微生物学报

Fc effector function	Method	Detection principle	Application scenario	Advantages and limitations	References
ADCC	Cytoplasmic-content release assay (e.g.,⁵¹Cr, LDH, or glucose-6-phosphate dehydrogenase)	Assessment of ADCC activity by measuring intracellular components released upon target-cell lysis	High-throughput screening and historically validated method	High sensitivity and wide adoption; radioactive contamination or cumbersome labeling, with background interference	[48,72-74]
Cell viability assay	Live/dead dye-based flow cytometry to determine the fraction of apoptotic/necrotic target cells	Clinical-sample analysis and antibody functional profiling	Radio-free and simple; dye non-specific binding and cell-type variability	[75-77]
Luciferase reporter gene assay	FcγRIIIa signaling in Jurkat-NFAT-Luc-CD16 reporter cells drives luciferase expression	High-throughput functional screening for vaccine or antibody evaluation	High specificity and automation-ready; relies on specific cell line and reports FcγR signaling rather than lysis	[70-71]
Effector cell activation method	Quantify NK-cell activation markers (e.g., CD107a, IFN-γ, MIP-1β)	Clinical immune monitoring and functional validation	Enables functional profiling of effector-cell subsets; indirect ADCC readout, limited assessment of target-cell lysis	[78-80]
CDC	Cytoplasmic content release assay	Quantify released intracellular contents (e.g., ⁵¹Cr, LDH) after target-cell lysis	Functional assessment of vaccine- or antibody-elicited antibodies	Well-established and quantifiable; cannot distinguish lysis mechanism, serum background interference	[72,81]
Cell viability assay	Live/dead dye staining followed by flow cytometric quantification of target-cell apoptosis/necrosis	Cell-line-level CDC evaluation	Radio-free and convenient; slightly lower sensitivity, dye selection requires optimization	[82]
C5b-9 assay	Quantify membrane attack complex (MAC) deposition on target cells as a surrogate of CDC activity	Mechanistic studies	High specificity and clinical-sample compatibility; does not directly report cell lysis	[83]
ADCD	Bead-based fluorescence assay	Detect C3/C4 deposition on antigen-coated microbeads after complement activation	Functional assessment of vaccine- or antibody-elicited antibodies	High-throughput and highly reproducible; reports complement binding	[84]
Virus-based assay	Quantify C3/C4b deposition on antigen-displaying viral particles after complement activation	Viral vaccine functional evaluation	Closer to authentic viral structure; experimentally complex	[85]
ADCP	Bead-based fluorescence assay	Antigen-antibody-coated beads co-cultured with macrophages; phagocytic uptake quantified by fluorescence intensity	Functional assessment of vaccine- or antibody-elicited antibodies	Quantitative, easy to standardize, high-throughput capable; requires optimization of bead-conjugated antigen quality	[86-87]
Sensitive fluorescent dye labeling assay	pH-sensitive dye-labeled antigen; fluorescence activated after lysosomal uptake	Mechanistic studies	Real-time imaging of phagocytic events, high-content screening; true endocytosis reflected, intuitive imaging; costly dyes, instrument-demanding, low throughput	[88-89]

Fc effector function	Method	Detection principle	Application scenario	Advantages and limitations	References
ADCC	Cytoplasmic-content release assay (e.g.,⁵¹Cr, LDH, or glucose-6-phosphate dehydrogenase)	Assessment of ADCC activity by measuring intracellular components released upon target-cell lysis	High-throughput screening and historically validated method	High sensitivity and wide adoption; radioactive contamination or cumbersome labeling, with background interference	[48,72-74]
Cell viability assay	Live/dead dye-based flow cytometry to determine the fraction of apoptotic/necrotic target cells	Clinical-sample analysis and antibody functional profiling	Radio-free and simple; dye non-specific binding and cell-type variability	[75-77]
Luciferase reporter gene assay	FcγRIIIa signaling in Jurkat-NFAT-Luc-CD16 reporter cells drives luciferase expression	High-throughput functional screening for vaccine or antibody evaluation	High specificity and automation-ready; relies on specific cell line and reports FcγR signaling rather than lysis	[70-71]
Effector cell activation method	Quantify NK-cell activation markers (e.g., CD107a, IFN-γ, MIP-1β)	Clinical immune monitoring and functional validation	Enables functional profiling of effector-cell subsets; indirect ADCC readout, limited assessment of target-cell lysis	[78-80]
CDC	Cytoplasmic content release assay	Quantify released intracellular contents (e.g., ⁵¹Cr, LDH) after target-cell lysis	Functional assessment of vaccine- or antibody-elicited antibodies	Well-established and quantifiable; cannot distinguish lysis mechanism, serum background interference	[72,81]
Cell viability assay	Live/dead dye staining followed by flow cytometric quantification of target-cell apoptosis/necrosis	Cell-line-level CDC evaluation	Radio-free and convenient; slightly lower sensitivity, dye selection requires optimization	[82]
C5b-9 assay	Quantify membrane attack complex (MAC) deposition on target cells as a surrogate of CDC activity	Mechanistic studies	High specificity and clinical-sample compatibility; does not directly report cell lysis	[83]
ADCD	Bead-based fluorescence assay	Detect C3/C4 deposition on antigen-coated microbeads after complement activation	Functional assessment of vaccine- or antibody-elicited antibodies	High-throughput and highly reproducible; reports complement binding	[84]
Virus-based assay	Quantify C3/C4b deposition on antigen-displaying viral particles after complement activation	Viral vaccine functional evaluation	Closer to authentic viral structure; experimentally complex	[85]
ADCP	Bead-based fluorescence assay	Antigen-antibody-coated beads co-cultured with macrophages; phagocytic uptake quantified by fluorescence intensity	Functional assessment of vaccine- or antibody-elicited antibodies	Quantitative, easy to standardize, high-throughput capable; requires optimization of bead-conjugated antigen quality	[86-87]
Sensitive fluorescent dye labeling assay	pH-sensitive dye-labeled antigen; fluorescence activated after lysosomal uptake	Mechanistic studies	Real-time imaging of phagocytic events, high-content screening; true endocytosis reflected, intuitive imaging; costly dyes, instrument-demanding, low throughput	[88-89]

抗体可结晶片段效应功能及其在疫苗免疫学检测中的应用进展

PDF下载

王亚伟 , 谭文杰 ^*

微生物学报 | 综述 2026,66(1): 18-33

收起

微生物学报 | 综述 2026, 66(1): 18-33

抗体可结晶片段效应功能及其在疫苗免疫学检测中的应用进展

全屏

王亚伟, 谭文杰^*

作者信息

中国疾病预防控制中心病毒病预防控制所，国家卫生健康委员会生物安全重点实验室，传染病溯源预警与智能决策全国重点实验室，北京

Progress in crystallizable fragment effector functions and its application in the evaluation of vaccines

Yawei WANG, Wenjie TAN^*

Affiliations

NHC Key Laboratory of Biosafety, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

出版时间: 2026-01-04 doi: 10.13343/j.cnki.wsxb.20250479

文章导航

摘要

收起

抗体是疫苗发挥保护作用的核心效应分子。传统观点认为抗体主要通过中和作用实现保护，即抗体的抗原结合片段(antigen-binding fragment, Fab)结构域能够阻断病毒与宿主细胞表面受体结合，从而抑制感染。近年来研究发现，抗体的可结晶片段(crystallizable fragment, Fc)结构域在调节免疫应答中同样发挥着关键作用。Fc结构域可与效应细胞[如自然杀伤细胞(natural killer cells, NK cells)、巨噬细胞、中性粒细胞和树突状细胞(dendritic cells, DCs)]表面的Fc受体(Fc receptors, FcRs)或补体受体结合，激活多条先天免疫信号通路，并介导多种非中和性抗病毒效应。这些效应功能包括抗体依赖的细胞介导的细胞毒作用(antibody-dependent cellular cytotoxicity, ADCC)、抗体依赖性细胞吞噬作用(antibody-dependent cellular phagocytosis, ADCP)、抗体依赖性补体沉积作用(antibody-dependent complement deposition, ADCD)以及补体依赖性细胞毒作用(complement-dependent cytotoxicity, CDC)等。尽管Fc效应功能的检测相较于中和抗体滴度测定更为复杂，但其在疫苗诱导的保护效应中的重要性日益凸显。本文系统综述了抗体Fc结构域介导的免疫效应机制，探讨了其在病毒性疫苗免疫保护过程中的功能，并总结了当前主流的Fc功能检测方法，为新型病毒性疫苗的研发与免疫效果评价提供理论依据与技术参考。

关键词

抗体 / 可结晶片段效应功能 / 抗体依赖的细胞介导的细胞毒作用 / 抗体依赖性细胞吞噬作用 / 抗体依赖性补体沉积作用 / 补体依赖性细胞毒作用 / 疫苗评价

Abstract

收起

Antibodies serve as critical effector molecules in mediating vaccine-induced protection. While antibody-mediated immunity has traditionally been attributed primarily to neutralization, where the fragment antigen-binding (Fab) domain blocks viral entry by preventing the interaction between viruses and host cells, accumulating evidence underscores the pivotal role of the crystallizable fragment (Fc) domain in orchestrating broader immune responses. By interacting with Fc receptors or complement receptors on effector cells such as natural killer cells, macrophages, neutrophils, and dendritic cells, the Fc domain activates multiple innate immune pathways and elicits a spectrum of non-neutralizing antiviral effector functions. These include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and complement-dependent cytotoxicity (CDC). Although the evaluation of Fc-mediated functions is more complex than the measurement of neutralizing antibody titers, the contribution of such functions to vaccine efficacy is increasingly recognized. This review provides a comprehensive overview of Fc-mediated immune effector mechanisms, highlights their critical roles in antiviral vaccine-induced protection, and summarizes recent advances in Fc function assays, with the aim of supporting the rational design and immunogenicity evaluation of next-generation viral vaccines.

