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重组抗体

Recombinant Antibodies

Section 1.

What is a recombinant antibody?

Section 2.

GeneTex’s Recombinant Monoclonal Antibody Production Initiative

Section 3.

Why GeneTex’s rabbit-based HL clones are a better choice.

 

section1

Section 1. What is a recombinant antibody?

A recombinant antibody (rAb) is generated from the cloned, in vitro-expressed heavy and light chains of a selected monoclonal antibody obtained through classical hybridoma methodology, display strategies alone or in combination, sorted single B cells from immunized animals, or by one of many other techniques. The fact that it is cloned means the rAb is defined by its primary sequence, which can be engineered to optimize affinity and be expressed in different binder formats. The numerous advantages of rAbs have been well-documented, with consistency of performance being of paramount significance.

GeneTex’s rAb protocol employs a multi-parameter FACS-based approach to isolate antigen-specific IgG+ memory B cells from an immunized animal, with subsequent cloning of the antibody variable-region genes into an IgG backbone and expression in mammalian cells (Fig. 1) (1). This protocol is very rapid and can be completed in weeks, and also affords the opportunity to identify antibodies with diverse capabilities in various applications. Importantly, it allows cloning of the heavy and light chains from the same B cell, thereby preserving natural pairing. And once cloned, the supply of a given rAb is inexhaustible with exceptional reproducibility.

 

(A)
(B)

Figure 1. (A) The GeneTex recombinant rabbit monoclonal antibody workflow (Starkie et al. (2016)). (B) Advantages of recombinant antibodies.

One outstanding advantage of this protocol is that the suitability of the individual clones for desired applications can be tested during screening. For example, GeneTex’s Iba1 rabbit recombinant antibody [HL22] (GTX635363) detects Iba1, a protein commonly used as an immunohistochemical marker of both quiescent and activated microglia. In its development and production, both paraffin- and frozen-IHC analyses (IHC-P, IHC-Fr, respectively) were conducted in the first screening process to identify the best clones for these applications, as shown below (Fig. 2). The clones were compared to a highly cited, market-leading commercial antibody to gauge their performance. This strategy provides us with valuable perspective on a clone’s market competitiveness during development. In addition, our recombinant antibody team has successfully expressed the antigen-binding regions of this antibody in the context of both mouse and rat IgG backbones, thus extending flexibility for multiplexed staining.

 

Figure 2. GeneTex's recombinant rabbit monoclonal Iba1 antibody [HL22] (GTX635363) is superior to a competitor's highly cited rabbit polyclonal antibody for both IHC-P (panels A vs. D) and IHC-Fr (panels B, C vs. E, F).

Another new recombinant rabbit monoclonal reagent is GeneTex’s RAS (G12D mutant) antibody [HL10] (GTX635362) (Fig. 3). It demonstrates a robust signal and specificity for the G12D mutation in wild-type (WT) and KRAS G12D mutant pancreatic tumor tissue sections (Fig. 3A) and in established cell line lysates (Fig. 3B), respectively. This RAS (G12D mutant) antibody (GTX635362) is the first commercial recombinant version that shows exceptional specificity by IHC-P for this key tumorigenic mutant protein on sequence-verified human pancreatic tumor samples.

 

Figure 3. (A) GeneTex's recombinant rabbit RAS (G12D mutant) antibody [HL10] (GTX635362) is sensitive and specific for the RAS G12D mutation by IHC of a KRAS G12D mutant pancreatic tumor tissue section (top) compared to a wild-type KRAS section (bottom). (B) The antibody is sensitive and specific for the RAS G12D mutation by WB of extracts from wild-type and mutant KRAS-confirmed human pancreatic cell lines. GAPDH for the loading control was detected by GTX100118. Lane1: HPDE. Lane2: HPNE. Lane3: AsPC1 (KRAS G12D). Lane4: BxPC3 (KRAS WT). Lane5: CFPAC1 (KRAS G12V). Lane6: HPAC (KRAS G12D). Lane7: HPAF-II (KRAS G12D). Lane8: MIA PaCa-22 (KRAS G12C). Lane9: PANC1 (KRAS G12D). Lane10: SU86.86 (KRAS G12D).

