鼠单克隆抗体的产生杂交瘤技术
Summary
An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.
Abstract
Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.
Introduction
在这个协议中提出的杂交瘤技术最早是由和Milstein 1在1975年描述的，除了一些技术改进，主要的程序没有显着在过去的40年2变化。该协议的目的是说明一个更合适的免疫策略，对于单克隆抗体的产生的标准方法，以及用于验证方法（ELISA）的例子。
抗体是难以置信的工具和向广泛的技术方法，如流式细胞术，磁细胞分选，或免疫荧光，以及用于疾病监测和治疗3的诊断和治疗方案。对于所需靶的单克隆抗体的商业供应是通过在24个不同的网络数据库的存在下，用抗体或抗体相关的产品4的几乎无数数量证明。 2015年，一ntibody分子的国际讨论5-7的部分原因是正确的验证和市售抗体的表征严重的问题。
它可能是困难和昂贵找到靶向抗原的特异性抗体，并且通常，它们不具有所需要的亲合性或特异性。虽然产生的抗体仍然是耗时且需要熟练人员开发和验证的抗体，单独地产生抗体可能比购买更好。
由于这样的事实：抗体的产生是耗费时间的，并且需要经验，为生产结合分子的替代方法被开发来克服这些问题。最常用的一种方法是通过噬菌体展示的重组生产单链抗体。用于可变结合区的基因是从细胞中提取，并用噬菌体的涂布蛋白质相结合。在S英格尔链然后噬菌体的表面上表达，并在几个平移步骤8进行筛选。生产的单链抗体的有点快，但它也需要一个熟练的实验者。一些重组单链抗体的缺点是稳定性差并用于体外诊断方面的缺乏适合的。在大多数诊断测试的Fc受体进行检测是必要的，这需要被添加到一个重组单链抗体之后。再次，这是费时和甚至比杂交瘤技术更加复杂。在体外诊断，全长单克隆小鼠和兔抗体已被证明是最好的选择。
所述免疫缀合物的设计：一个在产生单克隆抗体的主要步骤必须在实验室中的工作开始之前完成。需要解决的问题是：什么是目标在最终APPLICAT物理组合物离子，并且该基质是本？其中浓度将目标必须在应用程序？什么是最终的应用，什么是该抗体必须满足的要求？
总是考虑到，如果使用线性肽片段，它也有在所选择的目标的最后的表位是线性;否则，将抗体不结合。当然，独立的筛选方法的，抗体可被选择为识别在不同的应用不同的抗原的格式，但是这必须被非常精确地验证。这就是为什么抗体的开发和验证是这样雄心勃勃的过程的原因。
用于免疫的抗原格式的选择是对抗体发展的根本，并确定该过程的成功或失败。一旦小鼠表达相关的抗体滴度，所述脾细胞分离并用骨髓瘤细胞融合。最常见的骨髓瘤细胞系为鼠单克隆抗体的开发是X63-银8.653 9和来自Balb / c小鼠品系的Sp2 / 0-银14 10。将细胞从一个恶性B细胞淋巴瘤下降和被选择，因为他们不分泌任何自己重链或轻链。 1（脾细胞与骨髓瘤细胞）：细胞可以1:10和10之间适于比率。在这个协议中，将细胞适应于3：1的比率和由聚乙二醇（PEG）和电融合融合，根据Stoicheva和辉11。
B细胞和骨髓瘤细胞的融合是一个随机过程。因此，可以产生两个B淋巴细胞或两个骨髓瘤细胞的杂交体，但这些杂种将无法用于在培养很长时间存活。细胞经历一次黄嘌呤，氨基喋呤，和胸苷（HAT）的选择，其中只有融合杂交瘤细胞可存活由于使用嘧啶合成的从头合成途径的可能性。对于周一的产生oclonal抗体，它是必要的，以获得从一个母细胞始发的细胞系。的单克隆通过限制稀释技术和细胞生长的显微分析确保。杂交瘤培养上清液筛选特异性抗体的生产，主要是通过ELISA或流式细胞术，和最好的粘合剂来选择。大量培养和纯化后，该抗体分子可以最后被表征和验证为所需要的应用程序。
Protocol
Representative Results
Discussion
通过杂交瘤技术的单克隆抗体的产生需要的强烈和详细的表位分析，特别是对最终的应用中，当该抗体应当识别目标。这通常是由用户低估并导致弱表演抗体。融合过程总是随机的，这意味着特异杂交的结果在很大程度上取决于在该点的细胞比例和活力。有限稀释后，细胞是非常不稳定的，需要的细胞生长和抗体生产的严格监控。这些参数应排除故障的方法时，应仔细分析。
<p class="jove_content"…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge the German Federal Ministry of Education and Research (BMBF, Grant No: 03IPT7030X, 03IPT703A, and 03IP703) for funding our projects, “Artificial immune reactions,” “Camelid antibodies,” and “Antibody technologies.” We thank Prof. Burkhard Micheel for proofreading the manuscript and for the helpful comments.