Key words

antibody / Fc effector functions / antibody-dependent cellular cytotoxicity (ADCC) / antibody-dependent cellular phagocytosis (ADCP) / antibody-dependent complement deposition (ADCD) / complement-dependent cytotoxicity (CDC) / vaccine evaluation

引用本文

王亚伟, 谭文杰. 抗体可结晶片段效应功能及其在疫苗免疫学检测中的应用进展. 微生物学报, 2026 , 66 (1) : 18 -33 . DOI: 10.13343/j.cnki.wsxb.20250479

Yawei WANG, Wenjie TAN. Progress in crystallizable fragment effector functions and its application in the evaluation of vaccines[J]. Acta Microbiologica Sinica, 2026 , 66 (1) : 18 -33 . DOI: 10.13343/j.cnki.wsxb.20250479

正文

收起

疫苗接种是人类公共卫生史上最具成本效益的干预手段之一，在天花和脊髓灰质炎等高致死性传染病的防控中发挥了决定性作用。据世界卫生组织统计，全球每年通过疫苗接种可预防200万-300万例死亡^[1]。在病毒性疫苗诱导的免疫应答中，抗体产生是介导保护作用的核心机制^[2-3]。传统疫苗免疫效果评估常以中和抗体滴度作为关键免疫指标。中和抗体通过空间位阻阻止病毒颗粒与宿主细胞表面受体结合，或干扰病毒包膜与宿主细胞膜的融合过程，从而在感染早期构建免疫屏障^[4]。麻疹疫苗和脊髓灰质炎疫苗的成功应用证实了中和抗体在预防原发感染中的核心地位^[5-6]。

近年研究发现，中和抗体水平不能完全解释某些疫苗[如流感疫苗、人类免疫缺陷病毒(human immunodeficiency virus, HIV)疫苗]的临床保护效果差异^[7-9]，这促使研究者重新审视抗体可结晶片段(crystallizable fragment, Fc)介导的非中和功能在抗病毒免疫中的重要性。抗体Fc结构域可通过与免疫细胞表面Fc受体(Fc receptors, FcRs)及补体系统相互作用，介导多种效应功能，包括抗体依赖的细胞介导的细胞毒作用(antibody-dependent cellular cytotoxicity, ADCC)、抗体依赖性补体沉积作用(antibody-dependent complement deposition, ADCD)、抗体依赖性细胞吞噬作用(antibody-dependent cellular phagocytosis, ADCP)以及补体依赖性细胞毒性作用(complement-dependent cytotoxicity, CDC)^[4,7]。这些Fc效应功能已在HIV^[8]、流感^[10]、疟疾^[11]、结核病^[12]、严重急性呼吸综合征冠状病毒2型(severe acute respiratory syndrome-coronavirus-2, SARS-CoV-2)^[13]以及猴痘病毒(monkeypox virus, MPXV)^[14-15]等多种病毒感染模型和疫苗保护效应中得到验证。以流感病毒为例，部分针对血凝素(hemagglutinin, HA)的抗体或疫苗虽然体外缺乏中和活性，但其Fc结构域与FcRs结合后可有效募集自然杀伤细胞(natural killer cell, NK cell)通过ADCC作用抑制病毒感染^[10,16]。此外，ADCD和ADCP在流感病毒清除过程中也发挥重要功能^[17-18]，提示这些Fc效应机制可能通过协同作用提供免疫保护。

疫苗免疫效果评价若仅分析中和抗体水平则难以全面反映“保护性免疫”能力。系统解析抗体Fc结构域介导的效应功能，有助于更全面理解疫苗诱导的免疫保护机制，为疫苗设计和免疫策略优化提供新思路。本文旨在系统综述抗体Fc结构域在病毒性疫苗免疫中的作用机制，重点介绍主要Fc效应功能及其检测方法的最新研究进展，以期为新一代病毒性疫苗的研发与免疫效果评价提供全面的理论基础和技术参考。

1 抗体的分子结构与功能多样性

收起

抗体(immunoglobulin, Ig)是由抗原刺激的B细胞分化为浆细胞后分泌的一类免疫球蛋白，能够通过特异性结合抗原介导体液免疫应答。经典抗体结构呈“Y”字形，由2条相同的轻链(light chain, LC)和2条相同的重链(heavy chain, HC)通过二硫键连接而成(图1A)^[19-21]。轻链由一个可变区(variable region of light chain, V_L)和一个恒定区(constant region of light chain, C_L)组成；重链则包含一个可变区(variable region of heavy chain, V_H)和3个恒定区(constant regions of heavy chain, C_H1-C_H3)。根据重链恒定区的差异及其理化性质和功能，人类抗体可分为IgG、IgA、IgM、IgD和IgE 5类^[19-21]。

从功能结构域来看，抗体可分为抗原结合片段(antigen-binding fragment, Fab)和Fc (图1A)。Fab结构域包含V_L、V_H、C_L和C_H1区域，其可变区内含互补决定区(complementarity-determining regions, CDRs)，负责抗原特异性识别；Fc结构域由C_H2和C_H3构成，能与FcRs或补体受体(complement receptors, CRs)结合以激活下游免疫应答^[19]。以IgG为例，其Fc段主要与Fcγ受体家族相互作用，包括高亲和力的CD64(FcγRI)、中低亲和力的CD32(FcγRII)和CD16(FcγRIII)；其中，FcγRIIb因胞内段含免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibition motif, ITIM)，可抑制其他激活型FcγR介导的信号通路(图1B)^[20]。此外，IgG亚型对FcγR的亲和力也存在差异，IgG1和IgG3与FcγR的结合能力显著强于IgG2和IgG4 (图1B)^[21]。除IgG外，其他抗体类型也通过其特异性FcRs介导免疫功能，如IgA通过FcαRI(CD89)、IgE通过FcεRI参与信号转导^[22-23]。

抗体介导的Fc效应功能的类型受FcRs固有特性及其在不同免疫细胞中的表达模式的影响^[24-25]。免疫效应细胞根据发育来源与功能特性可分为固有免疫细胞(如吞噬细胞、NK细胞等)和适应性免疫细胞(如T细胞和B细胞)。Fc结构域主要通过与固有免疫细胞表面的FcRs结合，激活快速且非特异性的免疫防御反应^[25]。不同固有免疫细胞可按介导的效应机制进一步划分为多个功能亚群。例如，吞噬细胞(如巨噬细胞和中性粒细胞)主要通过清除病原体发挥免疫防御功能：巨噬细胞高表达FcγRI(CD64)和FcγRIIa(CD32a)，可通过ADCP清除被抗体包被的病原体，同时分泌白细胞介素(interleukin, IL)-1β、肿瘤坏死因子(tumor necrosis factor, TNF)-α等促炎因子增强局部免疫应答^[26]；中性粒细胞主要表达FcγRIIIb(CD16b)，通过诱导中性粒细胞胞外诱捕网形成(neutrophil extracellular trap formation, NETosis)释放和产生活性氧(reactive oxygen species, ROS)，在抗真菌免疫中起重要作用^[27]。NK细胞组成性表达FcγRIIIa(CD16a)能识别被抗体结合的病毒感染细胞，并通过释放穿孔素(perforin)和颗粒酶(granzyme)介导ADCC^[28-29]。树突状细胞依赖FcγRIIa(CD32a)摄取免疫复合物，通过主要组织相容性复合体(major histocompatibility complex, MHC)-I和MHC-II分子进行抗原交叉提呈，激活CD8⁺和CD4⁺ T细胞，并上调CD80、程序性死亡受体配体1(programmed cell death-ligand 1, PD-L1)等共刺激及免疫调节分子，诱导T细胞向Th1或Th2亚群分化^[30]。

除FcRs外，抗原呈递细胞(antigen-presenting cells, APCs)表面的CRs也在免疫效应中发挥关键作用。补体蛋白C3b和C4b在C1q介导下沉积于免疫复合物表面，可与中性粒细胞、单核细胞和DCs等细胞表面的CRs结合，诱导调理作用并促进抗原呈递，从而增强T细胞应答^[31]。此外，C3b及其衍生物iC3b、C3dg和C3d可与B细胞表面的CRs结合，促进B细胞向生发中心迁移、增强其存活能力，进而促进记忆B细胞生成并增强体液免疫应答^[32-33]。

