An overview and practical guide on the applications of recombinant antibodies as reagents in biomedical experiments and a summary of recombinant antibodies among the over 60,000 formal publications in Labome's Validated Antibody Database. However, since 2015, a number of suppliers have started to provide recombinant antibodies for researchers. The suppliers often use the same catalog numbers or clone names for the antibody preparations both from hybridoma and from expression hosts. This practice makes impossible to survey the citation data for recombinant antibodies. The article hence will no longer be updated.
While there are numerous research articles and reviews on the generation of recombinant antibodies and their therapeutic potentials, few articles have addressed the current state of recombinant antibodies and their potentials as they are applied in typical biomedical experimentations such as Western blot, flow cytometry, and immunochemistry. This review is intended to examine the citation of recombinant antibodies in research reports and review the current availability of recombinant antibodies as reagents. DNA sequences for recombinant antibodies can be obtained through cloning of immunogen-specific antibody genes or selection from a phage library. Recombinant antibodies are monoclonal and differ from regular monoclonal antibodies and polyclonal antibodies since they are produced in an expression host such as E. coli or CHO cell line. Major benefits of recombinant antibodies include 1) the sequence of a recombinant antibody can be modified to tailor to specific needs; 2) the generation of an antibody does not depend on the immunogenicity of a host animal; 3) recombinant antibodies are free from animal pathogens; 4) in vitro-expressed antibodies have excellent lot-to-lot consistency, especially since a significant number of hybridomas contain non-specific yet productive additional variable chains [1] ; 5) the antibody chains can be engineered to enable, for example, consistent conjugation [2]. In 2015, Drs. Andrew Bradbury and Andreas Plückthun, along with 110 co-signatories, proposed to transition the provision antibody reagents from the current state of confusion to a system with only sequenced well-characterized recombinants [3]. Several commercial entities have undertaken large-scale efforts to generate recombinant antibody reagents. Some of them are discussed below. In addition, academic groups and government institution are also getting involved. One common area of research that employs recombinant antibodies (or nanobodies) is the structural biology, where a small-size recombinant nanobody is used to stabilize a protein complex. For example, Warne T et al used recombinant conformation-specific nanobodies to determine the active-state structures of the β1-adrenoceptor [4]. Another area is the rapid generation of recombinant neutralizing antibodies against infectious disease pathogens from B cells of infected patients, for example, those against SARS-CoV 2 [5, 6].
As of September 2019, Labome product database contains 319647 monoclonal antibody products from 209 suppliers, 541380 polyclonal antibody products from 130 suppliers, and 14719 recombinant antibody products from 10 suppliers. The actual number of recombinant antibody products is likely to be higher since some suppliers do not distinguish recombinant antibody products from monoclonal antibody products and we lack an accurate counting.
Labome manually curates formal publications to develop Validated Antibody Database. As of September 2019, Labome has curated 62121 formal publications and collected 311855 instances of antibody applications. The vast majority of antibodies cited in the literature are either monoclonal antibodies produced through hybridoma culture or ascites or polyclonal antibodies generated from host animals (Table 1).
Antibody type | Num | |
---|---|---|
2018 | 2014 | |
polyclonal | 2949 | 10928 |
monoclonal | 25089 | 28881 |
recombinant | 115 | 34 |
Table 2 lists some of the commonly cited recombinant antibodies. The list is not exhaustive. The earliest citation that we can identify is the biotin-conjugate anti-phosphotyrosine antibody recombinant clone 4G10® ( 16-204) from Upstate (now part of MilliporeSigma) in 2005 [7]. This clone and its different conjugates are the most cited recombinant antibody so far. Other cited recombinant antibodies come from four large-scale recombinant antibody commercial offerings: ABfinity from Life Technologies, REAfinity from Miltenyi, HuCAL from AbDSerotec, and antibodies from Creative Diagnostics. They are discussed below. In addition, individual recombinant antibodies, apart from the systematical efforts, have been used as reagents in literature as well. For example, a recombinant antibody against sulfotyrosine [8], commercially offered by MilliporeSigma, with the clone name Sulfo-1C-A2, has been cited by at least five publications [9-14]. Another recombinant antibody against H3K9me3, produced in response to a large number of problematic histone antibodies that had been on the market [15], is also commercially available (Diagenode, C15500003). Recombinant antibodies (scFv) have been reported for the widely used c-myc clone 9E10 [16-18] ; however, we failed to identify any citations for the recombinant version.
