📚 Monoclonal Antibodies: Production and Applications | 单克隆抗体的制备与应用
Monoclonal antibodies (mAbs) are identical antibodies produced by a single clone of hybridoma cells, all recognising the same specific epitope on an antigen. Their remarkable specificity makes them powerful tools in medicine and research.
单克隆抗体（mAbs）是由单一杂交瘤细胞克隆产生的完全相同的抗体，它们都识别抗原上同一个特异性表位。它们卓越的特异性使其成为医学和研究领域的重要工具。
1. The Nature of Antibodies | 抗体的本质
Antibodies (immunoglobulins) are Y-shaped proteins produced by B lymphocytes (plasma cells) in response to antigens. Each antibody has a unique variable region — the antigen-binding site — that binds specifically to one antigen. The constant region determines the antibody’s class (IgG, IgM, IgA, etc.) and its effector functions.
抗体（免疫球蛋白）是由B淋巴细胞（浆细胞）在抗原刺激下产生的Y形蛋白质。每个抗体都有独特的可变区——即抗原结合位点——能特异性结合一种抗原。恒定区决定了抗体的类别（IgG、IgM、IgA等）及其效应功能。
In the immune system, polyclonal antibodies — a mixture of antibodies from many different B cell clones — are naturally produced. These recognise multiple epitopes on an antigen. Monoclonal antibodies, by contrast, are identical and recognise a single epitope.
在免疫系统中，自然产生的是多克隆抗体——来自许多不同B细胞克隆的抗体混合物，识别抗原上的多个表位。相比之下，单克隆抗体是相同的，只识别单一表位。
2. Why Monoclonal Antibodies? | 为什么需要单克隆抗体？
Polyclonal antibodies are limited by batch-to-batch variability. Different animals, or even different bleeds from the same animal, yield different mixtures of antibodies with varying affinities and specificities. This inconsistency hampers reproducibility in diagnostics and therapy.
多克隆抗体受限于批次间的差异。不同动物，甚至同一动物不同次采血，得到的抗体混合物在亲和力和特异性上都有差异。这种不一致性妨碍了诊断和治疗中的可重复性。
Monoclonal antibodies overcome these limitations: they are homogeneous, can be produced indefinitely, have well-defined specificity, and are renewable. Once a hybridoma cell line is established, it can serve as a permanent source of identical antibodies.
单克隆抗体克服了这些局限性：它们具有同质性、可以无限期生产、特异性明确且可再生。一旦建立了杂交瘤细胞系，它就可以成为完全相同的抗体的永久来源。
3. The Hybridoma Technique | 杂交瘤技术
The most common method for producing mAbs is the hybridoma technique, developed by Köhler and Milstein in 1975 (Nobel Prize in 1984). This technique fuses a short-lived antibody-producing B cell with an immortal myeloma (cancer) cell line to create a hybridoma cell that produces antibodies indefinitely.
生产单克隆抗体最常用的方法是杂交瘤技术，由 Köhler 和 Milstein 于1975年开发（1984年获诺贝尔奖）。该技术将寿命短暂的产抗体B细胞与永生化的骨髓瘤（癌细胞）细胞系融合，创造出能无限产生抗体的杂交瘤细胞。
The process begins by immunising a mouse with the target antigen. The mouse’s B cells produce antibodies against the antigen; these B cells are then harvested from the spleen.
该过程首先用目标抗原免疫小鼠。小鼠的B细胞会产生针对该抗原的抗体，随后从小鼠脾脏中采集这些B细胞。
4. Cell Fusion and Selection | 细胞融合与筛选
Isolated B cells are mixed with myeloma cells in the presence of polyethylene glycol (PEG), which promotes cell fusion. The resulting mixture contains: unfused B cells, unfused myeloma cells, B-cell–B-cell fusions, myeloma–myeloma fusions, and the desired B-cell–myeloma fusions (hybridomas).
分离的B细胞与骨髓瘤细胞在聚乙二醇（PEG）存在下混合，PEG促进细胞融合。所得混合物包含：未融合的B细胞、未融合的骨髓瘤细胞、B细胞-B细胞融合体、骨髓瘤-骨髓瘤融合体，以及所需的B细胞-骨髓瘤融合体（杂交瘤）。
Unfused B cells die quickly in culture because they have a limited lifespan. However, unfused myeloma cells and myeloma–myeloma fusions will proliferate indefinitely. To eliminate these, the culture is grown in HAT medium (containing Hypoxanthine, Aminopterin, and Thymidine).
未融合的B细胞在培养中因寿命有限而迅速死亡。然而，未融合的骨髓瘤细胞和骨髓瘤-骨髓瘤融合体会无限增殖。为了消除这些细胞，培养使用HAT培养基（含次黄嘌呤、氨基蝶呤和胸苷）。
Aminopterin blocks the de novo nucleotide synthesis pathway. Cells must therefore use the salvage pathway, which requires the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase). Myeloma cells used in this technique are HGPRT-deficient, so they die in HAT medium. Hybridomas inherit HGPRT from the normal B cell, survive, and grow.