FcRs的多样性使抗体Fc结构域具备动态调控免疫应答的能力。Fc结构域识别病原体后通过与不同FcRs结合启动免疫信号级联反应，介导ADCC、ADCP、ADCD和CDC等多种免疫效应^[7,34-35]。这些Fc介导的效应功能不仅在抗病毒免疫中发挥关键作用，还参与感染后炎症反应、免疫稳态维持及免疫病理过程的调控。综上所述，FcRs的结构多样性与组织特异性表达构成了不同抗体亚型协同调控免疫反应的机制基础，决定了Fc结构域介导的固有免疫应答的广度与强度。

2 抗体Fc结构域介导的免疫效应功能及检测方法

收起

抗体在抗病毒免疫中通过多种机制发挥作用。其Fc结构域可介导抗体与免疫细胞表面FcRs或CRs相互作用，启动一系列非中和性抗病毒免疫效应。目前研究已明确的Fc效应功能包括ADCC、CDC、ADCD以及ADCP等。

2.1 抗体依赖的细胞介导的细胞毒性作用(ADCC)

ADCC是指抗体的Fab段特异性识别病毒感染细胞表面抗原，并通过其Fc段与NK细胞、巨噬细胞、中性粒细胞等效应细胞表面的FcγRIIIa(CD16a)结合，从而激活效应细胞释放穿孔素与颗粒酶等细胞毒性物质，最终导致靶细胞裂解并伴随炎性细胞因子释放的过程^[29,36] (图2A)。在ADCC效应中，NK细胞是最主要的效应细胞，其特异性高表达FcγRIIIa，且胞内富含穿孔素和颗粒酶，具有快速杀伤能力且无须预先致敏的特点^[19,25]。在人类抗体亚型中，IgG1和IgG3与FcγRIIIa的亲和力最高，是介导NK细胞ADCC最有效的抗体亚型。近年研究发现，IgA和IgE可分别与其特异性受体FcαR和FcεRI结合介导免疫细胞发挥ADCC等效应功能，该发现在肿瘤免疫治疗领域展现出潜在应用价值^[37-38]。

ADCC在HIV、流感病毒、SARS-CoV-2等多种病原体感染的免疫防御中发挥重要作用^[39-42]。其作用机制既可通过抗体调理作用间接清除感染细胞，也能直接杀伤某些病原体。多项临床研究证实，HIV自然控制者体内ADCC活性抗体水平显著高于病情进展期患者^[43]；流感亚单位疫苗接种者及感染康复者体内ADCC活性的抗HA抗体水平与病毒清除及症状减轻显著相关^[44]。靶向流感病毒HA茎部的广谱中和抗体及相关疫苗，其交叉保护机制也主要依赖ADCC^[45-46]。基于上述发现，诱导ADCC功能抗体已成为新型疫苗设计的重要策略，特别是对于“广谱”或“通用”疫苗的开发^[40,45]。RV144 HIV疫苗试验中，V2区特异性IgG3抗体通过ADCC机制使感染风险显著降低^[47]。本课题组前期构建了检测甲型流感病毒M2蛋白诱导ADCC的方法，并验证了M2 mRNA疫苗免疫小鼠血清诱发的ADCC效应与疫苗保护相关^[48]；M2与NP蛋白联用时可产生对流感病毒的交叉保护^[49]。

2.2 补体依赖性细胞毒作用(CDC)和抗体依赖性补体沉积作用(ADCD)

抗体Fc结构域除可通过与先天免疫细胞表面FcRs结合介导免疫效应外，还能通过激活补体级联反应参与免疫防御。CDC是指抗体的Fab段与靶细胞膜表面的特异性抗原结合后，Fc段激活补体经典途径，形成攻膜复合物(membrane attack complex, MAC)，最终导致靶细胞裂解的过程^[50]。补体系统由40余种可溶性蛋白组成，可通过经典途径、凝集素途径和替代途径3种方式激活^[32]。其中，IgM、IgG3和IgG1等抗体亚型可有效激活经典补体途径^[20]。在经典途径中，IgM或IgG六聚体与C1q结合触发补体级联反应，生成C3转化酶(C4bC2a)，进一步将C3裂解为C3a与C3b^[50-51]。C3b可沉积于靶细胞表面，一方面参与MAC的组装，介导CDC效应导致病原体或感染细胞的裂解^[52]；另一方面C3b和C4b等补体裂解产物可作为“补体标记”共价结合于靶细胞表面，介导ADCD作用，增强巨噬细胞和树突状细胞等通过CRs介导的吞噬作用^[53]。此外，补体激活产物C3a和C5a作为重要的炎症介质，不仅能促进免疫复合物沉积并增强吞噬细胞对病原体的摄取能力，还可通过与T细胞和APCs表面的CRs相互作用调节适应性免疫应答^[53]。在感染早期，低亲和力的IgM抗体能够高效募集C1q，快速启动补体级联反应清除病原体，构成抗病毒初期的重要防线^[54]。

补体系统通过ADCD和CDC双重机制在抗病毒免疫中发挥重要作用。研究表明在补体缺陷小鼠模型中，经典、凝集素和替代3条补体激活途径对控制西尼罗病毒感染均不可或缺^[55]。此外，补体系统还参与痘苗病毒的中和过程，证实了抗体Fab结构域介导的中和作用与Fc结构域介导的补体激活功能的协同保护效应^[56-57]。在恒河猴模型中，基于刺突(spike, S)蛋白的DNA疫苗诱导的保护效果与ADCD及ADCP功能显著相关^[58]。类似地，采用Ad26载体表达S蛋白变异体的疫苗，其保护效力与ADCD水平及抗体Fc段与FcγRIIa的结合能力密切相关^[59]。这些发现为开发通过增强补体激活的新型疫苗提供了理论依据。

2.3 抗体依赖性细胞吞噬作用(ADCP)

抗体依赖性细胞吞噬作用(ADCP)是指巨噬细胞、单核细胞、DC和中性粒细胞等吞噬细胞通过FcRs识别抗原-抗体复合物或病毒感染细胞后介导的吞噬过程^[60-61]。该机制依赖于抗体Fc段与吞噬细胞表面FcRs的特异性结合，从而促进抗体包被的病毒颗粒或感染细胞的吞噬与清除(图2C)。ADCP不仅可直接清除病毒及感染细胞，还能通过增强抗原呈递和炎症因子释放进一步激活下游的适应性免疫反应^[33]。ADCP效应功能在不同吞噬细胞中表现出多样性。中性粒细胞可通过FcγRIIa、FcγRIIIb及FcαRI等受体对抗体复合物作出快速反应，执行病原体降解、NETosis以及抗原交叉呈递等功能^[27]。树突状细胞则借助FcRn依赖的pH机制摄取免疫复合物，并通过MHC I/II通路进行抗原呈递以诱导体液与细胞免疫应答^[19]。不同抗体同种型与FcRs结合可引发多种免疫效应，IgA与FcαRI结合可高效诱导NETosis^[62]，而IgG活化中性粒细胞常导致脱颗粒反应及炎性细胞因子的释放。

尽管ADCP在细菌感染防御中的作用已被广泛研究^[61,63]，但其在抗病毒感染中的作用机制尚未完全阐明。近年研究发现，ADCP在流感病毒、HIV等多种病毒感染及疫苗诱导保护中起关键作用^[61]。例如，在2019冠状病毒病(coronavirus disease 2019, COVID-19)住院患者中，S蛋白特异性抗体介导的ADCP水平与生存率呈正相关；急性期患者血清可诱导中性粒细胞NETosis反应^[64-65]；RV144 HIV疫苗人体临床试验显示，尽管疫苗诱导的V1/V2区IgG抗体不具中和活性，但其水平与感染风险下降显著相关，提示其可能通过ADCP机制发挥保护作用^[8,66-67]。Huber等^[68]通过小鼠疫苗免疫联合病毒攻击模型证实ADCP在流感病毒防护中的关键作用，其研究发现缺乏Fc受体γ链的基因敲除小鼠因无法介导ADCP而对流感病毒高度易感，且该过程与NK细胞介导的ADCC作用无关。此外，ADCP也可能参与人类重症流感感染后的恢复过程^[17,69]。

2.4 抗体Fc效应功能检测方法

抗体Fc结构域介导的免疫效应功能是疫苗研发和抗体药物评价中的重要指标。针对这些功能，研究人员已建立多种体外检测方法，通常涉及三大要素：靶细胞、效应细胞(或补体)以及特异性抗体。根据检测机制的差异，这些方法可大致分为2类：一类以靶细胞的损伤或裂解为核心指标，另一类则聚焦于效应细胞的激活状态或功能响应。