As mentioned above, the antigen-binding regions of a recombinant antibody can be inserted into various host IgG backbones or expressed in different binder formats (e.g., Fab fragments or scFvs). Here, GeneTex’s cloned TSG101 mouse monoclonal antibody (GTX70255) was converted to a rabbit IgG backbone (GTX635396) with preservation of performance. No cross-reaction was observed when the converted TSG101 rabbit IgG recombinant antibody was used in combination with an anti-mouse IgG secondary antibody (Fig. 4).

 

Figure 4. Backbone switch from a cloned mouse IgG antibody to a rabbit IgG antibody.

Literature:

Flyer - Recombinant Abs

Reference:

  1. PLoS One.2016; 11(3): e0152282.

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section2

Section 2. GeneTex’s Recombinant Monoclonal Antibody Production Initiative

GeneTex’s production focus is the creation of novel recombinant rabbit monoclonal antibodies validated using enhanced protocols defined by the International Working Group for Antibody Validation (IWGAV).

Over the last decade, studies published in many of the top scientific journals have clearly described the significant impact of poorly validated antibody reagents on the reproducibility of biomedical research. The unfortunate reality is that the commercial antibody market is compromised by a substantial subset of products that have been demonstrated to be nonspecific. This means that there are two major issues that must be addressed in order to improve the overall quality of the commercial antibody market and therefore the integrity and reproducibility of the biomedical research dependent on antibodies. The first is more widespread adoption of production platforms that emphasize recombinant antibody technology (please see “Section 1: What is a recombinant antibody?”) while the second involves incorporation of improved and more coherent validation strategies. GeneTex is making major strides in both of these endeavors. 

With the goal of providing better products for its customers through innovative technology, GeneTex has now shifted all of its new production to recombinant rabbit monoclonal antibodies (please see “Section 3: Why GeneTex’s rabbit-based HL clones are a better choice.”). Fully recombinant antibodies are defined by their primary sequence, their consistent performance and supply (issues that plague traditional polyclonals and hybridoma-based monoclonals with regard to lot-to-lot variability), and their ability to be engineered (i.e., backbone switching) to meet researcher requirements. These benefits may even evoke more favorable impressions by grant review and journal editorial committees. Other outstanding advantages of recombinant rabbit antibodies stem from the favorable biological characteristics of the rabbit immune system itself.

In conjunction with its pivot to recombinant antibody production, GeneTex’s second commitment to antibody reliability is its incorporation of enhanced validation protocols, based on the IWGAV proposal mentioned above, into its manufacturing and quality assurance (QA) workflow (1). This involves evaluation of each antibody using at least one, and preferably more, of five validation “pillars” defined in the IWGAV study. GeneTex’s version of the IWGAV plan follows the five standard strategies and includes (1) Knockout/Knockdown; (2) Comparable Antibodies; (3) Immunoprecipitation followed by Mass Spectrometry (IP/MS); (4) Biological and Orthogonal Validation; and (5) Recombinant Protein Expression (Fig. 1). The company refers to this as its “5+1” Pillar Plan when referencing new recombinant antibody production, with the “+1” designation referring to the pre-validation that is inherent in the application-specific assessment that occurs with clone selection during the recombinant antibody production process (please see “New Validated Recombinant Monoclonal Antibodies from GeneTex” below).

The new recombinant rabbit monoclonal antibodies that have been generated and evaluated using the production and validation approaches described above (these products are identified by “HL” in their clone designations) are just the beginning. GeneTex is also working to replace its successful polyclonals with new recombinants. The company envisions the “HL” clones representing the highest level of GeneTex validation and manufacturing capability.

Antibody fidelity, application-dependent reliability, and performance and supply consistency are crucial for ensuring research integrity. GeneTex joins other reputable companies in leveraging recombinant antibody technology and enhanced validation to create antibodies that researchers can trust.

  

Figure 1.  GeneTex’s “5+1” Pillar Plan for recombinant antibody validation.

New Validated Recombinant Monoclonal Antibodies from GeneTex

  Knockout / Knockdown Validation

Complete removal or significant reduction of the endogenous signal following genetic strategies involving genome editing or RNA interference, respectively.