氨基蝶呤阻断核苷酸从头合成途径。因此细胞必须使用补救途径，这需要酶HGPRT（次黄嘌呤-鸟嘌呤磷酸核糖转移酶）。该技术中使用的骨髓瘤细胞缺乏HGPRT，因此在HAT培养基中死亡。杂交瘤从正常B细胞继承了HGPRT，能够存活并生长。
HAT 培养基筛选原理：骨髓瘤（HGPRT⁻）→ 死亡；B细胞（寿命短）→ 死亡；杂交瘤（HGPRT⁺，永生）→ 存活
5. Cloning and Screening | 克隆与筛选
Surviving hybridoma cells are diluted and cultured individually in separate wells so that each well contains cells derived from a single hybridoma cell — a clone. Each clone produces a single, specific antibody.
存活下来的杂交瘤细胞被稀释并在单独的孔中分别培养，使每个孔含有来自单个杂交瘤细胞的细胞——一个克隆。每个克隆只产生一种特异性抗体。
Screening is then performed to identify the clone producing antibodies with the desired specificity. The most common method is ELISA (Enzyme-Linked Immunosorbent Assay). The culture supernatant is tested for antibodies that bind the target antigen. Positive clones are expanded and cryopreserved.
然后进行筛选，以鉴定产生具有所需特异性的抗体的克隆。最常用的方法是ELISA（酶联免疫吸附测定）。检测培养上清液中是否存在能结合目标抗原的抗体。阳性克隆被扩增并冷冻保存。
A crucial step is limiting dilution cloning, performed at least twice, to ensure monoclonality — that all cells in the final culture are truly derived from one parental cell. Without this, the culture may contain multiple different antibody-producing clones.
关键步骤是有限稀释克隆法，至少进行两次，以确保单克隆性——最终培养物中的所有细胞确实来自一个亲本细胞。如果不这样做，培养物中可能含有多个产生不同抗体的克隆。
6. Large-Scale Production | 大规模生产
Once a suitable hybridoma clone is established, antibodies can be produced in two main ways:
一旦建立了合适的杂交瘤克隆，可以通过两种主要方式生产抗体：
- In vitro: Hybridoma cells are cultured in bioreactors; antibodies are harvested from the culture medium.
- In vivo: Hybridoma cells are injected into the peritoneal cavity of mice, producing ascites fluid rich in antibodies; this method is less ethical and less commonly used now.
- 体外法：杂交瘤细胞在生物反应器中培养；从培养基中收获抗体。
- 体内法：将杂交瘤细胞注射到小鼠腹腔中，产生富含抗体的腹水；该方法伦理问题较多，现在较少使用。
For human therapeutic use, fully human or humanised antibodies are preferred. Mouse antibodies (e.g., muromonab-CD3) can trigger an HAMA (Human Anti-Mouse Antibody) response — the patient’s immune system attacks the foreign mouse antibodies. Genetic engineering techniques create chimeric (part mouse, part human) or fully human antibodies to minimise this problem.
对于人类治疗用途，优先选择全人源或人源化抗体。小鼠抗体（如莫罗单抗-CD3）可能触发HAMA（人抗小鼠抗体）反应——患者的免疫系统攻击外来小鼠抗体。基因工程技术可创建嵌合（部分小鼠、部分人）或全人源抗体以最大程度减少该问题。
7. Diagnostic Applications: Pregnancy Testing | 诊断应用：妊娠检测
Home pregnancy tests are a classic example of monoclonal antibody technology. They detect human chorionic gonadotropin (hCG), a hormone produced by the placenta shortly after implantation.
家庭妊娠检测是单克隆抗体技术的经典应用。它们检测人绒毛膜促性腺激素（hCG），这是着床后不久由胎盘产生的激素。
The test strip contains immobilised monoclonal antibodies specific to hCG, conjugated to coloured nanoparticles. When urine containing hCG is applied, hCG binds to the conjugated antibodies. The complex migrates along the strip by capillary action and is captured by a second set of immobilised antibodies at the test line, producing a visible colour. A control line always appears to confirm the test worked.
测试条含有固定在条上的、特异性针对hCG并与彩色纳米颗粒偶联的单克隆抗体。当含hCG的尿液滴加后，hCG与偶联抗体结合。该复合物通过毛细作用沿试纸条迁移，并被测试线处的第二组固定化抗体捕获，产生可见的颜色。对照线始终出现以确认检测有效。
双抗体夹心法：hCG + 标记抗体 → 复合物 → 被捕获抗体结合 → 显色
8. Diagnostic Applications: ELISA | 诊断应用：ELISA
ELISA is a widely used laboratory technique that employs enzyme-linked antibodies to detect and quantify antigens or antibodies in a sample. Two main types exist:
ELISA是一种广泛使用的实验室技术，利用酶标记抗体来检测和定量样品中的抗原或抗体。主要有两种类型：
In direct ELISA, the antigen is immobilised on a plate, an enzyme-linked antibody is added, and a colourless substrate is converted to a coloured product. The intensity of colour is proportional to the amount of antigen present.