在Fc效应功能的体外评估中，靶细胞与效应细胞的选择对实验结果的生理相关性和可重复性具有关键影响。靶细胞一般为可稳定表达目标抗原的细胞系，应尽可能贴近疾病模型或临床应用场景，并便于表征与标记。抗体结合靶细胞后，其Fc结构域介导与效应细胞或补体的相互作用，进而诱导下游免疫效应的激活。不同Fc功能依赖的效应细胞类型有所不同。例如，ADCC主要依赖表达FcγRIIIa的NK细胞^[48]，而ADCP则多采用单核-巨噬细胞系[如人单核细胞白血病细胞系(THP-1)、人组织细胞淋巴瘤细胞系(U937)]或外周血单核细胞(peripheral blood mononuclear cell, PBMC)来源的原代巨噬细胞。效应细胞的来源大致可分为3类：(1) 原代细胞，如外周血中分离的NK细胞和巨噬细胞等，其受体表达谱与功能状态更贴近体内环境，适用于疫苗或治疗性抗体的体内功能评估，但其获取难度大、个体差异显著，操作相对复杂；(2) 永生化细胞系，如NK-92和THP-1等，具备良好的操作稳定性和实验重复性，适用于机制研究和高通量筛选，但其Fc受体表达可能与原生细胞不同，且功能有限；(3) 工程化报告细胞，如表达特定Fcγ受体和荧光素酶的Jurkat报告系统^[70-71]，适用于标准化和定量化的功能检测，但缺乏完整的免疫效应功能，如吞噬和脱颗粒能力。不同细胞模型在Fcγ受体表达谱、信号转导能力以及对不同抗体亚类的响应方面存在差异。因此，应根据具体的研究目标与检测需求，合理选择适用的效应细胞类型。

以ADCC检测为例，其体外模型通常由靶细胞、效应细胞与待测抗体共同组成。常见方法包括以靶细胞为核心的检测策略，如⁵¹Cr释放法、乳酸脱氢酶(lactate dehydrogenase, LDH)释放法、荧光素酶报告法和流式细胞术活/死染色，以及以效应细胞激活为核心的功能检测方法，如检测CD107a表达或胞内细胞因子的产生等^[39]。CDC的功能评估主要基于靶细胞死亡率或存活率的量化，常用方法包括荧光染料标记、LDH或三磷酸腺苷(adenosine triphosphate, ATP)释放检测等；此外，还可通过流式细胞术或酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)检测C5b-9复合物的表达以间接反映MAC导致的细胞裂解水平。ADCD的检测通常采用荧光微珠、带抗原病毒颗粒或靶细胞为载体，结合流式细胞术、ELISA和免疫荧光染色技术测定表面C3b、C4b等补体成分的沉积程度。ADCP的评估则侧重于吞噬细胞对抗原的内化效率，主要采用流式细胞术或荧光成像等技术分析巨噬细胞对荧光标记抗原的摄取能力。表1从检测原理、应用场景、优缺点等方面系统总结了ADCC、CDC、ADCD和ADCP的体外检测策略。

3 讨论

收起

在疫苗诱导的免疫保护过程中，抗体不仅能够通过中和作用阻断病毒入侵，还可借助其Fc结构域与FcRs或CRs结合，激活先天免疫系统，进而发挥多种抗病毒效应。Fc介导的效应功能涵盖ADCC、ADCD、CDC及ADCP等，这些功能不仅可直接清除病毒或杀伤感染细胞，还能通过调节炎症微环境、增强抗原呈递能力以及促进T细胞活化间接参与免疫调控。

然而，Fc结构域的免疫功能具有双重性。在介导抗病毒反应的同时，它也可能诱发抗体依赖性增强(antibody-dependent enhancement, ADE)等免疫病理效应。ADE是指在非中和或亚中和抗体水平下，抗体未能有效中和病毒，反而通过FcRs与免疫细胞结合，形成抗体-病毒复合物并介导病毒内化与扩散，甚至诱导强烈的炎症反应，导致病毒感染增强并加重疾病的现象^[45,90-91]。该机制在登革热病毒(dengue virus, DENV)感染中已得到证实^[92-93]。在DENV感染中，病毒通过Fc介导通路感染FcRs阳性但不表达病毒天然受体的细胞，形成一种特殊的致病机制^[93-94]。具有既往感染史或疫苗接种史的人群因体内存在循环抗体，更易发生ADE。因此，疫苗研发与抗体疗法需评估相关风险。此外，补体系统在病毒感染时若被过度激活也可能加重疾病。例如，在DENV和HIV感染者血清中观察到补体激活水平与疾病严重程度呈正相关^[95-96]；SARS-CoV-2感染个体的IgG可通过经典或替代途径激活补体系统，释放C3a/C5a等炎性效应因子，其水平与COVID-19严重程度密切相关^[97]。这些结果表明，Fc介导的免疫反应在抗感染防御与免疫病理损伤之间存在动态平衡。需注意的是，ADE现象与疾病本身的免疫反应高度重叠，且缺乏统一检测标准，使得临床识别与判定存在挑战。尽管在SARS-CoV、中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)和寨卡病毒等多种病毒的体外模型中观察到ADE现象，但体内直接证据不足，分子机制仍需进一步阐明^[90]。

抗体Fc功能的激活受多种因素调控，包括抗体识别的表位特征、FcγR亚型与分布、抗体同种型、Fc区糖基化修饰、抗体与FcRs或抗原的亲和力以及免疫复合物稳定性^[7,35,98]。研究表明疫苗接种与自然感染所诱导抗体的Fc糖基化模式存在差异，这会影响ADCC、ADCP等效应功能的激活效率。在疫苗应答中，中和与非中和抗体的比例及其动态变化是决定抗体效应偏向保护性还是致病性的关键因素^[7]。值得强调的是，Fc效应功能不仅在“非中和机制”中发挥作用，其还可调节中和抗体的生成，Fc-FcR可通过影响抗原呈递效率、激活滤泡辅助T细胞及B细胞亲和力成熟等途径，促进高亲和力中和抗体的生成^[99]。例如，猴痘疫苗mRNA-1769在非人类灵长类动物中可诱导与改良安卡拉株疫苗相当甚至更优的保护效果，其在抑制MPXV病毒血症的发生及减轻组织病理损伤方面的作用与中和抗体及Fc介导的效应功能密切相关^[14,100]。进一步分析表明该疫苗免疫后所产生的针对胞外病毒的特异性抗体介导的Fc效应功能(ADCC、ADCP、ADCD)与中和活性具有协同效应，二者共同阻断病毒复制并减轻病程。这表明中和抗体与Fc效应功能并非相互排斥，而是协同构建体液免疫防线。

目前，Fc效应功能在多种病毒感染中的保护作用已得到广泛验证，但其在疫苗设计与免疫评估中的应用价值直到近年才受到充分重视。系统评估不同疫苗平台、抗原结构、佐剂类型、递送系统及给药方式对Fc功能诱导的影响有助于提升疫苗设计的科学性^[35,98]。新一代疫苗研发需深入理解免疫保护机制，统筹中和作用与非中和性Fc效应功能在体液免疫中的互补作用。此外，在病毒感染过程中可能会有多种Fc效应协同发挥作用，不同的效应功能和检测方法也具有相关性。因此，在疫苗或抗体的功能性评价中，常需综合采用多种检测方法，以全面反映其Fc依赖的免疫效应特征。随着技术发展，可通过Luminex多因子检测、流式细胞术、荧光素酶报告基因系统等方法，同步评估IgG亚型分布与FcR结合活性，实现对Fc效应功能的多维分析。尽管当前Fc功能研究多基于人类临床样本，但小鼠模型仍是机制研究不可替代的工具，由于人类与小鼠在FcR表达谱、Fc-FcR结合亲和力及下游信号通路方面存在显著物种差异^[24]，因此动物实验结果解读要充分考虑外推局限。

4 结论与展望

收起

综上所述，Fc结构域介导的效应功能是疫苗诱导免疫应答中不可忽视的关键组成部分，在增强免疫保护、解释疫苗效能差异及预测个体免疫反应等方面具有应用前景。未来疫苗设计应立足于系统免疫机制，整合中和抗体与Fc效应功能，探索其调控网络与机制基础。这对于提升疫苗免疫原性与保护效果，尤其是应对新发病毒及变异株具有重要意义。

基金

收起

国家自然科学基金(82061138008)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

World Health Organization. Immunization coverage[Z]. 2020. https://wwwwhoint/news-room/fact-sheets/detail/immunization-coverage

[2]

SUN

, LING

, YANG

, SUN

. Broad neutralizing antibody-based strategies to tackle influenza[J]. Current Opinion in Virology, 2022, 53: 101207.

[3]

BURTON

. Antiviral neutralizing antibodies: from in vitro to in vivo activity[J]. Nature Reviews Immunology, 2023, 23(11): 720-734.

[4]

ZOHAR

, ALTER

. Dissecting antibody-mediated protection against SARS-CoV-2[J]. Nature Reviews Immunology, 2020, 20(7): 392-394.

[5]

HARALAMBIEVA

, KENNEDY

, OVSYANNIKOVA

, SCHAID

, POLAND

. Current perspectives in assessing humoral immunity after measles vaccination[J]. Expert Review of Vaccines, 2019, 18(1): 75-87.