Target and clone name | Catalog number | Num | Sample References |
---|---|---|---|
CD133/1-VioBright FITC, human | 130-111-085 | 42 | [19, 20] |
beta Amyloid Recombinant Rabbit Monoclonal Antibody (H31L21) | 700254 | 31 | [21, 22] |
Phospho-FAK (Tyr397) Recombinant Rabbit Monoclonal Antibody (31H5L17) | 700255 | 19 | [23, 24] |
CD303 (BDCA-2)-VioBright FITC, human | 130-114-182 | 18 | [25, 26] |
phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)Antibody, recombinant clone AW39R, rabbit monoclonal | 05-797R | 16 | [27, 28] |
Caspase 3 Recombinant Rabbit Monoclonal Antibody (9H19L2) | 700182 | 16 | [29, 30] |
CD1c (BDCA-1)-VioBright FITC, human | 130-110-598 | 16 | [26, 31] |
CD141 (BDCA-3)-APC-Vio770, human | 130-110-362 | 13 | [31, 32] |
Vinculin Recombinant Rabbit Monoclonal Antibody (42H89L44) | 700062 | 12 | [33, 34] |
CD159a (NKG2A)-PE, human | 130-114-092 | 12 | [35, 36] |
Anti-HLA-DR, DP, DQ-Biotin, human | 130-104-869 | 11 | [37, 38] |
CD133/2-PE, human | 130-112-315 | 11 | [39] |
CD56 pure, human | 130-108-016 | 11 | [40, 41] |
Phospho-AKT1 (Ser473) Recombinant Rabbit Monoclonal Antibody (98H9L8) | 700392 | 10 | [42, 43] |
Anti-TRA-1-60-PE, human | 130-100-347 | 9 | [44, 45] |
Anti-LGR5-FITC, human | 130-112-508 | 9 | [46, 47] |
APC anti-human/mouse Granzyme B Recombinant | 372204 | 8 | [48, 49] |
CD304 (BDCA-4)-FITC, human | 130-112-044 | 8 | [32, 50] |
CD317 (PDCA-1)-FITC, mouse | 130-112-376 | 8 | [51, 52] |
Anti-SSEA-4-VioBlue, human | 130-098-366 | 7 | [44, 53] |
CD185 (CXCR5)-APC, human | 130-098-422 | 7 | [54, 55] |
CD244 (2B4)-Biotin, human | 130-099-038 | 7 | [56, 57] |
Anti-AN2-PE, human and mouse | 130-116-440 | 7 | [58, 59] |
Anti-F4/80-PE-Vio770, mouse | 130-118-320 | 7 | [60, 61] |
Anti-IL-17A-FITC, human | 130-118-396 | 7 | [62, 63] |
CD197 (CCR7) pure, human | 130-108-057 | 7 | [64, 65] |
CD34-PE, human | 130-120-520 | 7 | [66, 67] |
Sulfotyrosine Antibody, Clone Sulfo-1C-A2 | 05-1100 | 6 | [13, 68] |
Phospho-FAK (Tyr576) Recombinant Rabbit Monoclonal Antibody (2H74L24) | 700013 | 6 | [69, 70] |
Claudin 18 Recombinant Rabbit Monoclonal Antibody (34H14L15) | 700178 | 6 | [71, 72] |
Digoxigenin Recombinant Rabbit Monoclonal Antibody (9H27L19) | 700772 | 6 | [73, 74] |
Occludin Recombinant Rabbit Monoclonal Antibody (6H10L9) | 701161 | 6 | [75, 76] |
Anti-HLA Class I Bw4-Biotin, human | 130-103-919 | 6 | [77, 78] |
Anti-Ki-67-PerCP-Vio700, human and mouse | 130-100-293 | 6 | [79, 80] |
Anti-TCRβ-VioBlue, mouse | 130-104-861 | 6 | [81, 82] |
CD94-APC-Vio770, human | 130-101-146 | 6 | [83, 84] |
MARVELD2 Recombinant Rabbit Monoclonal Antibody (54H19L38) | 700191 | 6 | [85, 86] |
Anti-CCR10-PE, human | 130-120-547 | 6 | [87, 88] |
Anti-FoxP3-PE, mouse | 130-111-678 | 6 | [89, 90] |
Anti-ROR1-APC, human | 130-118-015 | 6 | [91, 92] |
Anti-TCR Vδ1-FITC, human | 130-118-498 | 6 | [35, 93] |
CD196 (CCR6)-PE, human | 130-120-598 | 6 | [94, 95] |
CD314 (NKG2D)-PE, human | 130-111-723 | 6 | [96, 97] |
CD314 (NKG2D)-PE, human | 130-124-349 | 6 | [96, 97] |
Phospho-ERK1/ERK2 (Thr185, Tyr187) Recombinant Rabbit Monoclonal Antibody (15H10L7) | 700012 | 5 | [98, 99] |
Phospho-JNK1/JNK2 (Thr183, Tyr185) Recombinant Rabbit Monoclonal Antibody (D12H7L17) | 700031 | 5 | [100, 101] |
RAB11A Recombinant Rabbit Monoclonal Antibody (3H18L5) | 700184 | 5 | [102, 103] |
Phospho-p53 (Ser15) Recombinant Rabbit Monoclonal Antibody (14H61L24) | 700439 | 5 | [104, 105] |
Anti-TCR Vα7.2-FITC, human | 130-100-190 | 5 | [106, 107] |
CD158a (KIR2DL1)-PE-Vio770, human | 130-103-969 | 5 | [108, 109] |
CD158e1/e2-Biotin, human | 130-104-483 | 5 | [110, 111] |
CD177-FITC, human | 130-101-521 | 5 | [112, 113] |
CD183 (CXCR3)-APC, human | 130-101-378 | 5 | [95, 107] |