在直接ELISA中，抗原固定在板上，加入酶联抗体，无色的底物被转化为有色产物。颜色的强度与抗原的量成正比。
In sandwich ELISA, a capture antibody is immobilised on the plate, the sample antigen binds to it, and then a second, enzyme-linked detection antibody binds to the antigen. This method increases specificity and sensitivity. ELISA is used in HIV testing, food allergen detection, and hormone assay.
在夹心ELISA中，捕获抗体固定在板上，样品中的抗原与其结合，然后第二种酶联检测抗体与抗原结合。该方法提高了特异性和灵敏度。ELISA用于HIV检测、食物过敏原检测和激素测定。
9. Therapeutic Applications: Cancer Treatment | 治疗应用：癌症治疗
Monoclonal antibodies have revolutionised cancer therapy. Trastuzumab (Herceptin) targets HER2 receptors, which are overexpressed on certain breast cancer cells. It blocks growth signalling and marks the cancer cells for destruction by the immune system.
单克隆抗体彻底改变了癌症治疗。曲妥珠单抗（赫赛汀）靶向HER2受体，该受体在某些乳腺癌细胞上过度表达。它阻断生长信号传导，并标记癌细胞以供免疫系统摧毁。
Rituximab targets CD20 on B-cell lymphomas, triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Antibody-drug conjugates (ADCs) deliver toxic drugs specifically to cancer cells, reducing systemic side effects — for example, trastuzumab emtansine (Kadcyla).
利妥昔单抗靶向B细胞淋巴瘤上的CD20，触发抗体依赖性细胞介导的细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）。抗体-药物偶联物（ADCs）将毒性药物特异地递送到癌细胞，减少全身副作用——例如曲妥珠单抗-艾坦辛（Kadcyla）。
10. Therapeutic Applications: Autoimmune Diseases | 治疗应用：自身免疫病
Autoimmune diseases result from the immune system attacking self-tissues. Monoclonal antibodies can modulate this response. Adalimumab (Humira) and infliximab (Remicade) are anti-TNFα antibodies that neutralise tumour necrosis factor-alpha, a key inflammatory cytokine in rheumatoid arthritis and Crohn’s disease.
自身免疫病是免疫系统攻击自身组织所致。单克隆抗体可以调节这种反应。阿达木单抗（修美乐）和英夫利昔单抗（类克）是抗TNFα抗体，能中和肿瘤坏死因子-alpha，这是类风湿关节炎和克罗恩病中的关键炎症细胞因子。
By binding TNFα, these mAbs prevent it from interacting with its receptors, thereby reducing inflammation, joint damage, and disease progression. Patients typically receive regular subcutaneous or intravenous injections over months or years.
通过结合TNFα，这些单克隆抗体阻止其与受体相互作用，从而减少炎症、关节损伤和疾病进展。患者通常在数月或数年内定期接受皮下或静脉注射。
11. Other Applications | 其他应用
Beyond diagnostics and therapy, mAbs have diverse applications:
除了诊断和治疗，单克隆抗体还有多种应用：
- Targeted drug delivery — ADCs transport chemotherapy agents directly to tumour cells.
- Immunosuppression in transplant medicine — e.g., muromonab-CD3 (OKT3) targets T cells to prevent graft rejection.
- Neutralising infectious agents — e.g., palivizumab (Synagis) against RSV (respiratory syncytial virus).
- Research — Western blotting, flow cytometry, immunofluorescence and immunoprecipitation for studying protein expression and localisation.
- Purification — affinity chromatography uses immobilised mAbs to purify proteins.
- 靶向药物递送——ADC将化疗药物直接运送到肿瘤细胞。
- 移植医学中的免疫抑制——例如莫罗单抗-CD3（OKT3）靶向T细胞以预防移植物排斥。
- 中和传染因子——例如帕利珠单抗（Synagis）用于抗RSV（呼吸道合胞病毒）。
- 研究——用于Western blotting、流式细胞术、免疫荧光和免疫沉淀，研究蛋白质表达和定位。
- 纯化——亲和层析使用固定化单克隆抗体来纯化蛋白质。
12. CIE Examination Focus | CIE考试重点
For CIE A-Level Biology, students should memorise the key stages of hybridoma production in chronological order: immunisation → spleen removal → B cell isolation → fusion with myeloma cells (PEG) → HAT selection → screening (ELISA) → cloning → large-scale production.
对于CIE A-Level生物学，学生应按时序记忆杂交瘤生产的关键步骤：免疫→取脾→分离B细胞→与骨髓瘤细胞融合（PEG）→HAT筛选→筛选（ELISA）→克隆→大规模生产。
Common exam questions test: why HAT medium is used, how selection works, why cloning is necessary, and why mAbs are an improvement over polyclonal antibodies. Be able to explain how the pregnancy test works step-by-step, and to describe the therapeutic mechanisms (neutralisation, ADCC, CDC, drug delivery).
常见考题测试：为什么使用HAT培养基、筛选如何运作、为什么需要克隆，以及为什么单克隆抗体比多克隆抗体更好。要能逐步解释妊娠检测的工作原理，并描述治疗机制（中和、ADCC、CDC、药物递送）。
核心流程记忆口诀：免疫 → 融合 → HAT筛选 → 克隆筛选 → 大规模培养
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