Integrin beta 1 / CD29 antibody [HL1255] (GTX636657)

  Comparable Antibodies

Independent antibodies against the same target but to different epitopes, often including distinct samples with different target protein expression levels.

Influenza A Virus Nucleoprotein antibody [HL1089] (GTX636247)

  Biological and Orthogonal Validation

Alterations in the endogenous level of the target protein in accordance with specific preparation conditions corresponding to defined biological characteristics, or comparison between antibody-dependent and -independent methods.

PARP antibody [HL1364] (GTX636804)

  Protein Overexpression

Tagged target proteins overexpressed in transfected cells are used as a positive control for validation.

AKT1 antibody [HL1142] (GTX636413)

  Recombinant Antibody

Antibody is generated using recombinant technology.

Gli1 antibody [HL247] (GTX635619)

Reference:

  1. Nat Methods. 2016 Oct;13(10):823-7.

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section3

Section 3. Why GeneTex’s rabbit-based HL clones are a better choice.

GeneTex’s recombinant rabbit monoclonal antibody production process is the platform for all new rabbit-based antibody generation (1). These products are distinguished by the “HL” designation in their respective clone numbers (e.g., “[HL1089]”). The rabbit, and specifically its immune system, is known to confer the following outstanding benefits for monoclonal antibody creation: broader antibody range, less complex immunoglobulin (Ig) structure, higher binding affinities, larger pool of lymphocytes to select antibodies (2). Each of these advantages is described in more detail below:

 

1. Broader antibody repertoire:

  1. Rabbits belong to the order Lagomorpha, which is evolutionarily distinct from the order Rodentia. This means that human antigens present epitopes that are often more immunogenic in rabbits than in mice, expanding the number of generated antibodies that may in turn cross-react with murine protein orthologs (2-5).
  2. The rabbit immune system is more successful in mounting an immune response to hapten or small molecule antigens, which is frequently not the case with the murine immune system (2-5).
  3. Mice used to generate monoclonal antibodies are from inbred lines and offer less diversity in their immunogenic responses than do rabbits. In addition, mouse spleens are much smaller than rabbit spleens (2-5).
  4. The rabbit immune system is different from those of mice and humans in that it utilizes somatic gene conversion (SGC) to expand its antibody range in addition to performing the same VDJ and VJ recombination and somatic hypermutation (SHM) mechanisms noted in mice and humans. Antibody diversity is further enhanced by other processes that include a kappa light chain variable gene structure distinctive for variability in the length of complementarity determining region 3 (LCDR3), as well as successive Ig development in the bone marrow, the gut-associated lymphoid tissues (GALT), and spleen and lymph node germinal centers (3, 5).

 2. Simpler Ig structure:

  1. The European rabbit (Oryctolagus cuniculus) is known to have single genes for IgE and IgM, and no genes for IgD. Interestingly, its genome contains 13 IGHA genes coding for at least 10 functional IgA subclasses. Importantly, though there are allelic variants, rabbits have only one IgG gene (and thus no subclass) and therefore differ from mice and humans (each with four IgG subclasses) (4, 5).

  2. In addition to the disulfide bonds found in a human or mouse IgG (linking the heavy chain pair or one heavy chain to its associated light chain), rabbit IgG antibodies demonstrate a unique intrachain disulfide bond in their K1 light chain. This is significant as perhaps 90% of IgGs in New Zealand White rabbits are IgG-k (K1), and this extra bond is thought to confer stability onto the rabbit IgG (2, 5).

3. Enhanced binding affinity:

  1. Rabbit monoclonal antibodies have very high affinities with Kd values characteristically in the picomolar range, with some possessing exceptionally low picomolar Kd values (2, 5).

  2. Picomolar Kds translate to increased sensitivity with consistent specificity.

4. Larger lymphocyte pool:

  1. Rabbits have a longer life span and their larger size (perhaps a 100-fold weight differential between a three-month-old rabbit and a six-week-old mouse) greatly facilitates blood and tissue sampling during antibody production (5).

  2. Consistent with the statements above, rabbit spleens allow isolation of 50-fold more B cells compared to murine spleens, meaning more clones recognizing a wider selection of epitopes (2-5).

