重组抗体作为实验试剂的研究
Mary Johnson1 (mary at labome dot com), Andrew Bradbury2
1 Synatom Research, Princeton, New Jersey, United States. 2 Biosciences Division, Los Alamos National Laboratory, USA
译者
朱婷婷 (ttzhu14 at fudan dot edu dot cn)
中国复旦大学生物医学研究院
DOI
http://dx.doi.org/10.13070/mm.cn.5.1297
日期
更新 : 2016-06-12; 原始版 : 2015-01-13
引用
实验材料和方法 2015;5:1297
摘要

本文是重组抗体作为实验试剂在生物医学中应用的综述和使用指南。

英文摘要

An overview and practical guide on the applications of recombinant antibodies as reagents in biomedical experiments.

虽然关于重组抗体的生产和治疗潜力有大量的研究和综述文章，但是只有极少的几篇文章提及重组抗体在典型的生物医学实验：例如免疫印迹，流式细胞术和免疫化学中的研究现状和发展潜力。这篇综述的目的是调查重组抗体在科学研究中的使用情况，研究目前重组抗体作为实验试剂的可行性。重组抗体的DNA序列可以通过克隆抗原特异的抗体基因，或从噬菌体文库中筛选获得。重组抗体本质上是单克隆抗体，并和常规的单克隆抗体和多克隆抗体都不同，因为它们是在表达宿主，诸如大肠杆菌或CHO细胞系中产生。重组抗体的主要优点包括：1）重组抗体的序列可以被修饰以适应于具体需要； 2）抗体的产生不依赖于宿主动物的免疫特性；3）重组抗体不含有动物病原体；4）体外表达的抗体在批次之间具有非常好的一致性。

文献中有关重组抗体作为实验试剂的研究
文献中有关重组抗体作为实验试剂的研究非常少

有关抗体的绝大多数文献，要么是关于通过杂交瘤培养或腹水方法生产单克隆抗体，要么是关于通过宿主动物产生多克隆抗体。截止2015年1月13日，来邦网调查了 9748 篇正式文献 中的43219个抗体应用实例，其中848篇文献中的2049个实例的抗体目录号（catalog number）和来邦网抗体数据库中所收录的89家优质供应商的目录号相匹配（其余的实例没有显示抗体目录号）。只有一个实例（2049个实例之一）表明其是重组抗体，其余的则是755种单克隆抗体和1293种多克隆抗体 (表一)这个唯一实例是来自Life Technologies的 GFP 抗体 (G10362) ，它用于免疫沉淀实验，去检测脱酰氨基作用在Bcl-xL降解中的调节作用 [1] 。

抗体类型数目
多克隆1293
单克隆755
重组1
表一. 来邦网已调查的正式出版物中，具有目录号的2049个抗体类型统计，这表明重组抗体在生物医学实验很少使用。
文献中被经常引用的重组抗体总结

有关于重组抗体被引用的研究结果列于表2。我们能够查证的最早的引用是，2005年Upsate公司（现在是EMD Millipore公司的一部分）的重组克隆号为4G10® ( 16-204) ，生物素偶联的磷酸化酪氨酸抗体 [2] 。这个克隆号及其不同偶联物的抗体是迄今为止被引用最多的重组抗体。其他重组抗体则是四个大型的重组抗体公司的产品： Life Technologies公司的ABfinity、AbDSerotec 公司的HuCAL、Miltenyi公司的REAfinity、和Creactive Diagnostics 公司的抗体。它们将在下面进行更详细地讨论。此外，个别重组抗体已被用作实验试剂。例如，硫代酪氨酸的重组抗体 [3] ，由EMD Millipore公司生产的，克隆号为Sulfo-1C-A2的抗体，至少被四篇文献引用 [4-7] 。另一个重组抗体，H3K9me3重组抗体，是为了应对市场上大量有问题的组蛋白抗体而生产的 [8] ，也是可以商业供给（Diagenode, C15500003）。重组抗体（scFv）被报道的使用最广泛的是c-myc的抗体，克隆号为9E10 [9-11] ，然而，我们并没有查证到其被引用的线索。已经有接近40种重组抗体由兔初级B细胞产生以用于immuo-MRM [12] 。SH2结构域的重组抗体用于筛选和鉴定杂交瘤品质和噬菌体展示技术，以用于大规模抗体生产和效用评估 [13] 。

