IL-2, tumor necrosis factor, interferon-α, etc. It inhibits tumor cell division and induces the production of antiviral proteins.

中文摘要目的    建立和验证全自动凝血分析仪检测人抗凝血酶Ⅲ（human antithrombin Ⅲ，AT-Ⅲ）活性的方法。方法    参照全自动凝血分析仪操作说明书进行AT-Ⅲ活性的检测，并对方法的线性、检测范围、精密度和准确度进行方法学验证。结果    全自动凝血分析仪检测AT-Ⅲ活性的方法稳定，仪器自动运行标准曲线的决定系数均>0.980，定标血浆标准曲线线性范围为0.12～1.23 IU/ml，定标血浆高、中、低值和定量下限稀释样品的效价回收率均在80%～120%范围内，且高、中、低值效价回收率的相对标准偏差（relative standard deviation，RSD）均<15%，定量下限稀释样品的效价回收率的RSD<20%，供试品的批内精密度RSD均<5%，批间精密度RSD均<10%，对3批AT-Ⅲ制品及其中间品进行活性检测，每批样品分别测定3次的平均RSD均＜5%。结论    全自动凝血分析仪检测AT-Ⅲ活性的方法具有较好的线性、检测范围、准确度和精密度，可用于AT-Ⅲ生产工艺中活性的检测。正文       人抗凝血酶Ⅲ（human antithrombin Ⅲ，AT-Ⅲ）是人体内一种重要的丝氨酸蛋白酶抑制剂，也是血液中最重要的抗凝血因子，主要抑制人血液中的凝血酶Ⅱa和凝血因子Ⅹa（factor Ⅹa，FⅩa）的活性[1-3]。AT-Ⅲ能与凝血酶或一些丝氨酸蛋白酶抑制剂按1∶1反应形成复合物，这种抗凝血酶-凝血酶复合物能够快速被肝脏细胞清除，在血液中的半衰期通常不超过5 min，可有效阻止血栓的形成。在肝素存在的情况下，AT-Ⅲ抑制凝血酶的活性增强1 000倍以上。AT-Ⅲ的主要生物学功能是抗凝血作用[4-7]。目前，AT-Ⅲ在国内还没有产品上市，因此中国药典2020年版尚未收录AT-Ⅲ的活性检测方法。本研究采用以AT-Ⅲ与肝素形成的复合物抑制凝血酶为基础的动力学方法，即AT-Ⅲ在适量肝素存在的情况下与FⅩa过量结合，剩余的FⅩa与FⅩa发色底物反应显色，检测波长405 nm处吸光度值（A405），建立AT-Ⅲ活性值与A450之间的回归曲线，依此检测人AT-Ⅲ生产工艺中AT-Ⅲ的活性，并对该检测方法的线性及检测范围、精密度和准确度进行方法学验证。1材料与方法1.1样品  AT-Ⅲ制品（批号201808004、201808005、201808006）、AT-Ⅲ原液（批号B20180608、B20180618、B20180627）、组分Ⅳ沉淀滤液（批号F20180608、F20180618、F20180627）、凝胶过滤亲和层析解离液（批号D20180608、D20180618、D20180627） 、肝素亲和层析解离液（批号H20180608、H20180618、H20180627）均由兰州生物制品研究所有限责任公司第四研究室制备。1.2主要试剂及仪器定标血浆（批号：315171，复溶后AT-Ⅲ标示效价为 0.92 IU/ml）购自美国Thermo公司，AT-Ⅲ测定试剂盒（批号：F1600679P1）购自法国HYPHEN BioMed公司，稀释液（批号：N1142958，规格：100 ml/瓶）、清洗液（批号：N0248417，规格：500 ml/瓶）、样品杯（批号：0005575100）、样品盘（批号：0006800000）均购自美国Instrumentation Laboratory公司；ACL7000全自动凝血分析仪购自美国贝克曼公司。1.3AT-Ⅲ活性检测方法的建立将稀释液放在全自动凝血分析仪样品盘的Dil位，依次将AT-Ⅲ测定试剂盒里的FⅩa及底物放在反应盘2和3位；复溶后的定标血浆放入样品盘的pool位，用于制定标准曲线；复溶后的待测AT-Ⅲ制品用稀释液稀释至1 IU/ml左右，放入样品盘的1—15位；按照AT-Ⅲ测定试剂盒说明书进行操作，记录每个样品的效价和A405。1.4方法验证由于AT-Ⅲ测定试剂盒规定的检测范围为0.10～1.50 IU/ml，因此将定标血浆分别用0.60、0.75、1.00、1.50、3.00及7.50 ml稀释液溶解，获得理论效价分别为1.53、1.23、0.92、0.61、0.31、0.12 IU/ml的稀释样品。1.4.1   线性    用1 ml注射用水充分溶解定标血浆，取0.5 ml放入样品盘的pool位，在1—12、17、18位放入14个空样品杯，将稀释液加入样品杯并置于16位，取4 ml稀释液加入4 ml样品杯并置于Dil位，运行仪器AT-Ⅲ检测程序下的CALIBRATION程序，设备自动将复溶的定标血浆再进行2倍系列稀释至3个浓度，将3个稀释度的A405和理论效价进行标准曲线的线性拟合。