Recombinant antibodies offer several key advantages compared to traditional antibodies. These include superior lot-to-lot consistency, continuous supply, and animal-free manufacturing. As such, recombinant antibodies are seeing increased use for scientific research, especially as a means of addressing the ongoing reproducibility crisis.
Traditional polyclonal and monoclonal antibodies are the product of normal B cell development and genetic recombination. They are generated by immunizing an animal with an antigen to elicit an immune response. While polyclonal antibodies are secreted by many different B cell clones and recognize multiple antigenic epitopes, monoclonals originate from a single B cell clone and are specific for just one epitope.
Recombinant antibodies are monoclonal, but their production involves in vitro genetic manipulation. After cloning the antibody genes into an expression vector, this is then transfected into an appropriate host cell line for antibody expression. Mammalian cell lines are most commonly used for recombinant antibody production, although cell lines of bacterial, yeast, or insect origin are also suitable.
Because recombinant antibody production involves sequencing the antibody light and heavy chains, it is a highly controlled and reliable process. In contrast, hybridoma-based systems for producing monoclonal antibodies are subject to genetic drift and instability, increasing the potential for lot-to-lot variability or loss of antibody expression. Recombinant antibodies are highly consistent from lot to lot, thereby ensuring reproducible experimental results.
In vitro methods for producing antibodies are amenable to large-scale production, meaning antibody availability is unlikely to become a limiting factor. Moreover, since the recombinant antibody sequence is known, continuity of supply is assured; in situations where an antibody will be used to support large, long-term studies, this can be an especially critical factor.
Unlike traditional methods for antibody production, recombinant approaches avoid the need to use animals. Where polyclonal antibodies are purified directly from the serum of the immunized host, and monoclonals are purified from either hybridoma-derived tissue culture supernatant or ascites, recombinant antibodies are instead purified from the tissue culture supernatants of transfected host cell lines. Regardless of whether an antibody is polyclonal, monoclonal or recombinant, it must always be properly validated in the intended application prior to experimental use. At CST, we adhere to the Hallmarks of Antibody Validation™, six complementary strategies for determining the specificity, sensitivity, and functionality of an antibody in any given assay. By carefully tailoring these strategies to each antibody product, we guarantee that CST antibodies will work as expected, to help you achieve results you can trust.
Immunohistochemical analysis of paraffin-embedded CT26 syngeneic tumor using CD177 (E1V7N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic mammary tumor using CD177 (E1V7N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded Renca syngeneic tumor using CD177 (E1V7N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse ovary using CD177 (E1V7N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse colon using CD177 (E1V7N) Rabbit mAb.
| Cat. # | Size | Qty. | Price |
|---|---|---|---|
| 28740S | 100 µl |
|
| REACTIVITY | M |
| SENSITIVITY | Endogenous |
| MW (kDa) | 105 |
| Source/Isotype | Rabbit IgG |
Product Information
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunohistochemistry (Paraffin) | 1:50 - 1:200 |
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
|
RECOMMENDED DETECTION REAGENTS |
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 | SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653 |
|---|---|---|
|
COMPATIBLE CHROMOGEN |
SignalStain® DAB Substrate Kit #8059 | SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713 |
| SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 | SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824 | |
| SignalStain® Deep Black Peroxidase Substrate Kit #72986 | ||
| SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644 |
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 283
Mouse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp314 of mouse CD177 protein.
Cluster of differentiation 177 antigen (CD177, also known as human neutrophil alloantigen 2a and NB1), is a glycosyl-phosphatidylinositol (GPI)-linked N-glycosylated transmembrane protein expressed primarily on the surface of neutrophils (1,2). Expression of CD177 can be induced by granulocyte-colony-stimulating factor and during pregnancy or bacterial infection (3-5). CD177 mediates adhesion and transendothelial migration of neutrophils via interaction with CD31/PECAM-1 and integrin β2 on endothelial cells (6,7). CD177 also promotes the cell surface expression of proteinase 3 (PR3), which may play a role in enhancing endothelial cell junctional integrity and thus vascular integrity during neutrophil diapedesis (8-10). In association with CD11b/ITGAM, integrin β2, and PR3, CD177 promotes activation of TNF-α primed neutrophils including degranulation and superoxide production (11). CD177 is a polymorphic gene that has been linked to several important clinical diseases, including polycythemia vera, Wegener’s granulomatosis, and immune mediated neonatal neutropenia (12-14).
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
PhosphoSitePlus®