抗原选择和制备
Optimal selection and preparation of the antigen is crucial to obtaining high-quality antibodiesin both polyclonal and monoclonal antibody production. Although the importance of antigen selection and preparation is not immediately apparent to some in the field, Envigo’s experience has shown that it is a critical consideration.
该first step - selecting the antigen
该过程的第一步是选择感兴趣的抗原。需要考虑的特征包括免疫原性潜力;许多抗原不是高度免疫原性的。除了问题组织代表或者自我抗原，the antigen type, source, purity, 3-D structure, and the intended final use of the antibody product are also important up front considerations.由于一些应用更适合多克隆而不是单克隆的生产，而不是单克隆的生产，因此第一个问题之一是：“您打算如何使用抗体？“
制备免疫抗原
选择合适的抗原后，下一步骤正在制备抗原immunization并且选择刺激的B细胞或抗体收集。因为对天然抗原的访问通常是有限的，所以重组蛋白表达通常用于产生足够的抗原。这个process可以使用真核和/或原核表达系统。
Consideration when using eukaryotic expression is the possible impact on the proper folding and post-translational modifications of the antigen, which may influence whether the epitope is presented properly when antigen presentation occurs. On the other hand, the presence of post-translational modifications may not be important for the presentation of some antigens, such as small antigens or peptides in which the 3D structure is not an issue.
In addition to recombinant proteins, synthetic peptides can be employed as an抗原来源。他们上诉的一部分是他们易于获得，廉价才能产生并且可以用来生产针对不同蛋白质同种型或特异性磷酸化蛋白的抗体。由于大多数肽缺乏二级结构，it is important to avoid epitopes that may be unavailable due to a protein’s tertiary structure因为所得抗体可能无法识别天然蛋白质。
另一种考虑是抗原是否自然被发现为孤独的分子或作为蛋白质的较大络合物的一部分。关于原生蛋白是否经历的变化和翻译后修饰等的见解，或者如何折叠可能是非常有帮助的;这些变化可能会影响现实世界环境中检测的表位的可用性。
Enhancing an immune response
为了增强动物的免疫应答并产生针对小肽的抗体，研究人员通常将肽耦合到载体蛋白质，如匙孔颗粒血糖（KLH），甲状蛋白酶（Tg），卵烧蛋白（OVA）或牛血清白蛋白（BSA）。该se proteins are themselves immunogenic which leads to the generation of antibodies directed against the carrier so antibody screening and characterization is important。
该procedure to produce synthetic peptides or recombinant antigens can also be a source of impurities. For example, reaction byproducts, media components, and/or contamination with other peptides is possible; these impurities can have a dramatic effect on the immune response following immunization. Alternative approaches to using standard peptide-carrier conjugates have also been developed.
全长（本机）蛋白抗原通常被认为是相对于其他抗原策略的优异选择，主要是在实证数据上，其表明它们倾向于在识别天然靶蛋白的引发抗体时更好地表现更好。在一项研究中，使用三种免疫方法：肽抗原，DNA免疫和全长蛋白，调查人员针对10个血清蛋白质的抗体引起抗体。该作者据报道，总体上，用全长蛋白质免疫始终产生最佳效果。
然而,获得完整的-length proteins in the quantities needed for successful immunization is often a limiting factor and thus not appropriate in all cases. In addition, the processes necessary to acquire sufficient amounts of full-length antigens may introduce impurities such as detergents, denaturants, solvents, or altered pH conditions that can affect the health of animals and result in poor titer antiserum or B cell stimulation.
考虑
Once an appropriate antigen has been generated in sufficient quantities, further steps in its preparation must be optimized to provide the best opportunity for inducing a potent and appropriate immune response. In particular, the抗原的纯度对抗体产量产生显着影响;如果用于抗体生产过程的原料以其他方式被污染或劣等，例如细菌抗原的存在或不适当的缓冲液组合物（这会影响抗原的溶解度和结构），则最终产品处于增加风险performing poorly。
虽然获得高纯度的抗原可能是艰苦的过程，但实施最小化抗原污染的方案是最佳实践，有助于确保抗体特异性。免疫系统可以对杂质安装重大反应，其可以用误导的抗体或B细胞污染抗体制剂。
结论
增加出色的抗体的机会需要仔细规划最早的阶段，包括选择和制备抗原。这一前期投资将使抗体成功，节省时间，努力和金钱在长远来看。
根据您的抗体的最终应用，应该以敞开的思想开始，并考虑是否是一个多克隆抗体ormonoclonal antibody是您申请的最佳拟合。
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