GeneTex hopes that the distinct advantages of rabbit monoclonal antibodies discussed above will encourage you to consider our HL clones for your research needs. As GeneTex has now shifted all of its new antibody production to the creation of these monoclonal antibodies, our product inventory will continue to expand rapidly moving forward.

 

 

GeneTex’s Well-validated HL Clone Antibodies

ACE2 antibody [HL1092]   (GTX636265)

Vimentin antibody [HL1506] (GTX636980)

ERK1 (phospho Thr202/Tyr204) + ERK2 (phospho Thr185/Tyr187) antibody [HL173] (GTX635617)

NRF2 antibody [HL1021] (GTX635826)

p70 S6K (phospho Thr421/Ser424) antibody [HL129] (GTX635621)

PD-L1 antibody [HL1041] (GTX635975)

 

References:

  1. PLoS One. 2016 Mar 29;11(3):e0152282. doi: 10.1371/journal.pone.0152282. eCollection 2016.
  2. Am J Transl Res. 2011 May 15;3(3):269-74. Epub 2011 Apr 23.
  3. Comp Immunol.2006;30(1-2):137-53. doi: 10.1016/j.dci.2005.06.017.
  4. Immunogenetics. 2016 Feb;68(2):83-107. doi: 10.1007/s00251-015-0868-8.
  5. Exp Mol Med.2017 Mar; 49(3): e305. doi: 10.1038/emm.2017.23.

 

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雄激素受体(AR-V7 剪接变体特异性)抗体

雄激素受体(AR-V7 剪接变体特异性)抗体

在晚期前列腺癌中,雄激素受体剪接变体 7 (AR-V7) 是最丰富的雄激素受体 mRNA 剪接变体...
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通过敲除验证的整合素 β-1抗体

通过敲除验证的整合素 β-1抗体

整合素是由α和β亚基组成的异二聚体蛋白,已在哺乳动物中发现了至少 18 个 α 亚基和 8 个 β 亚基...
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SARS-CoV-2-directed T cell Response in Convalescent and Vaccinated Individuals

SARS-CoV-2-directed T cell Response in Convalescent and Vaccinated Individuals

SARS-CoV-2-directed T cell Response in Convalescent and Vaccinated Individuals
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NRF2激活的癌症治疗新范式

NRF2激活的癌症治疗新范式

NRF2激活的癌症治疗新范式
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Article alert: Proteomic Analysis Reveals How RNA Viruses Remodel Host Polysomes

Article alert: Proteomic Analysis Reveals How RNA Viruses Remodel Host Polysomes

Article alert: Proteomic Analysis Reveals How RNA Viruses Remodel Host Polysomes
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甲型流感和乙型流感重组单克隆抗体

甲型流感和乙型流感重组单克隆抗体

GeneTex推出一系列针对甲型流感病毒(Flu A)和乙型流感病毒 (Flu B) 核蛋白(NP)的重组兔单克隆抗体...
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IHC- and FCM-validated Antibodies for SARS-CoV-2 Research

IHC- and FCM-validated Antibodies for SARS-CoV-2 Research

IHC- and FCM-validated Antibodies for SARS-CoV-2 Research
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新冠肺炎中和性抗体与病毒结合位的结构解析

新冠肺炎中和性抗体与病毒结合位的结构解析

新冠肺炎中和性抗体与病毒结合位的结构解析
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COVID-19

新冠病毒抗体来了!

GeneTex分析抗原序列与抗原特性,率先与合作实验室完成测试,GTX632604抗体可于western blot实验下, 辨识新冠病毒的刺突蛋白
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Skin Regeneration without Scar Formation

Skin Regeneration without Scar Formation

Skin Regeneration without Scar Formation
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GeneTex推出SARS-CoV-2 (COVID-19) RBD ACE2结合活性/中和测定套组

GeneTex推出SARS-CoV-2 (COVID-19) RBD ACE2结合活性/中和测定套组

GeneTex推出SARS-CoV-2 (COVID-19) RBD ACE2结合活性/中和测定套组
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mRNA Vaccine-induced Antibody Profiles against SARS-CoV-2 Variants

mRNA Vaccine-induced Antibody Profiles against SARS-CoV-2 Variants

mRNA Vaccine-induced Antibody Profiles against SARS-CoV-2 Variants
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SARS-CoV-2 can infect the human brain

