Monoclonal Antibodies | 单克隆抗体

📚 Monoclonal Antibodies | 单克隆抗体

Antibodies are Y-shaped proteins produced by B-lymphocytes (B-cells) that bind specifically to antigens, such as pathogens or toxins, to neutralise them or mark them for destruction. A monoclonal antibody (mAb) is an antibody engineered to recognise only one specific epitope on an antigen, produced by identical immune cells that are all clones of a single parent cell. Because of their exquisite specificity and consistency, monoclonal antibodies have become indispensable tools in diagnostics, therapy, and research. This article explores the principles, production, and applications of monoclonal antibodies for Cambridge International A-Level Biology.

抗体是由 B 淋巴细胞（B 细胞）产生的 Y 形蛋白，能特异性地与抗原（如病原体或毒素）结合，从而中和它们或标记它们以供破坏。单克隆抗体（mAb）是一种经过设计、仅识别抗原上一个特定表位的抗体，由同一免疫细胞的克隆群体产生，所有细胞均源自同一个亲本细胞。因其极高的特异性和一致性，单克隆抗体已成为诊断、治疗和科研中不可或缺的工具。本文探讨单克隆抗体在剑桥国际 A-Level 生物学中的原理、生产及应用。

1. What Are Monoclonal Antibodies? | 什么是单克隆抗体？

Monoclonal antibodies are homogeneous antibody molecules, all identical in structure and antigen-binding specificity. They are produced by fusing a B-lymphocyte that makes the desired antibody with a myeloma cell (cancerous B-cell) to create a hybridoma. The hybridoma divides indefinitely and secretes large quantities of a single type of antibody. This contrasts with the polyclonal antibodies naturally produced in an immune response, which are a mixture of antibodies binding to multiple epitopes on the same antigen.

单克隆抗体是均一的抗体分子，所有分子在结构和抗原结合特异性上完全相同。它们通过将能产生目标抗体的 B 淋巴细胞与骨髓瘤细胞（癌变的 B 细胞）融合，形成杂交瘤。杂交瘤能无限分裂并大量分泌单一类型的抗体。这与体内免疫应答中自然产生的多克隆抗体不同，后者是能识别同一抗原上多个不同表位的抗体混合物。

In Cambridge A-Level Biology, students are expected to understand that a monoclonal antibody is specific to a single antigenic determinant, making it a highly precise reagent. The term ‘monoclonal’ means derived from a single clone, ensuring uniformity. This precision is critical for applications where cross-reactivity must be avoided, such as in targeted cancer therapies and immunoassays.

在剑桥 A-Level 生物学中，学生需要理解单克隆抗体针对的是单一的抗原决定簇，因而是一种高度精准的试剂。“单克隆”一词指源自单一克隆，确保了均一性。在必须避免交叉反应的应用中，例如靶向癌症治疗和免疫测定，这种精准性至关重要。

2. Polyclonal vs Monoclonal Antibodies | 多克隆抗体与单克隆抗体

In vivo, when an animal is exposed to an antigen, many different B-cell clones are activated, each producing an antibody that binds to a distinct epitope on the antigen. The serum collected from such an animal contains a mixture of antibodies, known as polyclonal antibodies. Polyclonal antibodies can recognise multiple epitopes, which can be advantageous for detecting antigens that may have variable conformations, but they lack batch-to-batch consistency.

在体内，当动物接触抗原时，多种不同的 B 细胞克隆被激活，每种 B 细胞产生结合抗原上不同表位的抗体。从该动物收集的血清含有这种抗体混合物，即多克隆抗体。多克隆抗体可识别多个表位，这有利于检测构象可能变化的抗原，但批间一致性较差。

Monoclonal antibodies, in contrast, are derived from a single B-cell clone. They recognise only one epitope, show high specificity, and are consistent from batch to batch. This makes them ideal for standardised diagnostic tests and therapeutic agents. The trade-off is that if the target epitope is altered or masked, the mAb may fail to bind, whereas a polyclonal mix might still recognise other parts of the antigen.

