📚 Monoclonal Antibodies: Production, Uses and Evaluation | 单克隆抗体：制备、应用与评价
Monoclonal antibodies are laboratory-produced antibody molecules that all recognise the same antigenic determinant. They have transformed diagnosis, immunotherapy and biomedical research because of their remarkable specificity. In A-Level Biology you need to explain how they are made using hybridoma technology, and how their uniformity creates both advantages and limitations.
单克隆抗体是实验室制备的、均一识别同一抗原决定簇的抗体分子。它们因高度特异性而彻底改变了疾病诊断、免疫治疗和生物医学研究。在A-Level生物中，你需要解释如何通过杂交瘤技术制备单克隆抗体，以及这种均一性如何带来优点和局限性。
1. Antibody Structure and the Polyclonal Baseline | 抗体结构与多克隆基础
Each B lymphocyte displays a membrane-bound antibody that binds one specific epitope. When an animal is exposed to a natural antigen, many different B cell clones are stimulated, so the serum contains a polyclonal mixture of antibodies that target different epitopes of the same antigen.
每个B淋巴细胞表面展示一种膜结合抗体，只结合一个特定表位。当动物接触天然抗原时，许多不同的B细胞克隆被激活，因此血清中含有针对同一抗原不同表位的多克隆抗体混合物。
Polyclonal antibodies are valuable in immune defence, but their batch-to-batch variation and mixed specificity make them less reliable for standardised diagnostic tests or targeted therapy.
多克隆抗体在免疫防御中很有用，但它们的批间差异和混合特异性使其在标准化诊断检测或靶向治疗中可靠性较低。
2. What Is a Monoclonal Antibody? | 什么是单克隆抗体
A monoclonal antibody is a single molecular species derived from one clone of B cells. All molecules have identical variable regions, so they bind the same epitope with the same affinity. This uniformity allows precise detection or blockade of a chosen antigen.
单克隆抗体是来自一个B细胞克隆的单一分子种类。所有分子具有相同的可变区，因此以相同亲和力结合同一表位。这种均一性使得能够精确检测或阻断选定的抗原。
Monoclonal antibodies are not naturally produced in large amounts; they are engineered by fusing an antibody-secreting B cell with a cancer cell to create a hybridoma.
单克隆抗体不会在体内大量自然产生；它们通过将分泌抗体的B细胞与癌细胞融合形成杂交瘤，从而人工制备。
3. The Hybridoma Principle | 杂交瘤原理
The key challenge is that normal antibody-secreting B cells survive only a short time in culture, while myeloma cells divide indefinitely but do not produce the desired antibody. Fusion combines the B cell’s specific antibody production with the myeloma’s immortality.
关键难题在于正常分泌抗体的B细胞在培养中存活时间很短，而骨髓瘤细胞能无限分裂却不产生所需抗体。融合将B细胞的特异性抗体生产能力与骨髓瘤的无限增殖能力结合起来。
The resulting hybrid cell is called a hybridoma. It can be cloned to give a stable cell line that secretes large quantities of one monoclonal antibody.
融合产生的杂交细胞称为杂交瘤。它可以被克隆成稳定细胞系，大量分泌一种单克隆抗体。
4. Immunisation and Harvest of B Cells | 免疫接种与B细胞获取
A mouse is injected with the target antigen, often combined with an adjuvant to enhance the immune response. After a few weeks, the spleen contains many activated B cells producing antibodies against the antigen. The spleen is removed aseptically and its cells are dispersed.
给小鼠注射目标抗原，通常与佐剂混合以增强免疫应答。数周后，脾脏中含有许多针对该抗原产生抗体的活化B细胞。在无菌条件下取出脾脏并分散细胞。
These splenic B cells are then mixed with myeloma cells in the presence of polyethylene glycol, which encourages cell membranes to fuse. Some cells fuse successfully, but many remain unfused.
然后将这些脾B细胞与骨髓瘤细胞混合，在聚乙二醇存在下促进细胞膜融合。部分细胞成功融合，但许多仍未融合。
5. Selection in HAT Medium | HAT培养基筛选
The mixture is transferred to HAT medium containing hypoxanthine, aminopterin and thymidine. Aminopterin blocks the de novo synthesis of nucleotides. Myeloma cells used in this technique lack the enzyme HGPRT, so they cannot use the salvage pathway and die. Unfused spleen B cells have HGPRT but cannot divide indefinitely and soon die.
将混合物转移到含有次黄嘌呤、氨基蝶呤和胸腺嘧啶的HAT培养基中。氨基蝶呤阻断核苷酸的从头合成。该技术中使用的骨髓瘤细胞缺乏HGPRT酶，因此不能利用补救途径而死亡。未融合的脾B细胞具有HGPRT，但不能无限分裂，很快死亡。
Hybridoma cells survive because they inherit the spleen cell’s functional HGPRT and the myeloma cell’s ability to divide continuously. Thus only fused cells multiply.