目的基因和克隆号产品目录号数目引用文献
phosphotyrosine, 4G10® 及其偶联物 05-77717 [2, 14-29]
GFP, ABfinity G1036213 [30-40]
amyloid beta [1-42] H31L21, ABfinity 7002549 [41-49]
sulfotyrosine, Sulfo-1C-A2 05-11004 [4-7]
CASP3 [D175] 9H19L2, ABfinity 7001823 [50-52]
tricellulin, 54H19L38, ABfinity 7001913 [53-55]
claudin-18, 34H14K15, ABfinity 7001783 [56-58]
JNK1/2 [pT183/pY185], D12H7L17, ABfinity 7000312 [59, 60]
T-bet, 16H4L5, ABfinity 7000592 [61, 62]
interferon gamma, HuCAL HCA0431 [63]
dj-1 (oxidized at C106), HuCAL HCA0241 [64]
farnesyl diphosphate synthetase, HuCAL HCA0121 [65]
hemoglobin, HuCAL HCA0421 [66]
ki67, HuCAL HCA0061 [67]
desmin, HuCAL HCA071A1 [68]
CD282, HuCAL HCA1511 [69]
ERK1/2 [pT185/pY187], 15H10L7, ABfinity 7000121 [70]
EGFR [pY1068], 2H9L3, ABfinity 7010531 [71]
TNF-alpha, 17H1L4, ABfinity 7011351 [72]
SMAD1/5 [pS463/465], 31H14L11, ABfinity 7000471 [73]
MNK [pT197/202], 18H4L11, ABfinity 7002421 [74]
PKC-theta [pT538], F4H4L1, ABfinity 7000431 [75]
SUMO-3, 1H9L17, ABfinity 7001861 [76]
RANBP3, B24H15L41, ABfinity 7000761 [77]
vinculin, 42H89L44, ABfinity 7000621 [78]
EGFR, 30H45L48, ABfinity 7003081 [79]
MDA5, 33H12L34, ABfinity 7003601 [80]
digoxigenin, 9H27L19, ABfinity 7007721 [81]
GST, 1H14L28, ABfinity 7007751 [82]
trastuzumab, HuCAL HCA1671 [83]
CD282, HuCAL HCA1521 [69]
trastuzumab, HuCAL HCA1771 [83]
LC3B, 2H30L32, ABfinity 7007121 [84]
IL6, 4H16L21, ABfinity 7010281 [85]
SNCA, 14H2L1, ABfinity 7010851 [86]
FGFB, 7HCLC, ABfinity 7101481 [87]
ANGPTL4, 1HCLC, ABfinity 7101861 [88]
integrin alpha 6, 5HCLC, ABfinity 7102091 [89]
claudin-8 (C-Term), 6HCLC, ABfinity 7102231 [90]
TNF, Creative BiomartMOM-18006-S(P)1 [91]
CCR3, REAfinity REA1221 [92]
CCR6, REAfinity REA2771 [92]
CD123-APC, REAfinity REA1141 [93]
CCR7-PE, REAfinity REA1081 [94]
STAT1 (pY701), REAfinity REA3451 [95]
表二.正式出版物中重组抗体的引用情况总结。
重组抗体作为实验试剂的大规模使用

利用文库生产的重组抗体，具有特异识别多种抗原的潜力。然而现状是，重组抗体作为实验试剂未被充分利用。几个供应商提供的重组抗体，作为实验试剂，往往是通过专有程序（proprietary processes）生产的。它们中的四个产品已被文献引用（表二）。