CD66b-PE, human | 130-104-414 | 5 | [114, 115] |
CD44v6-APC, human | 130-111-425 | 5 | [116, 117] |
CD335 (NKp46)-APC, human | 130-112-122 | 5 | [118, 119] |
CD16-APC, human | 130-113-389 | 5 | [36, 120] |
Anti-DLL4-FITC, human | 130-118-378 | 5 | [121, 122] |
Anti-FcεRIα-FITC, human | 130-110-863 | 5 | [123, 124] |
Anti-T-bet-APC, human and mouse | 130-119-821 | 5 | [125, 126] |
CD146-PE, human | 130-111-508 | 5 | [127, 128] |
CD184 (CXCR4)-PE-Vio615, human | 130-109-890 | 5 | [129, 130] |
CD226 (DNAM-1)-FITC, human | 130-117-637 | 5 | [131, 132] |
CD44-FITC, human | 130-113-903 | 5 | [116, 117] |
Generation of recombinant antibodies through libraries has the potential to provide specific antibodies for many antigens. However, it appears that recombinant antibodies remain under-utilized as reagents. Several suppliers offer recombinant antibodies, often generated through proprietary processes, as research reagents. Table 2 lists some of the products.
ABfinity has been cited quite often, for example, ABfinity GFP antibody G10362 was cited by over a hundred publications (e.g. [133, 134] ). ABfinity antibody is an intact IgG molecule with both light and heavy chains. Here is a more informative, and earlier, version of Invitrogen description about Abfinity antibodies: "ABfinity™ antibodies are recombinant antibodies developed by immunizing animals, screening for functionality, and cloning the immunogen-specific antibody genes into high-level expression vectors. The antibodies are produced on a large scale by expressing them in mammalian cells, and purifying them with protein A. These recombinant antibodies are expressed in mammalian expression systems but appear just like their counterparts isolated from serum or produced by hybridomas. Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain". There are a total of 1063 Abfinity antibodies. At least 118 of them have been cited in the literature.
SGC (Structural Genomics Consortium), a not-for-profit, public-private partnership, generated over 1,124 different recombinant antibodies targeting 152 chromatin-related proteins, among which, most of the best-performing ones for each target protein are distributed through Invitrogen [135] and thus can be searched at Labome.
Miltenyi Biotec offers 361 recombinant clones sold under the brand name of REAfinity antibodies (as of September 2018). Each REAfinity antibody is comprised of a single type of heavy chain and a single type of light chain, with mutated human IgG1 parts for Fc regions to eliminate any affinity with endogenous Fcgamma receptors. REAfinity antibodies are commonly used for flow cytometry. For example, Miltenyi Biotec REAfinity anti-CCR3 (REA122) and anti-CCR6 (REA277) antibodies were used in flow cytometry to study the role of transcription factor IRF1 in the anti-tumor function of TH9 cells [136], anti-CD123-APC (REA114) was used to investigate the distribution and function of plasmacytoid dendritic cells in peripheral blood and gut mucosa of HIV infected patients [137], and CCR7-PE (REA108) antibody was used to identify immunogenic antigens from Aspergillus fumigatus [138]. Clone REA345 anti-STAT1 (pY701) antibody was used to investigate the role of IL-28R in NK cell functions [139]. Table 3 lists the number of citations of REAfinity antibodies over the years that Labome has identified. Although our search is not exhaustive (it is likely about half of the citations have been identified by Labome), except in the case of two clones REA110 and REA169 for which exhaustive manual searches were undertaken, the increase in the number of citations of REAfinity antibodies over the years is illustrative of the adoption of recombinant antibodies.