[6]

BIANCHI

, LAROCCA

AMV

, BOZZI

, SPINELLI

, GERMINARIO

, TAFURI

, STEFANIZZI

. Long-term persistence of poliovirus neutralizing antibodies in the era of polio elimination: an Italian retrospective cohort study[J]. Vaccine, 2021, 39(22): 2989-2994.

[7]

ZHANG

, STACEY

, D’AGOSTINO

, TUGG

, MARZOK

, MILLER

. Beyond neutralization: Fc-dependent antibody effector functions in SARS-CoV-2 infection[J]. Nature Reviews Immunology, 2023, 23(6): 381-396.

[8]

HAYNES

, GILBERT

, McELRATH

, ZOLLA-PAZNER

, TOMARAS

, ALAM

, EVANS

, MONTEFIORI

, KARNASUTA

, SUTTHENT

, LIAO

, DeVICO

, LEWIS

, WILLIAMS

, PINTER

, FONG

, JANES

, DeCAMP

, HUANG

, RAO

, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial[J]. New England Journal of Medicine, 2012, 366(14): 1275-1286.

[9]

OHMIT

, PETRIE

, CROSS

, JOHNSON

, MONTO

. Influenza hemagglutination-inhibition antibody titer as a correlate of vaccine-induced protection[J]. The Journal of Infectious Diseases, 2011, 204(12): 1879-1885.

[10]

BOUDREAU

, ALTER

. Extra-neutralizing FcR-mediated antibody functions for a universal influenza vaccine[J]. Frontiers in Immunology, 2019, 10: 440.

[11]

SUSCOVICH

, FALLON

, DAS J, DEMAS

, CRAIN

, LINDE

, MICHELL

, NATARAJAN

, AREVALO

, BROGE

, LINNEKIN

, KULKARNI

, LU

, SLEIN

, LUEDEMANN

, MARQUETTE

, MARCH

, WEINER

, GREGORY

, COCCIA

, et al. Mapping functional humoral correlates of protection against malaria challenge following RTS, S/AS01 vaccination[J]. Science Translational Medicine, 2020, 12(553): eabb4757.

[12]

COLER

, DAY

, ELLIS

, PIAZZA

, BECKMANN

, VERGARA

, ROLF

, LU

, ALTER

, HOKEY

, JAYASHANKAR

, WALKER

, SNOWDEN

, EVANS

, GINSBERG

, REED

. The TLR-4 agonist adjuvant, GLA-SE, improves magnitude and quality of immune responses elicited by the ID93 tuberculosis vaccine: first-in-human trial[J]. NPJ Vaccines, 2018, 3: 34.

[13]

TAUZIN

, NAYRAC

, BENLARBI

, GONG

, GASSER

, BEAUDOIN-BUSSIÈRES

, BRASSARD

, LAUMAEA

, VÉZINA

, PRÉVOST

, ANAND

, BOURASSA

, GENDRON-LEPAGE

, MEDJAHED

, GOYETTE

, NIESSL

, TASTET

, GOKOOL

, MORRISSEAU

, ARLOTTO

, et al. A single dose of the SARS-CoV-2 vaccine BNT162b2 elicits Fc-mediated antibody effector functions and T cell responses[J]. Cell Host & Microbe, 2021, 29(7): 1137-1150.e6.

[14]

FREYN

, ATYEO

, EARL

, AMERICO

, CHUANG

, NATARAJAN

, FREY

, GALL

, MOLIVA

, HUNEGNAW

, ARUNKUMAR

, OGEGA

, NASIR

, SANTOS

, LEVIN

, MENI

, JORQUERA

, BENNETT

, JOHNSON

, DURNEY

, et al. An mpox virus mRNA-lipid nanoparticle vaccine confers protection against lethal orthopoxviral challenge[J]. Science Translational Medicine, 2023, 15(716): eadg3540.

[15]

MUCKER

, FREYN

, BIXLER

, CIZMECI

, ATYEO

, EARL

, NATARAJAN

, SANTOS

, FREY

, LEVIN

, MENI

, ARUNKUMAR

, STADLBAUER

, JORQUERA

, BENNETT

, JOHNSON

, HARDCASTLE

, AMERICO

, COTTER

, KOEHLER

, et al. Comparison of protection against mpox following mRNA or modified vaccinia Ankara vaccination in nonhuman Primates[J]. Cell, 2024, 187(20): 5540-5553.e10.

[16]

, TAN

, MULLARKEY

, LEE

, LAM

, KRAMMER

, HENRY

, WILSON

, ASHKAR

, PALESE

, MILLER

. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(42): 11931-11936.

[17]

ANA-SOSA-BATIZ

, VANDERVEN

, JEGASKANDA

, JOHNSTON

, ROCKMAN

, LAURIE

, BARR

, READING

, LICHTFUSS

, KENT

. Influenza-specific antibody-dependent phagocytosis[J]. PLoS One, 2016, 11(4): e0154461.

[18]

O’BRIEN

, MORRISON

, DUNDORE

, HEISE

, SCHULTZ-CHERRY

. A protective role for complement C3 protein during pandemic 2009 H1N1 and H5N1 influenza A virus infection[J]. PLoS One, 2011, 6(3): e17377.

[19]

PINCETIC

, BOURNAZOS

, DiLILLO

, MAAMARY

, WANG

, DAHAN

, FIEBIGER

, RAVETCH

. Type I and type II Fc receptors regulate innate and adaptive immunity[J]. Nature Immunology, 2014, 15(8): 707-716.

[20]

VIDARSSON

, DEKKERS

, RISPENS

. IgG subclasses and allotypes: from structure to effector functions[J]. Frontiers in Immunology, 2014, 5: 520.

[21]

BRUHNS

, IANNASCOLI

, ENGLAND

, MANCARDI

, FERNANDEZ

, JORIEUX

, DAËRON

. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses[J]. Blood, 2009, 113(16): 3716-3725.

[22]

MKADDEM S

BEN

, BENHAMOU

, MONTEIRO

. Understanding Fc receptor involvement in inflammatory diseases: from mechanisms to new therapeutic tools[J]. Frontiers in Immunology, 2019, 10: 811.

[23]

BOURNAZOS

, WOOF

, HART

, DRANSFIELD

. Functional and clinical consequences of Fc receptor polymorphic and copy number variants[J]. Clinical & Experimental Immunology, 2009, 157(2): 244-254.

[24]

BRUHNS

, JÖNSSON

. Mouse and human FcR effector functions[J]. Immunological Reviews, 2015, 268(1): 25-51.

[25]

NIMMERJAHN

, RAVETCH

. Fcgamma receptors as regulators of immune responses[J]. Nature Reviews Immunology, 2008, 8(1): 34-47.

[26]

SHAPOURI-MOGHADDAM

, MOHAMMADIAN

, VAZINI

, TAGHADOSI

, ESMAEILI

, MARDANI

, SEIFI

, MOHAMMADI

, AFSHARI

, SAHEBKAR

. Macrophage plasticity, polarization, and function in health and disease[J]. Journal of Cellular Physiology, 2018, 233(9): 6425-6440.

[27]

KOLACZKOWSKA

, KUBES

. Neutrophil recruitment and function in health and inflammation[J]. Nature Reviews Immunology, 2013, 13(3): 159-175.

[28]

CRINIER

, NARNI-MANCINELLI

, UGOLINI

, VIVIER

. SnapShot: natural killer cells[J]. Cell, 2020, 180(6): 1280-1280.e1.

[29]

De TAEYE

, BENTLAGE

AEH

, MEBIUS

, MEESTERS

, LISSENBERG-THUNNISSEN

, FALCK

, SÉNARD

, SALEHI

, WUHRER

, SCHUURMAN

, LABRIJN

, RISPENS

, VIDARSSON

. FcγR binding and ADCC activity of human IgG allotypes[J]. Frontiers in Immunology, 2020, 11: 740.

[30]

GUILLIAMS

, GINHOUX

, JAKUBZICK

, NAIK

, ONAI

, SCHRAML

, SEGURA

, TUSSIWAND

, YONA

. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny[J]. Nature Reviews Immunology, 2014, 14(8): 571-578.

[31]

OLIVEIRA

, KRETZSCHMAR

, dos SANTOS

ACM

, CAMARGO

, NISIHARA

, FARIAS

TDJ

, FRANKE

, WITTIG

, SCHMIDT

, BUSCH

, PETZL-ERLER

, BOLDT

ABW

. Complement receptor 1 (CR1, CD35) polymorphisms and soluble CR1: a proposed anti-inflammatory role to quench the fire of “fogo selvagem” Pemphigus foliaceus[J]. Frontiers in Immunology, 2019, 10: 2585.

[32]

SARMA

, WARD

. The complement system[J]. Cell and Tissue Research, 2011, 343(1): 227-235.

[33]

ROSSBACHER

, SHLOMCHIK

. The B cell receptor itself can activate complement to provide the complement receptor 1/2 ligand required to enhance B cell immune responses in vivo [J]. The Journal of Experimental Medicine, 2003, 198(4): 591-602.