相反，单克隆抗体源自单一的 B 细胞克隆。它们仅识别一个表位，特异性高，且批间一致。这使得它们成为标准化诊断测试和治疗药物的理想选择。其代价在于，如果目标表位发生改变或被掩蔽，单克隆抗体可能无法结合，而多克隆混合物仍可能识别抗原的其他部位。

Feature 特征	Polyclonal 多克隆	Monoclonal 单克隆
Epitope specificity 表位特异性	Multiple epitopes 多表位	Single epitope 单一表位
Batch consistency 批间一致性	Variable 可变	Highly consistent 高度一致
Production 生产	Immunise animal, collect serum 免疫动物，收集血清	Hybridoma technology 杂交瘤技术
Risk of cross-reactivity 交叉反应风险	Higher 较高	Lower 较低

3. Production of Monoclonal Antibodies: Hybridoma Technology | 单克隆抗体的生产：杂交瘤技术

The classic method for producing monoclonal antibodies is hybridoma technology, developed by Kohler and Milstein in 1975 (Nobel Prize in Physiology or Medicine 1984). The technique involves fusing an antibody-producing B-lymphocyte from an immunised animal (usually a mouse) with a tumour cell from a B-cell lineage (myeloma). The resulting hybrid cell, called a hybridoma, possesses two key properties: the ability to produce a specific antibody, and the capacity to divide indefinitely in culture.

生产单克隆抗体的经典方法是杂交瘤技术，由 Kohler 和 Milstein 于 1975 年发展（1984 年诺贝尔生理学或医学奖）。该方法将一只免疫动物（通常是小鼠）中的抗体生成 B 淋巴细胞与 B 细胞谱系的肿瘤细胞（骨髓瘤）融合。形成的杂合细胞称为杂交瘤，具有两种关键特性：产生特定抗体的能力，以及在培养中无限分裂的能力。

A-Level candidates should be able to describe the four main stages: immunisation, cell fusion, selection, and screening. The choice of myeloma cell line is critical; it must lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) so that unfused myeloma cells can be eliminated in selective HAT medium. The fused hybridomas, however, receive the HGPRT gene from the B-cell partner and survive. This elegant selection strategy is a key concept in the syllabus.

A-Level 考生应能描述四个主要阶段：免疫、细胞融合、选择培养和筛选。骨髓瘤细胞系的选择至关重要；它必须缺乏次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT），以便未融合的骨髓瘤细胞能在 HAT 选择培养基中被清除。然而融合的杂交瘤从 B 细胞伙伴处获得 HGPRT 基因，因此可以存活。这种精妙的选择策略是考纲中的一个关键概念。

4. Step 1: Immunisation and B-Cell Harvesting | 第一步：免疫与 B 细胞采集

A mouse (or other mammal) is injected with the antigen of interest, often along with an adjuvant to boost the immune response. Over several weeks, the mouse receives booster injections to stimulate B-lymphocytes that produce antibodies against the antigen. Once a sufficient antibody titre is detected in the blood, the spleen is removed. The spleen is a rich source of activated B-cells, each making a specific antibody.

给小鼠（或其他哺乳动物）注射目标抗原，通常与佐剂一起注射以增强免疫应答。在数周内，小鼠接受加强注射，以刺激产生针对该抗原的抗体的 B 淋巴细胞。一旦血液中检测到足够的抗体滴度，便取出脾脏。脾脏是活化的 B 细胞（每种 B 细胞产生特定抗体）的丰富来源。

The harvested spleen cells are then dissociated into a single-cell suspension. This suspension contains many different B-cells, each producing antibodies of different specificities. The challenge now is to isolate the few B-cells that make the desired antibody and immortalise them. The next step, cell fusion, solves this problem by merging the short-lived B-cells with immortal cancer cells.

采集的脾脏细胞随后被分散成单细胞悬液。该悬液含有许多不同的 B 细胞，每种 B 细胞产生不同特异性的抗体。现在的挑战是分离出少数能产生目标抗体的 B 细胞并使其永生化。下一步细胞融合将通过将短寿命的 B 细胞与永生化的癌细胞融合来解决这个问题。

5. Step 2: Cell Fusion with Myeloma Cells | 第二步：与骨髓瘤细胞的融合

The spleen cells are mixed with myeloma cells in a ratio that favours fusion. A fusion agent, such as polyethylene glycol (PEG) or an electric field (electrofusion), is used to promote the merging of plasma membranes. PEG disrupts the cell membrane and allows adjacent cells to fuse. The result is a mixture of unfused spleen cells, unfused myeloma cells, hybridomas (fused B-cell + myeloma), and occasional fused myeloma–myeloma or spleen–spleen cells.