SARS-CoV-2 can infect the human brain

SARS-CoV-2 can infect the human brain
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GeneTex’s SARS-CoV-2 Spike RBD Antibodies Effectively Inhibit Viral Entry

GeneTex’s SARS-CoV-2 Spike RBD Antibodies Effectively Inhibit Viral Entry

GeneTex’s SARS-CoV-2 Spike RBD Antibodies Effectively Inhibit Viral Entry
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Neuropilins Facilitate SARS-CoV-2 Entry into Host Cells

Neuropilins Facilitate SARS-CoV-2 Entry into Host Cells

Neuropilins Facilitate SARS-CoV-2 Entry into Host Cells
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Zika Virus Infection Increases Risk of Subsequent Severe Dengue Disease

Zika Virus Infection Increases Risk of Subsequent Severe Dengue Disease
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Nuclear PD-L1 Mediates Pyroptosis in Cancer Cells

Nuclear PD-L1 Mediates Pyroptosis in Cancer Cells

Nuclear PD-L1 Mediates Pyroptosis in Cancer Cells
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2020诺奖-丙型肝炎病毒研究的重大意义

2020诺奖-丙型肝炎病毒研究的重大意义

2020诺奖-丙型肝炎病毒研究的重大意义
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GeneTex推出重组单克隆Iba1抗体

GeneTex推出重组单克隆Iba1抗体

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Profiling cytokine storm in COVID-19 patients by metatranscriptomic sequencing

Profiling cytokine storm in COVID-19 patients by metatranscriptomic sequencing
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2019 新型冠状病毒

2019 新型冠状病毒

最近发生于全球的一系列肺炎病例是由一种新型的β冠状病毒(Coronavirus)引起
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科学家对抗严重急性呼吸综合征冠状病毒2

严重急性呼吸综合征冠状病毒2与历史上流行过的严重急性呼吸综合征(SARS)和中东呼吸综合征(MERS)同为冠状病毒类,了解病毒的结合受体与交互作用方式,将有助于严重急性呼吸综合征冠状病毒2的药物开发。
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肥胖症与糖尿病

肥胖的普及说明了世界面临着全球营养危机,然而,关于最佳饮食重点和应对这些挑战的政策方法仍存在大量困惑和争议...
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NLRP3炎性体激活驱动tau病理

NLRP3炎性体激活驱动tau病理
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胆固醇载体也可以治疗阿尔茨海默氏症?

科学家最近在《自然医学》期刊上发表了一项令人兴奋的研究,确定ApoE是经典补体级联反应的主要检查点抑制剂。
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从禽流感看细胞因子风暴

近期欧洲与亚洲多处爆发了高致病性的禽流感疫情,主要是由H5N1, H5N6与H5N8亚型的A型流感病毒所引起,导致多数禽类死亡,除了对经济影响重大,也存在感染人体的可能风险。
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GeneTex推出RAS (G12D突变体) 重组抗体 [HL10]

GeneTex的RAS(G12D突变体)重组抗体适用于IHC染色和WB。
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细胞氧化还原的关键转录因子NRF2与疾病的治疗和预防!

NRF2大部分保留在胞质中;而在氧化刺激下,NRF2可以自由移动到细胞核,与DNA上的抗氧化剂反应元件(AREs)结合
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GeneTex Introduces Recombinant Monoclonal Iba1 Antibody

GeneTex Introduces Recombinant Monoclonal Iba1 Antibody

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Leaky blood-brain barrier triggers neural dysfunction that is reversible

In an exciting Science Translational Medicine paper, Senatorov et al. identify the progressive, age-dependent breakdown of the blood-brain barrier (BBB) as the trigger for subsequent neural pathology and pathophysiology
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GeneTex Partners With BenchSci to Augment Antibody Catalog Publication Data

GeneTex Partners With BenchSci to Augment Antibody Catalog Publication Data

Partnership will use BenchSci's artificial intelligence to help researchers run successful experiments with the right antibody
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SARS-CoV-2 Proteins and Antibodies for Lateral Flow-based Neutralization Assay

SARS-CoV-2 Proteins and Antibodies for Lateral Flow-based Neutralization Assay

for Lateral flow-based Neutralization Assay
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