[34]

GUNN

, BAI

. Building a better antibody through the Fc: advances and challenges in harnessing antibody Fc effector functions for antiviral protection[J]. Human Vaccines & Immunotherapeutics, 2021, 17(11): 4328-4344.

[35]

BOWMAN

, KAPLONEK

, McNAMARA

. Understanding Fc function for rational vaccine design against pathogens[J]. mBio, 2024, 15(1): e0303623.

[36]

SMYTH

, CRETNEY

, KELLY

, WESTWOOD

, STREET

SEA

, YAGITA

, TAKEDA

, van DOMMELEN

SLH

, DEGLI-ESPOSTI

, HAYAKAWA

. Activation of NK cell cytotoxicity[J]. Molecular Immunology, 2005, 42(4): 501-510.

[37]

LOHSE

, LOEW

, KRETSCHMER

, JANSEN

JHM

, MEYER

, BROEKE T

TEN

, RÖSNER

, DECHANT

, DERER

, KLAUSZ

, KELLNER

, SCHWANBECK

, FRENCH

, TIPTON

TRW

, CRAGG

, SCHEWE

, PEIPP

, LEUSEN

JHW

, VALERIUS

. Effector mechanisms of IgA antibodies against CD20 include recruitment of myeloid cells for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity[J]. British Journal of Haematology, 2018, 181(3): 413-417.

[38]

KARAGIANNIS

, JOSEPHS

, KARAGIANNIS

, GILBERT

, SAUL

, RUDMAN

, DODEV

, KOERS

, BLOWER

, CORRIGAN

, BEAVIL

, SPICER

, NESTLE

, GOULD

. Recombinant IgE antibodies for passive immunotherapy of solid tumours: from concept towards clinical application[J]. Cancer Immunology, Immunotherapy, 2012, 61(9): 1547-1564.

[39]

VINCKEN

, ARMENDÁRIZ-MARTÍNEZ

, RUIZ-SÁENZ

. ADCC: the rock band led by therapeutic antibodies, tumor and immune cells[J]. Frontiers in Immunology, 2025, 16: 1548292.

[40]

De VRIES

, HOSCHLER

, RIMMELZWAAN

. ADCC: an underappreciated correlate of cross-protection against influenza [J]. Frontiers in Immunology, 2023, 14: 1130725.

[41]

RICHARD

, PRÉVOST

, ALSAHAFI

, DING

, FINZI

. Impact of HIV-1 envelope conformation on ADCC responses[J]. Trends in Microbiology, 2018, 26(4): 253-265.

[42]

HAGEMANN

, RIECKEN

, JUNG

, HILDEBRANDT

, MENZEL

, BUNDERS

, FEHSE

, KOCH-NOLTE

, HEINRICH

, PEINE

, SCHULZE ZUR WIESCH

, BREHM

, ADDO

, LÜTGEHETMANN

, ALTFELD

. Natural killer cell-mediated ADCC in SARS-CoV-2-infected individuals and vaccine recipients[J]. European Journal of Immunology, 2022, 52(8): 1297-1307.

[43]

WREN

, CHUNG

, ISITMAN

, KELLEHER

, PARSONS

, AMIN

, COOPER

, INVESTIGATORS

ASC

, STRATOV

, NAVIS

, KENT

. Specific antibody-dependent cellular cytotoxicity responses associated with slow progression of HIV infection[J]. Immunology, 2013, 138(2): 116-123.

[44]

JEGASKANDA

, LUKE

, HICKMAN

, SANGSTER

, WIELAND-ALTER

, McBRIDE

, YEWDELL

, WRIGHT

, TREANOR

, ROSENBERGER

, SUBBARAO

. Generation and protective ability of influenza virus-specific antibody-dependent cellular cytotoxicity in humans elicited by vaccination, natural infection, and experimental challenge[J]. The Journal of Infectious Diseases, 2016, 214(6): 945-952.

[45]

KHURANA

, LOVING

, MANISCHEWITZ

, KING

, GAUGER

, HENNINGSON

, VINCENT

, GOLDING

. Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease[J]. Science Translational Medicine, 2013, 5(200): 200ra114.

[46]

ZHONG

, LIU

, WILSON

, HOLIDAY

, LI

, BAI

, TZENG

, STEVENS

, YORK

, LEVINE

. Antibody-dependent cell-mediated cytotoxicity to hemagglutinin of influenza a viruses after influenza vaccination in humans[J]. Open Forum Infectious Diseases, 2016, 3(2): ofw102.

[47]

HEGER

, SCHUETZ

, VASAN

. HIV vaccine efficacy trials: RV144 and beyond[J]. Advances in Experimental Medicine and Biology, 2018, 1075: 3-30.

[48]

LIANG

, GUO

, LI

, LIU

, ZHANG

, SUN

, LU

, ZHAI

, WANG

, NING

, TAN

. A novel method to assess antibody-dependent cell-mediated cytotoxicity against influenza A virus M2 in immunized murine models[J]. Biosafety and Health, 2024, 6(3): 178-185.

[49]

WANG

, HUANG

, WANG

, TAN

, RUAN

. Improving cross-protection against influenza virus using recombinant vaccinia vaccine expressing NP and M2 ectodomain tandem repeats[J]. Virologica Sinica, 2019, 34(5): 583-591.

[50]

BORDRON

, BAGACEAN

, TEMPESCUL

, BERTHOU

, BETTACCHIOLI

, HILLION

, RENAUDINEAU

. Complement system: a neglected pathway in immunotherapy[J]. Clinical Reviews in Allergy & Immunology, 2020, 58(2): 155-171.

[51]

WANG

, de JONG

, van den BREMER

ETJ

, BEURSKENS

, LABRIJN

, UGURLAR

, GROS

, SCHUURMAN

, PARREN

PWHI

, HECK

AJR

. Molecular basis of assembly and activation of complement component C1 in complex with immunoglobulin G1 and antigen[J]. Molecular Cell, 2016, 63(1): 135-145.

[52]

MERLE

, CHURCH

, FREMEAUX-BACCHI

, ROUMENINA

. Complement system part I: molecular mechanisms of activation and regulation[J]. Frontiers in Immunology, 2015, 6: 262.

[53]

WEST

, KOLEV

, KEMPER

. Complement and the regulation of T cell responses[J]. Annual Review of Immunology, 2018, 36: 309-338.

[54]

JOHN

, HUNJADI

, HAWLIN

, REISER

, KUNERT

. Interaction studies of hexameric and pentameric IgMs with serum-derived C1q and recombinant C1q mimetics[J]. Life, 2024, 14(5): 638.

[55]

MEHLHOP

, NELSON

, JOST

, GORLATOV

, JOHNSON

, FREMONT

, DIAMOND

, PIERSON

. Complement protein C1q reduces the stoichiometric threshold for antibody-mediated neutralization of west Nile virus[J]. Cell Host & Microbe, 2009, 6(4): 381-391.

[56]

BENHNIA

, McCAUSLAND

, MOYRON

, LAUDENSLAGER

, GRANGER

, RICKERT

, KORIAZOVA

, KUBO

, KATO

, CROTTY

. Vaccinia virus extracellular enveloped virion neutralization in vitro and protection in vivo depend on complement[J]. Journal of Virology, 2009, 83(3): 1201-1215.

[57]

BENHNIA

, McCAUSLAND

, LAUDENSLAGER

, GRANGER

, RICKERT

, KORIAZOVA

, TAHARA

, KUBO

, KATO

, CROTTY

. Heavily isotype-dependent protective activities of human antibodies against vaccinia virus extracellular virion antigen B5[J]. Journal of Virology, 2009, 83(23): 12355-12367.

[58]

, TOSTANOSKI

, PETER

, MERCADO

, McMAHAN

, MAHROKHIAN

, NKOLOLA

, LIU

, LI

, CHANDRASHEKAR

, MARTINEZ

, LOOS

, ATYEO

, FISCHINGER

, BURKE

, SLEIN

, CHEN

, ZUIANI

, LELIS

FJN

, TRAVERS

, et al. DNA vaccine protection against SARS-CoV-2 in Rhesus macaques [J]. Science, 2020, 369(6505): 806-811.

[59]

MERCADO

, ZAHN

, WEGMANN

, LOOS

, CHANDRASHEKAR

, YU

, LIU

, PETER

, McMAHAN

, TOSTANOSKI

, HE

, MARTINEZ

, RUTTEN

, BOS R, van MANEN

, VELLINGA

, CUSTERS

, LANGEDIJK

, KWAKS

, BAKKERS

MJG

, et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in Rhesus macaques [J]. Nature, 2020, 586(7830): 583-588.

[60]

HUANG

, BARREDA

, WORTH

, INDIK

, KIM

, CHIEN

, SCHREIBER

. Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis[J]. Journal of Leukocyte Biology, 2006, 80(6): 1553-1562.

[61]

TAY MZ, WIEHE

, POLLARA

. Antibody-dependent cellular phagocytosis in antiviral immune responses[J]. Frontiers in Immunology, 2019, 10: 332.