将脾细胞与骨髓瘤细胞以有利于融合的比例混合。使用融合剂，如聚乙二醇（PEG）或电场（电融合），以促进细胞膜的融合。PEG 破坏细胞膜，使相邻细胞融合。结果得到的是未融合脾细胞、未融合骨髓瘤细胞、杂交瘤（融合的 B 细胞 + 骨髓瘤）以及少量骨髓瘤-骨髓瘤或脾-脾融合细胞的混合物。

At this stage, the culture contains a heterogeneous population. The key is to selectively kill unfused cells while allowing only the functional hybridomas to survive. Unfused spleen cells will naturally die within a few days in culture because normal B-cells are not immortal. Unfused myeloma cells, however, are immortal and would overgrow the culture unless eliminated. This is achieved by using a selective medium.

此时培养物中包含异质性群体。关键是要选择性杀死未融合的细胞，仅让功能性杂交瘤存活。未融合的脾细胞在培养几天后会自然死亡，因为正常 B 细胞并非永生化。然而未融合的骨髓瘤细胞是永生化的，若不加以清除，将过度生长。这通过使用选择培养基来实现。

6. Step 3: Selection of Hybridoma Cells in HAT Medium | 第三步：在 HAT 培养基中选择杂交瘤细胞

HAT medium contains hypoxanthine, aminopterin, and thymidine. Aminopterin blocks the de novo pathway of nucleotide synthesis. Cells can only survive if they possess a salvage pathway enzyme, HGPRT, to utilise hypoxanthine and thymidine from the medium. The myeloma cell line used is genetically deficient in HGPRT (HGPRT^-), so unfused myeloma cells cannot use the salvage pathway and die in HAT medium.

HAT 培养基含有次黄嘌呤（hypoxanthine）、氨基蝶呤（aminopterin）和胸腺嘧啶核苷（thymidine）。氨基蝶呤阻断核苷酸合成的从头途径。细胞只有具备补救途径酶 HGPRT，才能利用培养基中的次黄嘌呤和胸腺嘧啶核苷存活。所用的骨髓瘤细胞系在遗传上缺乏 HGPRT（HGPRT^-），因此未融合的骨髓瘤细胞无法利用补救途径，在 HAT 培养基中死亡。

B-cells do have HGPRT, but unfused B-cells cannot survive indefinitely in culture; they die after a few days because they are not immortalised. Only hybridoma cells survive: they inherit immortality from the myeloma partner and the HGPRT gene (and thus the salvage pathway) from the B-cell. This selective pressure enriches the culture for hybridomas, all of which produce antibodies, but not necessarily the desired antibody.

B 细胞拥有 HGPRT，但未融合的 B 细胞在培养中无法无限期存活；它们会在几天后死亡，因为它们并未永生化。只有杂交瘤细胞能存活：它们从骨髓瘤伙伴处继承了永生化，并从 B 细胞处继承了 HGPRT 基因（以及补救途径）。这种选择压力使培养物富集了杂交瘤，所有这些杂交瘤都产生抗体，但不一定产生目标抗体。

7. Step 4: Screening and Cloning | 第四步：筛选与克隆

After HAT selection, the surviving hybridomas are a mixed population, each secreting a different antibody. The next task is to identify those producing the antibody that specifically binds to the antigen of interest. This is typically done using an enzyme-linked immunosorbent assay (ELISA) or similar immunoassay, where the secreted antibody is tested against the immobilised antigen.

在 HAT 选择之后，存活的杂交瘤是一个混合群体，每个杂交瘤分泌不同的抗体。下一步任务是鉴定那些能产生特异性结合目标抗原的抗体的杂交瘤。这通常通过酶联免疫吸附测定（ELISA）或类似的免疫测定来完成，在这些测定中，将分泌的抗体与固定化的抗原进行测试。

Positive wells are identified, and the cells from those wells are subjected to cloning by limiting dilution. This ensures that the subsequent culture originates from a single hybridoma cell, generating a true monoclonal population. Cloning is repeated several times to guarantee monoclonality. The final stable monoclonal cell line can be expanded and cryopreserved for future antibody production.

鉴定出阳性孔后，将这些孔中的细胞通过有限稀释法进行克隆。这确保随后的培养源自单个杂交瘤细胞，产生真正的单克隆群体。克隆需重复数次以保证单克隆性。最终稳定的单克隆细胞系可以扩大培养并冷冻保存，用于日后抗体生产。

8. Large-Scale Production and Purification | 大规模生产与纯化

Once a hybridoma clone that secretes the desired monoclonal antibody is established, large quantities of the antibody can be produced in vitro using bioreactors or roller bottles. The hybridoma cells are grown in culture medium that is harvested and replaced regularly. The antibody is secreted into the supernatant, which is then collected for purification. Alternatively, the hybridoma can be injected into the peritoneal cavity of mice, where it grows as an ascites tumour, producing a high concentration of antibody in the ascitic fluid. However, this in vivo method raises ethical concerns.