Materials
| glutaraldehyde | Sigma Aldrich | G5882 | |
| N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) | Sigma Aldrich | 39391-10ML | |
| Sulfo-GMBS | Perbio Science Germany | 22324 | |
| ovalbumin | Sigma Aldrich | A5503 | |
| bovine serum albumin | Sigma Aldrich | A2153 | |
| keyhole limpet hemocyanin | Sigma Aldrich | H8283 | |
| Falcon tubes 15 mL | Biochrom GmbH | P91015 | |
| reaction vials, 1.5 mL | Carl Roth GmbH & C0.KG | CNT2.1 | |
| hollow needle | Carl Roth GmbH & C0.KG | C724.1 | |
| glass wool | Carl Roth GmbH & C0.KG | 6574.1 | |
| Sephadex G25 coarse | Sigma Aldrich | GE-17-0034-02 | |
| Freund´s adjuvant, complete | Sigma Aldrich | F5881-10ML | |
| ELISA plates, 96 well | Greiner bio-one | 655101 | |
| neonatal calf serum | Biochrom GmbH | S1025 | |
| TipOne Tips 1000 µL | Starlab | S1111-2021 | |
| Pipette tips 200 µL | Greiner bio-one | 739291 | |
| HRP-conjugated goat-anti-mouse IgG antibody | Dianova | 115-035-003 | |
| tetramethylbenzidine | Carl Roth GmbH & C0.KG | 6350.2 | |
| Natriumdihydrogenphosphat | Carl Roth GmbH & C0.KG | K300.2 | |
| peroxide/urea | |||
| sulphuric acid | Carl Roth GmbH & C0.KG | 4623.3 | |
| RPMI 1640 | Life technologies GmbH | 31870074 | |
| L-glutamine | Carl Roth GmbH & C0.KG | HN08.2 | |
| beta-mercaptoethanol | Sigma Aldrich | M6250 | |
| fetal calf serum | Invitrogen | 10270106 | |
| TC-flask 25 cm2 | Peske GmbH | 86-V025 | |
| TC-flask 75 cm2 | Peske GmbH | 86-V075 | |
| ethanol, 96% | Carl Roth GmbH & C0.KG | P075.1 | |
| cell strainer | VWR international | 734-0002 | |
| Falcon tubes 50 mL | Biochrom GmbH | P91050 | |
| PEG 8000 | Sigma Aldrich | 1546605 | |
| electroporation cuvette, 2mm | Biodeal Handelsvertretung Edelmann e.K. | EKL2,25 | |
| hypoxanthine | Sigma Aldrich | H9636-25G | |
| azaserine | Sigma Aldrich | A4142 | |
| thymidine | USB Europa GmbH | 22305 1 GM | |
| TC-plates 96 well | Biochrom GmbH | P92696 | |
| TC-plates 24 well | Biochrom GmbH | P92424 | |
| cryotubes, 1mL | Sigma Aldrich | V7384-1CS | |
| dimethylsulfoxid | Carl Roth GmbH & C0.KG | 4720.1 | |
| protein A sepharose | Sigma Aldrich | P3391-1G | |
| SDS sample loading buffer, Roti-Load 1 | Carl Roth GmbH & C0.KG | K929.1 | |
| unstained protein ladder | BioRad Laboratories | 161-0363 | |
| comassie brilliant blue R-250 | BioRad Laboratories | 161-0406 |
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