[62]

ALEYD

, van HOUT

MWM

, GANZEVLES

, HOEBEN

, EVERTS

, BAKEMA

, van EGMOND

. IgA enhances NETosis and release of neutrophil extracellular traps by polymorphonuclear cells via Fcα receptor I[J]. Journal of Immunology, 2014, 192(5): 2374-2383.

[63]

LEY

, HOFFMAN

, KUBES

, CASSATELLA

, ZYCHLINSKY

, HEDRICK

, CATZ

. Neutrophils: new insights and open questions[J]. Science Immunology, 2018, 3(30): eaat4579.

[64]

ZUO

, YALAVARTHI

, SHI

, GOCKMAN

, ZUO

, MADISON

, BLAIR

, WEBER

, BARNES

, EGEBLAD

, WOODS

, KANTHI

, KNIGHT

. Neutrophil extracellular traps in COVID-19[J]. JCI Insight, 2020, 5(11): e138999.

[65]

SKENDROS

, MITSIOS

, CHRYSANTHOPOULOU

, MASTELLOS

, METALLIDIS

, RAFAILIDIS

, NTINOPOULOU

, SERTARIDOU

, TSIRONIDOU

, TSIGALOU

, TEKTONIDOU

, KONSTANTINIDIS

, PAPAGORAS

, MITROULIS

, GERMANIDIS

, LAMBRIS

, RITIS

. Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis[J]. The Journal of Clinical Investigation, 2020, 130(11): 6151-6157.

[66]

CHUNG

, KUMAR

, ARNOLD

, YU

, SCHOEN

, DUNPHY

, SUSCOVICH

, FRAHM

, LINDE

, MAHAN

, HOFFNER

, STREECK

, ACKERMAN

, McELRATH

, SCHUITEMAKER

, PAU MG, BADEN

, KIM

, MICHAEL

, BAROUCH

, et al. Dissecting polyclonal vaccine-induced humoral immunity against HIV using systems serology[J]. Cell, 2015, 163(4): 988-998.

[67]

RERKS-NGARM

, PITISUTTITHUM

, NITAYAPHAN

, KAEWKUNGWAL

, CHIU

, PARIS

, PREMSRI

, NAMWAT

, de SOUZA

, ADAMS

, BENENSON

, GURUNATHAN

, TARTAGLIA

, McNEIL

, FRANCIS

, STABLEIN

, BIRX

, CHUNSUTTIWAT

, KHAMBOONRUANG

, THONGCHAROEN

, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand[J]. New England Journal of Medicine, 2009, 361(23): 2209-2220.

[68]

HUBER

, LYNCH

, BUCHER

, LE

, METZGER

. Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections[J]. The Journal of Immunology, 2001, 166(12): 7381-7388.

[69]

VANDERVEN

, LIU

, ANA-SOSA-BATIZ

, NGUYEN

, WAN

, WINES

, HOGARTH

, TILMANIS

, REYNALDI

, PARSONS

, HURT

, DAVENPORT

, KOTSIMBOS

, CHENG

, KEDZIERSKA

, ZHANG

, XU

, KENT

. Fc functional antibodies in humans with severe H7N9 and seasonal influenza[J]. JCI Insight, 2017, 2(13): e92750.

[70]

GARVIN

, STECHA

, GILDEN

, WANG

, GRAILER

, HARTNETT

, FAN

, CONG

, CHENG

. Determining ADCC activity of antibody-based therapeutic molecules using two bioluminescent reporter-based bioassays[J]. Current Protocols, 2021, 1(11): e296.

[71]

CHENG

, GARVIN

, PAGUIO

, MORAVEC

, ENGEL

, FAN

, SUROWY

. Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies[J]. Journal of Immunological Methods, 2014, 414: 69-81.

[72]

KUMAR

, NAGARAJAN

, UCHIL

. Analysis of cell viability by the lactate dehydrogenase assay[J]. Cold Spring Harbor Protocols, 2018, 2018(6): 465-468.

[73]

BRUNNER

, MAUEL

, CEROTTINI

, CHAPUIS

. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs[J]. Immunology, 1968, 14(2): 181-196.

[74]

NERI

, MARIANI

, MENEGHETTI

, CATTINI

, FACCHINI

. Calcein-acetyoxymethyl cytotoxicity assay: standardization of a method allowing additional analyses on recovered effector cells and supernatants[J]. Clinical and Diagnostic Laboratory Immunology, 2001, 8(6): 1131-1135.

[75]

GÓMEZ-ROMÁN

, FLORESE

, PATTERSON

, PENG

, VENZON

, ALDRICH

, ROBERT-GUROFF

. A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity[J]. Journal of Immunological Methods, 2006, 308(1/2): 53-67.

[76]

SHEEHY

, McDERMOTT

, FURLAN

, KLENERMAN

, NIXON

. A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis[J]. Journal of Immunological Methods, 2001, 249(1/2): 99-110.

[77]

BEAUDOIN-BUSSIÈRES

, RICHARD

, PRÉVOST

, GOYETTE

, FINZI

. A new flow cytometry assay to measure antibody-dependent cellular cytotoxicity against SARS-CoV-2 Spike-expressing cells[J]. STAR Protocols, 2021, 2(4): 100851.

[78]

ALTER

, MALENFANT

, ALTFELD

. CD107a as a functional marker for the identification of natural killer cell activity[J]. Journal of Immunological Methods, 2004, 294(1/2): 15-22.

[79]

KRZEWSKI

, GIL-KRZEWSKA

, NGUYEN

, PERUZZI

, COLIGAN

. LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity[J]. Blood, 2013, 121(23): 4672-4683.

[80]

SCHÖNBERG

, HEJAZI

, UHRBERG

. Protocol for the clonal analysis of NK cell effector functions by multi-parameter flow cytometry[J]. Methods in Molecular Biology, 2012, 903: 381-392.

[81]

DECKER

, LOHMANN-MATTHES

. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity[J]. Journal of Immunological Methods, 1988, 115(1): 61-69.

[82]

HIEMSTRA

, SANTEGOETS

KCM

, JANMAAT

, de

GOEIJ BECG

, HAGEN W

TEN

, van DOOREMALEN

, BOROSS

, van den BRAKEL

, BOSGRA

, ANDRINGA

, van KESSEL-WELMERS

, VERZIJL

, HIBBERT

, FRERICHS

, MUTIS

, van de DONK NWCJ, AHMADI

, SATIJN

, SASSER

, BREIJ

ECW

. Preclinical anti-tumour activity of HexaBody-CD38, a next-generation CD38 antibody with superior complement-dependent cytotoxic activity[J]. EBioMedicine, 2023, 93: 104663.

[83]

MOLLNES

, LEA

, FRØLAND

, HARBOE

. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex[J]. Scandinavian Journal of Immunology, 1985, 22(2): 197-202.

[84]

FISCHINGER

, FALLON

, MICHELL

, BROGE

, SUSCOVICH

, STREECK

, ALTER

. A high-throughput, bead-based, antigen-specific assay to assess the ability of antibodies to induce complement activation[J]. Journal of Immunological Methods, 2019, 473: 112630.

[85]

SPENCER

, GOLDBERG

, PANDEY

, ORDONEZ

, DUFLOO

, BARNETTE

, SUTTON

, HENDERSON

, AGNOR

, GAO

, BRUEL

, SCHWARTZ

, HAIGWOOD

, ACKERMAN

, HESSELL

. Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis[J]. Nature Communications, 2022, 13: 662.

[86]

ACKERMAN

, MOLDT

, WYATT

, DUGAST

, McANDREW

, TSOUKAS

, JOST

, BERGER

, SCIARANGHELLA

, LIU

, IRVINE

, BURTON

, ALTER

. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples[J]. Journal of Immunological Methods, 2011, 366(1/2): 8-19.

[87]

KARSTEN

, MEHTA

, SHIN

, DIEFENBACH

, SLEIN

, KARPINSKI

, IRVINE

, BROGE

, SUSCOVICH

, ALTER

. A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis[J]. Journal of Immunological Methods, 2019, 471: 46-56.

[88]

MIKSA

, KOMURA

, WU

, SHAH

, WANG

. A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester[J]. Journal of Immunological Methods, 2009, 342(1/2): 71-77.

[89]

KAMEN

, MYNENI

, LANGSDORF

, KHO E, ORDONIA

, THAKURTA

, ZHENG

, SONG

, CHUNG

. A novel method for determining antibody-dependent cellular phagocytosis[J]. Journal of Immunological Methods, 2019, 468: 55-60.

[90]

TAYLOR

, FOO SS, BRUZZONE

, DINH

, KING

NJC

, MAHALINGAM

. Fc receptors in antibody-dependent enhancement of viral infections[J]. Immunological Reviews, 2015, 268(1): 340-364.

[91]

HALSTEAD

, MAHALINGAM

, MAROVICH

, UBOL

, MOSSER

. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes[J]. The Lancet Infectious Diseases, 2010, 10(10): 712-722.