一旦建立了分泌所需单克隆抗体的杂交瘤克隆，就可以使用生物反应器或转瓶体外大量生产抗体。杂交瘤细胞在培养基中生长，定期收集并更换培养基。抗体分泌到上清液中，收集上清液进行纯化。另一种方法是，可将杂交瘤注射到小鼠腹腔内，以腹水瘤形式生长，在腹水中产生高浓度抗体。然而这种体内方法引发了伦理问题。

Purification typically involves protein A or protein G affinity chromatography, which binds the constant region of antibodies, or ion-exchange chromatography. The purified mAb is then characterised for concentration, purity, specificity, and affinity before being formulated for use. Scale-up must maintain the genetic stability of the hybridoma to avoid loss of antibody production.

纯化通常涉及蛋白 A 或蛋白 G 亲和层析，结合抗体的恒定区，或离子交换层析。纯化的单克隆抗体随后进行浓度、纯度、特异性和亲和力鉴定，再配制成成品。扩大培养中必须维持杂交瘤的遗传稳定性，以避免丧失抗体生产能力。

9. Applications in Diagnosis: ELISA and Pregnancy Testing | 诊断应用：ELISA 与妊娠测试

Monoclonal antibodies are widely used in diagnostic immunoassays because of their high specificity. In an indirect ELISA for detecting antibodies against a pathogen (e.g., HIV), the antigen is coated on a microtitre plate. The patient’s serum is added, and if specific antibodies are present, they bind. A monoclonal anti-human antibody linked to an enzyme is then added. This mAb recognises human antibodies and, upon addition of a substrate, produces a colour change proportional to the amount of bound antibody.

单克隆抗体因其高度特异性而广泛用于诊断免疫测定。在检测针对病原体（如 HIV）的抗体的间接 ELISA 中，将抗原包被在微量滴定板上。加入患者血清，如果存在特异性抗体，它们会结合。然后加入连接酶的单克隆抗人抗体。该单克隆抗体识别人抗体，加入底物后产生与结合抗体量成正比的颜色变化。

A familiar application is the home pregnancy test (lateral flow immunoassay). Monoclonal antibodies specific to human chorionic gonadotropin (hCG), a hormone produced by the placenta shortly after implantation, are immobilised on a test strip. Urine flows along the strip by capillary action. Anti-hCG mAbs conjugated to coloured particles bind any hCG present. The complex moves further and is captured by a second immobilised anti-hCG mAb at the test line, producing a visible coloured line. A control line using a different mAb ensures the test is valid.

一个为人熟知的应用是家用妊娠测试（侧向流免疫测定）。针对人绒毛膜促性腺激素（hCG，一种着床后不久由胎盘产生的激素）的特异性单克隆抗体被固定在测试条上。尿液通过毛细作用沿试纸条流动。偶联了有色颗粒的抗 hCG 单克隆抗体结合任何存在的 hCG。该复合物继续迁移，在测试线处被第二种固定的抗 hCG 单克隆抗体捕获，产生可见的色线。使用另一种单克隆抗体的控制线确保测试有效。

Monoclonal antibodies are also used in blood typing, detection of cardiac markers (e.g., troponin) during heart attacks, and rapid tests for infectious diseases like influenza and SARS-CoV-2. Their consistent quality makes these tests reliable and standardised worldwide.

单克隆抗体还用于血型鉴定、心脏病发作时心脏标志物（如肌钙蛋白）的检测，以及流感、SARS-CoV-2 等传染病的快速检测。其稳定的质量使得这些测试可靠且在全球标准化。

10. Applications in Therapy: Cancer and Autoimmune Diseases | 治疗应用：癌症与自身免疫疾病

In cancer therapy, monoclonal antibodies can be designed to target tumour-specific antigens. For example, trastuzumab (Herceptin) binds to the HER2 receptor overexpressed in some breast cancers, blocking growth signals and marking the cancer cells for immune destruction (antibody-dependent cellular cytotoxicity). Rituximab targets CD20 on B-cell lymphomas, depleting malignant B-cells.