杂交瘤细胞能存活，因为它们继承了脾细胞的功能性HGPRT和骨髓瘤细胞的持续分裂能力。因此只有融合细胞才能增殖。
6. Screening and Cloning | 筛选与克隆
Not every hybridoma makes the desired antibody, because the mouse spleen contains B cells of many specificities. Supernatants from individual wells are tested by ELISA or a similar assay to identify wells containing the target antibody.
并非每个杂交瘤都能产生所需抗体，因为小鼠脾脏含有许多不同特异性的B细胞。可使用ELISA或类似方法检测各孔上清液，以鉴定含目标抗体的孔。
Positive hybridoma lines are then cloned by limiting dilution to guarantee that each cell line originates from a single hybridoma. This ensures the antibody is truly monoclonal.
然后通过有限稀释法对阳性杂交瘤进行克隆，以确保每个细胞系来自单个杂交瘤。这保证抗体确实是单克隆的。
7. Large-Scale Production | 大规模生产
Selected hybridoma cells are cultured in bioreactors where nutrients, pH, temperature and oxygen are controlled. The secreted antibody is harvested from the culture medium and purified by chromatography.
将选定的杂交瘤细胞在生物反应器中培养，营养成分、pH、温度和氧气均受到控制。分泌的抗体从培养基中收获，并通过色谱法纯化。
Modern therapeutic monoclonal antibodies are often produced in genetically engineered mammalian cells to improve glycosylation and reduce immunogenicity. Fully human or humanised antibodies are preferred for clinical use.
现代治疗性单克隆抗体通常在基因工程哺乳动物细胞中生产，以改善糖基化并降低免疫原性。临床使用中优选全人源或人源化抗体。
8. Diagnostic Applications | 诊断应用
Monoclonal antibodies are widely used in diagnostics because they bind a single known molecule. One key example is the pregnancy test, which detects human chorionic gonadotrophin (hCG) in urine. In a lateral flow test, mobile labelled antibodies bind hCG and the complex is captured by immobilised antibodies at the test line.
单克隆抗体广泛用于诊断，因为它们能结合单一已知分子。一个关键例子是妊娠试验，它检测尿液中的绒毛膜促性腺激素（hCG）。在侧向层析试验中，移动的标记抗体结合hCG，复合物被固定在检测线上的抗体捕获。
A control line contains immobilised antibodies that bind unbound labelled antibodies, showing that the liquid has flowed correctly. The same principle is used to detect pathogens, cardiac biomarkers and hormones in clinical laboratories.
对照线含有固定抗体，可结合未反应的标记抗体，显示液体已正确流动。同样原理也用于临床实验室检测病原体、心脏标志物和激素。
9. Therapeutic Applications | 治疗应用
In cancer treatment, monoclonal antibodies can bind to antigens overexpressed on tumour cells. They may mark the cancer cell for destruction by immune cells, block growth factor receptors, or deliver a toxic drug or radioisotope directly to the tumour.
在癌症治疗中，单克隆抗体可结合肿瘤细胞过度表达的抗原。它们可以标记癌细胞供免疫细胞杀伤、阻断生长因子受体，或将毒性药物或放射性同位素直接递送至肿瘤。
Examples include anti-HER2 therapy for certain breast cancers and anti-CD20 therapy for B-cell lymphomas. Monoclonal antibodies are also used in autoimmune diseases to neutralise inflammatory cytokines such as TNF-alpha.
例子包括用于某些乳腺癌的抗HER2疗法和用于B细胞淋巴瘤的抗CD20疗法。单克隆抗体还用于自身免疫病，以中和炎症细胞因子如TNF-α。
10. Advantages and Limitations | 优点与局限性
Monoclonal antibodies offer high specificity, reproducible batch quality and the ability to be mass-produced. They can detect vanishingly small quantities of antigen and can be engineered for different effector functions.
单克隆抗体具有高度特异性、批间质量重现性好，并且可以大规模生产。它们能检测极微量的抗原，并可被设计用于不同的效应功能。
However, production is expensive and requires sophisticated facilities. Mouse-derived antibodies may provoke an immune response in patients unless they are humanised. Tumours can lose the target antigen or become resistant, and storage usually requires a cold chain.
然而，生产成本高且需要先进设施。小鼠来源的抗体在患者体内可能引发免疫反应，除非经过人源化处理。肿瘤可能丢失靶抗原或产生耐药性，储存通常需要冷链。
Ethical questions also arise from the use of animals for immunisation and, historically, for ascites production. Current emphasis is on in vitro methods and humanised antibodies to reduce animal use.
使用动物进行免疫以及历史上通过腹水法生产还引发了伦理问题。目前重点在于体外方法和人源化抗体，以减少动物使用。
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