Year | Publications | Clones |
---|---|---|
2014 | 5 | 6 |
2015 | 17 | 19 |
2016 | 30 | 30 |
2017 | 60 | 51 |
2018 | 47 | 39 |
HuCAL recombinant antibodies have been cited since 2008 when HCA024, a human anti-human DJ-1 (oxidized at C106) antibody was cited in an article titled "Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1" [140]. The generation of HuCAL recombinant antibodies is based on a 2000 article by Knappik A et al "Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides" [141]. HuCAL platform and its successor have been employed to generate a number of therapeutic antibody candidates. HuCAL catalog products, along with other AbDSerotec products were sold by HuCAL owner MorphoSys to Bio-Rad in 2012. Currently, there are 292 HuCAL recombinant antibody products, including a number of antibodies against antibody drugs such as Adalimumab, Bevacizumab, Cetuximab, Infliximab, Rituximab, Trastuzumab, Natalizumab, Tocilizumab, Omalizumab, Alemtuzumab, Golimumab, and Ustekinumab. The recombinant antibody reagents are Fab monovalent. In terms of antibody affinity, the monovalent intrinsic affinity of clone 18654 against Adalimumab is 0.16 nM by real-time, label-free molecular interaction analysis using an Attana A200 instrument on immobilized Adalimumab.
Creative Biomart recombinant anti-human TNF antibody scFv fragment MOM-18006-S(P) was used in an ELISA assay to study the effects of methylprednisolone on inflammatory activity and oxidative stress in the lungs of brain-dead rats [142]. Its recombinant antibodies are generated from a process similar to HuCAL in the form of scFv fragments. Labome lists 413 recombinant antibodies from Creative Biomart.
The NIH-funded Protein Capture Reagents Program (http://proteincapture.org/) includes Rutgers antigen program, Johns Hopkins University monoclonal antibody program, and Recombinant Antibody Network (http://recombinant-antibodies.org/), starting in 2011. Recombinant Antibody Network (RAN) in turn is formed by an international consortium of three expert centers at the University of Chicago, University of Toronto, and the University of California at San Francisco (UCSF). The reagents from this program can be searched at Protein Capture Reagents Program website (http://proteincapture.org/) and purchased from three distributors (DSHB (http://dshb.biology.uiowa.edu/), DNASU (https://dnasu.org/DNASU/), and CDI Laboratories (http://cdi-lab.com/)). Please note some of the reagents are only available from one or two distributors. As of Jan 15, 2015, 754 recombinant antibodies are in the RAN catalog and it appears that most of the recombinant antibodies are in the validation/characterization phase. Seventeen DSHB recombinant antibodies from Protein Capture Reagents Program can be searched at Labome: Anaphase-promoting complex subunit 2 (PCRP-ANAPC2-RAB-C77), Histone chaperone Anti-silencing function 1B (PCRP-ASF1B-RAB-C68), Forkhead box D3 (PCRP-FOXD3-RAB-C99), Methyl-CpG binding domain protein 4 (PCRP-MBD4-RAB-C73), Histone-lysine N-methyltransferase SETD7 (PCRP-SETD7-RAB-C70), Histone-lysine N-methyltransferase SMYD3 (PCRP-SMYD3-RAB-C71), T-box transcription factor TBX4 (PCRP-TBX4-RAB-C78), T-box transcription factor TBX4 (PCRP-TBX4-RAB-C80), T-box transcription factor TBX4 (PCRP-TBX4-RAB-C81), VENTX homeobox protein (PCRP-VENTX-RAB-C43), Zinc finger and BTB domain containing 21 (PCRP-ZBTB21-RAB-C85), Zinc finger and BTB domain containing 39 (PCRP-ZBTB39-RAB-C86), Zinc finger, FYVE domain containing 20 (Rabenosyn-5) (PCRP-ZFYVE20-RAB-C12), Zinc fingers and homeoboxes 1 (PCRP-ZHX1-RAB-C59), Zinc finger protein 165 (PCRP-ZNF165-RAB-C92), Zinc finger protein 446 (PCRP-ZNF446-RAB-C26), Zinc finger and SCAN domain-containing protein 29 (PCRP-ZSCAN29-RAB-C3). These recombinant antibodies have dissociation constants ranging from < 1 nM to 64 nM.
The Protein Capture Reagents Program, as a pilot project, ended with no followup in 2015 [143].
Several other suppliers are providing recombinant antibodies. These providers include Yumab, Absolute Antibody, BBI Solutions, Adipogen, Randox, and Abcam. Abcam released its 18000th recombinant antibody according to its 2019 annual report. Absolute Antibody has generated over 4,000 recombinant antibody products.
Plasmids for recombinant anti-mouse and anti-rabbit IgG secondary nanobodies are available from Addgene (IDs 104157–104164) [144]. These nanobodies have the potential to replace widely used polyclonal secondary antibodies [144]. Recombinant antibodies against zebrafish cadherin 2 have been generated to distinguish highly homologous members of the same family and to replace commonly used, yet non-specific polyclonal antibodies [145].
Thanks are due to Dr. Kalpana Singh from Miltenyi Biotec for the REAfinity references.
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