在癌症治疗中，可设计单克隆抗体靶向肿瘤特异性抗原。例如曲妥珠单抗（Herceptin）与某些乳腺癌中过表达的 HER2 受体结合，阻断生长信号，并标记癌细胞供免疫清除（抗体依赖的细胞毒性）。利妥昔单抗靶向 B 细胞淋巴瘤上的 CD20，清除恶性 B 细胞。

Monoclonal antibodies can also be conjugated to drugs, radioisotopes, or toxins to deliver a cytotoxic payload directly to cancer cells while sparing normal tissue. These are referred to as antibody-drug conjugates (ADCs). In autoimmune diseases, mAbs can neutralise inflammatory cytokines: infliximab binds tumour necrosis factor-alpha (TNF-α) and is used to treat rheumatoid arthritis and Crohn’s disease. The specificity of mAbs reduces the side effects associated with conventional immunosuppressive drugs.

单克隆抗体还可以与药物、放射性同位素或毒素偶联，将细胞毒性有效载荷直接递送至癌细胞，同时避免损伤正常组织。这类抗体称为抗体-药物偶联物（ADC）。在自身免疫疾病中，单克隆抗体可中和炎性细胞因子：英夫利昔单抗结合肿瘤坏死因子-α（TNF-α），用于治疗类风湿关节炎和克罗恩病。单克隆抗体的特异性降低了传统免疫抑制药物相关的副作用。

11. Advantages and Limitations of Monoclonal Antibodies | 单克隆抗体的优势与局限性

The major advantages of monoclonal antibodies include extremely high specificity for a single epitope, reproducible and consistent production, and the ability to be produced in unlimited quantities. They can be engineered (chimeric or humanised antibodies) to reduce immunogenicity in patients. They also serve as exquisitely sensitive probes in research, such as in immunohistochemistry and flow cytometry.

单克隆抗体的主要优势包括对单个表位的极高特异性、可重复和一致的生产，以及可以无限量生产的能力。可通过工程化改造（嵌合或人源化抗体）降低在患者体内的免疫原性。它们还在研究中用作极其灵敏的探针，如免疫组织化学和流式细胞术。

However, there are limitations. Many therapeutic mAbs were originally derived from mice, and patients can develop human anti-mouse antibodies (HAMA), leading to rapid clearance of the mAb and possible allergic reactions. Production is costly and technically demanding, requiring sophisticated facilities and quality control. Monoclonal antibodies are also susceptible to small changes in the target epitope; a single point mutation in a viral antigen, for instance, can render a mAb ineffective. Additionally, their large size limits tissue penetration and they typically require intravenous administration.

然而存在局限性。许多治疗用单克隆抗体最初源自小鼠，患者可能产生人抗鼠抗体（HAMA），导致单克隆抗体被快速清除，可能引发过敏反应。生产成本高且技术要求高，需要先进的设施和质量控制。单克隆抗体也易受目标表位微小变化的影响；例如病毒抗原的单个点突变可能使单克隆抗体失效。此外，其体积较大限制了组织渗透，通常需要静脉给药。

12. Ethical Considerations and Modern Developments | 伦理考量与现代发展

The traditional method using mouse ascites fluid raises significant animal welfare concerns. Many countries now restrict the in vivo production and require in vitro methods wherever possible. The use of genetically modified animals and recombinant DNA technology has led to the creation of fully human monoclonal antibodies via phage display libraries or transgenic mice carrying human immunoglobulin genes. These methods reduce the need for animal immunisation and eliminate HAMA issues.

使用小鼠腹水的传统方法引发重大的动物福利关切。许多国家现在限制体内生产，并要求尽可能采用体外方法。转基因动物和重组 DNA 技术的使用催生了通过噬菌体展示文库或携带人免疫球蛋白基因的转基因小鼠生产全人源单克隆抗体的方法。这些方法减少了动物免疫的需求，消除了 HAMA 问题。

Modern developments also include bispecific antibodies that can bind two different targets simultaneously, and single-chain variable fragments (scFv) that retain binding capability but are smaller and penetrate tissues better. These advances promise more effective and safer biologics. As A-Level students, appreciating the ethical dimension and the continuous innovation in this field is essential for evaluating the societal impact of biotechnology.

现代发展还包括能同时结合两个不同靶点的双特异性抗体，以及保留结合能力但体积更小、组织穿透性更好的单链可变区片段（scFv）。这些进展有望带来更有效、更安全的生物制剂。作为 A-Level 学生，认识伦理维度及该领域的持续创新，对评估生物技术的社会影响至关重要。

Published by TutorHao | Biology Revision Series | aleveler.com

更多咨询请联系16621398022（同微信）

Monoclonal Antibodies | 单克隆抗体