[92]

HALSTEAD

, O’ROURKE

. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody[J]. The Journal of Experimental Medicine, 1977, 146(1): 201-217.

[93]

HALSTEAD

. In vivo enhancement of dengue virus infection in Rhesus monkeys by passively transferred antibody[J]. The Journal of Infectious Diseases, 1979, 140(4): 527-533.

[94]

HALSTEAD

, O’ROURKE

, ALLISON

. Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection[J]. The Journal of Experimental Medicine, 1977, 146(1): 218-229.

[95]

NASCIMENTO

EJM

, SILVA

, CORDEIRO

, BRITO

, GIL

LHVG

, BRAGA-NETO

, MARQUES

ETA

. Alternative complement pathway deregulation is correlated with dengue severity[J]. PLoS One, 2009, 4(8): e6782.

[96]

SENALDI

, PEAKMAN

, McMANUS

, DAVIES

, DE

TEE

, VERGANI

. Activation of the complement system in human immunodeficiency virus infection: relevance of the classical pathway to pathogenesis and disease severity[J]. The Journal of Infectious Diseases, 1990, 162(6): 1227-1232.

[97]

GEORG

, ASTABURUAGA-GARCÍA

, BONAGURO

, BRUMHARD

, MICHALICK

, LIPPERT

, KOSTEVC

, GÄBEL

, SCHNEIDER

, STREITZ

, DEMICHEV

, GEMÜND

, BARONE

, TOBER-LAU

, HELBIG

, HILLUS

, PETROV

, STEIN

, DEY

, PACLIK

, et al. Complement activation induces excessive T cell cytotoxicity in severe COVID-19[J]. Cell, 2022, 185(3): 493-512.e25.

[98]

NAWAB

. Vaccinal antibodies: Fc antibody engineering to improve the antiviral antibody response and induce vaccine-like effects[J]. Human Vaccines & Immunotherapeutics, 2021, 17(12): 5532-5545.

[99]

DiLILLO

, PALESE

, WILSON

, RAVETCH

. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection[J]. The Journal of Clinical Investigation, 2016, 126(2): 605-610.

[100]

MAYER

, WESKAMM

, ADDO

. Next-generation mpox vaccines: efficacy of mRNA-1769 compared to modified vaccinia virus Ankara in non-human Primates[J]. Signal Transduction and Targeted Therapy, 2024, 9: 327.

2026年第66卷第1期

PDF下载

281

108

引用本文

BibTeX

文章信息

doi: 10.13343/j.cnki.wsxb.20250479

接收时间：2025-06-19
首发时间：2026-01-12
出版时间：2026-01-04

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2025-06-19
录用日期：2025-07-10

基金

National Natural Science Foundation of China(82061138008)

国家自然科学基金(82061138008)

作者信息

中国疾病预防控制中心病毒病预防控制所，国家卫生健康委员会生物安全重点实验室，传染病溯源预警与智能决策全国重点实验室，北京

通讯作者:

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20250479

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Fc effector function	Method	Detection principle	Application scenario	Advantages and limitations	References
ADCC	Cytoplasmic-content release assay (e.g.,⁵¹Cr, LDH, or glucose-6-phosphate dehydrogenase)	Assessment of ADCC activity by measuring intracellular components released upon target-cell lysis	High-throughput screening and historically validated method	High sensitivity and wide adoption; radioactive contamination or cumbersome labeling, with background interference	[48,72-74]
Cell viability assay	Live/dead dye-based flow cytometry to determine the fraction of apoptotic/necrotic target cells	Clinical-sample analysis and antibody functional profiling	Radio-free and simple; dye non-specific binding and cell-type variability	[75-77]
Luciferase reporter gene assay	FcγRIIIa signaling in Jurkat-NFAT-Luc-CD16 reporter cells drives luciferase expression	High-throughput functional screening for vaccine or antibody evaluation	High specificity and automation-ready; relies on specific cell line and reports FcγR signaling rather than lysis	[70-71]
Effector cell activation method	Quantify NK-cell activation markers (e.g., CD107a, IFN-γ, MIP-1β)	Clinical immune monitoring and functional validation	Enables functional profiling of effector-cell subsets; indirect ADCC readout, limited assessment of target-cell lysis	[78-80]
CDC	Cytoplasmic content release assay	Quantify released intracellular contents (e.g., ⁵¹Cr, LDH) after target-cell lysis	Functional assessment of vaccine- or antibody-elicited antibodies	Well-established and quantifiable; cannot distinguish lysis mechanism, serum background interference	[72,81]
Cell viability assay	Live/dead dye staining followed by flow cytometric quantification of target-cell apoptosis/necrosis	Cell-line-level CDC evaluation	Radio-free and convenient; slightly lower sensitivity, dye selection requires optimization	[82]
C5b-9 assay	Quantify membrane attack complex (MAC) deposition on target cells as a surrogate of CDC activity	Mechanistic studies	High specificity and clinical-sample compatibility; does not directly report cell lysis	[83]
ADCD	Bead-based fluorescence assay	Detect C3/C4 deposition on antigen-coated microbeads after complement activation	Functional assessment of vaccine- or antibody-elicited antibodies	High-throughput and highly reproducible; reports complement binding	[84]
Virus-based assay	Quantify C3/C4b deposition on antigen-displaying viral particles after complement activation	Viral vaccine functional evaluation	Closer to authentic viral structure; experimentally complex	[85]
ADCP	Bead-based fluorescence assay	Antigen-antibody-coated beads co-cultured with macrophages; phagocytic uptake quantified by fluorescence intensity	Functional assessment of vaccine- or antibody-elicited antibodies	Quantitative, easy to standardize, high-throughput capable; requires optimization of bead-conjugated antigen quality	[86-87]
Sensitive fluorescent dye labeling assay	pH-sensitive dye-labeled antigen; fluorescence activated after lysosomal uptake	Mechanistic studies	Real-time imaging of phagocytic events, high-content screening; true endocytosis reflected, intuitive imaging; costly dyes, instrument-demanding, low throughput	[88-89]

Fc effector function

Method

Detection principle

Application scenario

Advantages and limitations

References

ADCC

Cytoplasmic-content release assay (e.g.,⁵¹Cr, LDH, or glucose-6-phosphate dehydrogenase)

Assessment of ADCC activity by measuring intracellular components released upon target-cell lysis

High-throughput screening and historically validated method

High sensitivity and wide adoption; radioactive contamination or cumbersome labeling, with background interference

[48,72-74]

Cell viability assay

Live/dead dye-based flow cytometry to determine the fraction of apoptotic/necrotic target cells

Clinical-sample analysis and antibody functional profiling

Radio-free and simple; dye non-specific binding and cell-type variability

[75-77]

Luciferase reporter gene assay

FcγRIIIa signaling in Jurkat-NFAT-Luc-CD16 reporter cells drives luciferase expression

High-throughput functional screening for vaccine or antibody evaluation

High specificity and automation-ready; relies on specific cell line and reports FcγR signaling rather than lysis

[70-71]

Effector cell activation method

Quantify NK-cell activation markers (e.g., CD107a, IFN-γ, MIP-1β)

Clinical immune monitoring and functional validation

Enables functional profiling of effector-cell subsets; indirect ADCC readout, limited assessment of target-cell lysis

[78-80]

CDC

Cytoplasmic content release assay

Quantify released intracellular contents (e.g., ⁵¹Cr, LDH) after target-cell lysis

Functional assessment of vaccine- or antibody-elicited antibodies

Well-established and quantifiable; cannot distinguish lysis mechanism, serum background interference

[72,81]

Cell viability assay

Live/dead dye staining followed by flow cytometric quantification of target-cell apoptosis/necrosis

Cell-line-level CDC evaluation

Radio-free and convenient; slightly lower sensitivity, dye selection requires optimization

[82]

C5b-9 assay

Quantify membrane attack complex (MAC) deposition on target cells as a surrogate of CDC activity

Mechanistic studies

High specificity and clinical-sample compatibility; does not directly report cell lysis

[83]

ADCD

Bead-based fluorescence assay

Detect C3/C4 deposition on antigen-coated microbeads after complement activation

Functional assessment of vaccine- or antibody-elicited antibodies

High-throughput and highly reproducible; reports complement binding

[84]

Virus-based assay

Quantify C3/C4b deposition on antigen-displaying viral particles after complement activation

Viral vaccine functional evaluation

Closer to authentic viral structure; experimentally complex

[85]

ADCP

Bead-based fluorescence assay

Antigen-antibody-coated beads co-cultured with macrophages; phagocytic uptake quantified by fluorescence intensity

Functional assessment of vaccine- or antibody-elicited antibodies

Quantitative, easy to standardize, high-throughput capable; requires optimization of bead-conjugated antigen quality

[86-87]

Sensitive fluorescent dye labeling assay

pH-sensitive dye-labeled antigen; fluorescence activated after lysosomal uptake

Mechanistic studies

Real-time imaging of phagocytic events, high-content screening; true endocytosis reflected, intuitive imaging; costly dyes, instrument-demanding, low throughput

[88-89]