要求标准曲线的决定系数（R2）≥0.980。1.4.2   检测范围    将配制的6个稀释样品作为待测样品，进行样品检测程序，每个样品重复3次。将每个样品3次检测的A405均值与理论效价进行标准曲线的线性拟合，并将各个稀释样品A405均值代入该线性方程获得对应稀释样品的回算值，计算每个稀释样品的效价回收率和变异系数（coefficient of variation，CV），确认标准曲线的线性范围。效价回收率为回算值与理论效价的百分比。接受标准：标准曲线各稀释样品A405的CV≤10.0%；标准曲线的R2≥0.980，高、中、低值和正常值的效价回收率为理论效价的80%～120%，检测下限的效价回收率为理论效价的80%～120%。1.4.3 精密度1.4.3.1 批内精密度    将稀释后的3批AT-Ⅲ制品、稀释液、AT-Ⅲ活性测定试剂分别进行活性检测，每批样品重复测定9次，记录检测结果，并计算每批样品9次检测结果的相对标准偏差（relative standard deviation，RSD），接受标准：每批样品重复检测9次的RSD＜10%。1.4.3.2 批间精密度    3批AT-Ⅲ制品在3个工作日进行活性检测，每批次分别测定5次，根据每批次在3个工作日的试验结果计算RSD，接受标准：每批样品在不同时间内活性检测结果的RSD＜10%。1.4.4 准确度    将配制的高、中、低值和定量下限的定标血浆样品作为待测样品，每个样品5个复孔，计算效价回收率。接受标准：每个样品的效价回收率均为理论效价的80%～120%，高、中、低值效价回收率的RSD均＜15%，定量下限效价回收率的RSD＜20%。1.5方法的初步应用用建立的活性检测方法检测AT-Ⅲ制品和中间品各3批，每个样品重复检测3次，计算RSD，接受标准：每批样品活性检测结果的RSD＜5%。2结果2.1线性全自动凝血分析仪自动运行6次的标准曲线见表1。每个稀释度血浆浓度的CV均<10%；标准曲线的R2均> 0.980。2.2检测范围定标血浆不同稀释样品拟合曲线为一次线性方程y=1.028 5x-0.020 9，R2＝0.998，各稀释样品的回收率在95%～120%之间，标准曲线的线性范围在0.12～1.23 IU/ml之间，见表2。2.3精密度2.3.1  批内精密度    3批AT-Ⅲ制品分别重复测定9次的RSD均＜5%，见表3。2.3.2  批间精密度    3批 AT-Ⅲ制品在3个工作日进行活性检测，每批测定5次，平均RSD分别为7.35%、7.69%、9.64%，见表4。2.4准确度定标血浆高、中、低值和定量下限的效价回收率均为理论效价的80%～120%，其中高、中、低值效价回收率的RSD均＜15%，定量下限效价回收率的RSD＜20%，见表5。2.5方法的初步应用对3批AT-Ⅲ制品及其中间品进行活性检测，测定3次的平均RSD均＜5%，见表6。3讨论目前，AT-Ⅲ活性的检测方法只有欧洲药典收录的手工法，其检测原理是发色底物法，但该方法的前期准备工作繁琐，要消耗大量的时间和人力[8-11]。本研究建立了利用全自动凝血分析仪检测AT-Ⅲ活性的方法，其检测原理采用的是发色底物法，通过向样品中加入过量的FⅩa检测AT-Ⅲ的活性。在检测过程中，仪器的性能确认所用样品为定标血浆，但由于AT-Ⅲ纯化过程中涉及各种缓冲液，其中某些组分可能对检测有干扰，另外，由于目前市场上无AT-Ⅲ制剂的国际标准品，仅能用血浆标准品的标准曲线检测纯化的AT-Ⅲ样品，因此，需对两者的检测一致性进行评判。本研究检测3批AT-Ⅲ制品，每批样品重复检测9次的RSD均＜5%，3批AT-Ⅲ制品在不同日期各重复检测5次，其RSD均＜10%，表明该方法具有良好的精密度；定标血浆高、中、低值及定量下限稀释品的效价回收率均在理论效价的80%～120%范围内，且高、中、低值效价回收率的RSD均＜15%，定量下限稀释品效价回收率的RSD＜20%，表明该方法的准确性良好；全自动凝血分析仪自动运行标准曲线的R2均>0.980，标准曲线的检测范围在0.12～1.23 IU/ml之间；对3批AT-Ⅲ制品及其中间品进行活性检测，每批样品分别测定3次的平均RSD均＜5%，表明该方法可用于AT-Ⅲ生产工艺中活性的检测。本研究建立的利用全自动凝血分析仪检测AT-Ⅲ活性的方法具有操作简便、快速、准确度和精密度高等优势，且不需要过多人工操作，减少了人为操作误差，弥补了手工法的不足，具有良好的应用前景。作者付艳丽  孔涛  郭玉婷  曹扬  梁凌宇兰州生物制品研究所有限责任公司第四研究室，甘肃省疫苗工程技术研究中心，兰州 730046通信作者：梁凌宇，Email：lyliang@vacmic.com引用本文：付艳丽, 孔涛, 郭玉婷, 等. 人抗凝血酶Ⅲ生产工艺中活性检测方法的建立及初步应用 [J]. 国际生物制品学杂志, 2022, 45(4): 187-190.  DOI：10.3760/cma.j.cn311962-20211111-00073