AbDSerotec公司的HuCAL抗体

自2008年以来，HuCAL重组抗体一直被引用。HCA024，人抗人源DJ-1抗体（C106被氧化）被一篇文章引用，这篇文章名为“NonO-组蛋白相互结合在肿瘤坏死因子α抑制脯氨酰-4-羟化酶α1中的作用” [64] 。HuCAL重组抗体的生产是基于一篇2000年，Knappik A等人发表的名为“基于模块保守框架和三核苷酸随机化CDRs而全合成的人源组合抗体文库（HuCAL）”的文章 [96] 。已应用HuCAL平台生产了大量潜在的治疗抗体 [97] 。在2012年，HuCAL目录产品，与其他AbDSerotec公司的产品一起 被HuCAL的持有者MorphoSys卖给了Bio-Rad公司 。当前，有292种HuCAL重组抗体产品，其中包括了一些识别抗体药物的抗体：如 阿达木单抗（Adalimumab）、 贝伐单抗（Bevacizumab）、 西妥昔单抗（Cetuximab）、 英夫利昔单抗​​ 抗体（Infliximab）、 利妥昔单抗（Rituximab）、 曲妥珠单抗（Trastuzumab）、 那他珠单抗（Natalizumab）、 托珠单抗（Tocilizumab）、 奥马珠单抗（Omalizumab）、 阿仑单抗（Alemtuzumab）、 戈利木单抗（Golimumab）、和 优特克单抗（Ustekinumab）。重组抗体本质是一价抗原结合片段。在抗体亲和力方面，克隆号为18654的一价抗体与阿达木单抗（Adalimumab）的亲和力为0.16 nM，这一实验结果是通过固定阿达木单抗（Adalimumab），并使用Attana A200仪器进行实时分析无标记的分子相互作用而获得的。

Life Technologies公司的Abfinity和SGC重组抗体

ABfinity经常被引用，例如， ABfinity GFP 抗体 G10362 至少被11个出版物引用 [30-40] 。 ABfinity抗体是具有两个轻链和重链的完整IgG分子的抗体。

“ABfinity™抗体具有以下特点：重组抗体；由免疫动物生产；具有筛选功能；并且可以将免疫原特异的抗体基因克隆进高表达量载体。这种抗体的生产方式是：在哺乳动物细胞中大规模表达，并利用蛋白A进行纯化。这些重组抗体在哺乳动物表达系统中表达，但是也出现抗体从血清中分离或由杂交瘤产生时的一些相似情况。完整的IgG大小为150 kDa，还原可生成〜25 kDa轻链和〜50 kDa的重链。” [98] 。 目前，来邦网统计的ABfinity重组抗体产品有398种。ABfinity产品的引用情况见表二。

SGC（结构基因组学联盟），一个非营利，公私合作，生产了作用于152种染色质相关蛋白的1124不同的重组抗体，其中，大多数的作用于单个目的蛋白的成效最佳的抗体生产商是Life Technologies公司 [99] ，这些信息可以在来邦网上搜索获得。

Creative Biomart 重组抗体

Creative Biomart重组抗体，人源TNF抗体的scFv片段MOM-18006-S（P）被用于ELISA实验，研究在脑死亡大鼠中，甲基强的松龙（methylprednisolone）在肺的炎症活性和氧化应激中的作用 [91] 。其重组抗体的生产方式和HuCAL的scFv片段生产的过程类似。来邦网列出了Creative Biomart的413种重组抗体。

Miltenyi Biotec公司的REAfinity抗体

Miltenyi Biotec旗下的品牌REAfinity抗体在售的大概有300种重组抗体。每个REAfinity抗体是由单一类型的重链和单一类型的轻链组成，并且使用突变的人源IgG1作为Fc区域以消除与内源Fcγ受体的亲和力。Miltenyi Biotec的REAfinity抗体：抗CCR3（REA122）和抗CCR6（REA277）抗体被用于流式细胞术，研究转录因子IRF1在TH9细胞中的抗肿瘤功能 [92] ；抗CD123-APC（REA114）抗体被用来研究浆细胞样树突细胞在HIV感染者的外周血和肠粘膜中的分布规律与功能 [93] ；抗CCR7-PE（REA108）抗体被用来鉴定烟曲霉菌中的免疫抗原 [94] 。克隆号为REA345的STAT1（pY701）抗体用来研究IL-28R在NK细胞的作用 [95].

蛋白质捕获试剂计划（Protein Capture Reagents Program）和重组抗体网络

美国国立卫生研究院（NIH）资助的蛋白质捕获试剂计划（http://proteincapture.org/）包括：罗格斯大学（Rutgers）抗原计划、约翰·霍普金斯大学（Johns Hopkins University）单克隆抗体计划、和重组抗体网络（http://recombinant-antibodies.org/），该计划从2011年开始。重组抗体网络（RAN）依次由隶属于一个国际财团的三个专家中心负责，这三个专家中心是：芝加哥大学、多伦多大学、加州大学旧金山分校（UCSF）。这项计划的试剂可以在蛋白质捕获试剂计划网站（http://proteincapture.org/）搜索获得，并且可以从三个经销商（DSHB（http://dshb.biology.uiowa.edu/）、DNASU（https： //dnasu.org/DNASU/）、和CDI实验室（http://cdi-lab.com/））处购买。请注意，部分试剂仅可从一个或两个经销商处购买。至2015年1月15日，RAN目录中有754重组抗体，但是似乎大多数重组抗体处在效价和特性验证阶段。在来邦网可以搜索到17种蛋白质捕获试剂计划中的DSHB重组抗体。分别为： 分裂后期促进复合体亚基2（Anaphase promoting complex subunit 2，PCRP-ANAPC2-RAB-C77）、 组蛋白分子伴侣反沉默功能蛋白1B（Histone chaperone Anti-silencing function 1B，PCRP-ASF1B-RAB-C68）、 叉头框蛋白D3（Forkhead box D3，PCRP-FOXD3-RAB-C99）、 甲基化CpG结合结构域蛋白4（Methyl-CpG binding domain protein 4 ，PCRP-MBD4-RAB-C73）、 组蛋白赖氨酸N-甲基转移酶SETD7（Histone-lysine N-methyltransferase SETD7，PCRP-SETD7-RAB-C70）、 组蛋白赖氨酸N-甲基转移酶SMYD3（Histone-lysine N-methyltransferase SMYD3，PCRP-SMYD3-RAB-C71）、 T-box转录因子TBX4（T-box transcription factor TBX4，PCRP-TBX4-RAB-C78）、 T-box转录因子TBX4（PCRP-TBX4-RAB-C80）、 T-box转录因子TBX4（PCRP-TBX4-RAB-C81）、 VENTX同源蛋白（PCRP-VENTX-RAB-C43）、 锌指和BTB结构域蛋白21（Zinc finger and BTB domain containing 21，PCRP-ZBTB21-RAB-C85）、 锌指和BTB结构域蛋白39（PCRP-ZBTB39-RAB-C86）、 锌指和FYVE结构域蛋白20（Rabenosyn-5，PCRP-ZFYVE20-RAB-C12）、 锌指和同源框1蛋白（Zinc fingers and homeoboxes 1，PCRP-ZHX1-RAB-C59）、 锌指蛋白165（PCRP-ZNF165-RAB-C92）、 锌指蛋白446（PCRP-ZNF446-rab-C26）、 锌指和SCAN结构域蛋白29（PCRP-ZSCAN29-RAB-C3）。这些重组抗体的解离常数从1 nM到64 nM。

蛋白质捕获试剂计划是一个试点项目，没有后续项目，结束于2015年 [100] 。

重组抗体的其他供应商

其他几个供应商也提供重组抗体。在已发表的正式出版物中，我们一直没能确定其引用情况。这些供应商包括Yumab、Absolute Antibody、 BBI Solutions、 Adipogen、 和 Randox。此外，Abcam公司基于其Epitomics RabMAb兔杂交瘤克隆产生了˜5000种重组抗体。

致谢

感谢Miltenyi Biotec公司的Kalpana Singh博士的REAfinity文献。

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