Recombinant Monoclonal Antibodies

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What is a Recombinant Monoclonal Antibody?

Through the years, the technology used to produce antibodies has evolved. Traditional polyclonal antibodies are produced through the immunization of a host species to produce antibody clones against multiple epitopes in an antigen. The production of traditional monoclonal antibodies also involves immunization of lab animals with antigens for the induction of antibody producing B-cells which are isolated and immortalized by the generation of hybridomas. Monoclonal antibodies bind to just one epitope, creating a higher level of specificity, and recombinant technology has allowed scientists to significantly increase the specificity and sensitivity of the antibodies produced. However, engineering a monoclonal antibody from a hybridoma for improved specificity is a challenge due to the lack of nucleotide sequence.

Different from polyclonal or traditional monoclonal antibody production, our recombinant rodent antibody production starts with a pre-existing hybridoma from which antibody sequences are isolated, cloned, and expressed for antibody production. Hybridomas are used to isolate a desired sequence and recombinant technology immortalizes the antibody sequence so that the exact sequence can be stored forever. The recombinant hybridoma conversions maintain the original antibody isotype and mimic the original clone with zero variation between batches of antibodies produced.

In addition to hybridoma conversions, recombinant antibodies can be produced by directly cloning antibody sequences from B-cells. This development process involves: (1) immunization of host animal (rabbit or goat) with desired antigen, (2) isolation of B cells from the host animal, (3) isolation, cloning, and expression of antibody sequence for small-scale production, (4) application testing (WB, ICC, ELISA, etc.) and screening of clones for highest specificity and sensitivity to isolate highly desired clones, and (5) sequencing of clones to scale up production after further purification. Application testing is continuous throughout the production process to ensure high quality products.


Why Choose Recombinant?

Using recombinant monoclonal antibodies reduces experimental variability, allows for higher reproducibility, and provides the capability to engineer these antibodies. Recombinant antibodies are immortal sequences that combine the specificity of traditional monoclonal antibodies with enhanced lot-to-lot consistency.


Benefits to Utilizing Recombinant Monoclonal Antibodies:

  1. Same Antibody, Every Time - Knowing the exact antibody sequence ensures identical clones are produced and used each time
  2. Flexible Format - Using recombinant technology, specific sequences can be modified to achieve different goals by adding a tag (e.g. HA, Flag, V5) or direct conjugation (e.g. biotinylation), thus conveniently converting the same binder tool to a format more amenable to particular tasks
  3. Improved Efficacy – Expression of antibody sequences into different cell types results in varying levels of post-translational modifications (e.g. glycosylation), creating antibodies with improved functionality
  4. Reduced Unspecific Binding - Sequence sizes can be altered to increase specificity in binding of the antibody to the desired antigen (e.g. eliminating Fc fragment to decrease interference with binding, engineering the CDR domains, etc.)
  5. Expanded Specificity - Binding sequences from different antibodies can provide bi-specific or multi-specific clones with enhanced binding properties

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Antibody Validation & Quality Assurance

Recombinant monoclonal antibodies are validated multiple times throughout the production process. When the desired sequence is first isolated from cloned B cells, sequences are tested and verified in small-scale, as well as at the pilot expression stage of the production process. Novus Biologicals relies on five antibody validation strategies to ensure the highest level of specificity and reproducibility. These strategies include using knockout cell lines, orthogonal validation, independent-antibody studies, expression of tagged antibodies, and biological validation. The validation standards used are in accordance with the International Working Group for Antibody Validation’s recommendations. Visit the 5 Pillars of Antibody Validation page to learn more about our validation standards and processes.

To prevent antibody cross-reactivity and inconsistent assays, many of our top-selling polyclonal antibodies are now sold in a recombinant form to assure there is no variation between batches – allowing you to reproduce your original results every time.

genetic strategic validation

Genetic Strategy Validation

biological strategies validation

Biological Strategies Validation


The LC3B antibody is available in polyclonal (left), and recombinant monoclonal (right) forms. The knockout and biological strategy validation of these products is shown below.

LC3B detection with LC3B Polyclonal Antibody in HeLa cells, parental or LC3B KO and with or without Chloroquine, WB LC3B detection with LC3B Recombinant Monoclonal Antibody in HeLa cells, parental or LC3B KO and with or without Chloroquine, WB

Knockout Validated: Left- LC3B Polyclonal Antibody [NB100-2220], and Right- LC3B Recombinant Monoclonal Antibody (1251A) [NBP2-46892]. Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (Catalog # NB100-2220), and 2.0 ug/mL of Recombinant Rabbit Anti-Human/Mouse/Rat LC3B Monoclonal Antibody (1251A) (Catalog # NBP2-46892) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog# HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa (as indicated) in the parental HeLa cell line but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.



biological strategies validation

Biological Strategies Validation

The IL-1β antibody is available in non-recombinant monoclonal (left), and recombinant monoclonal (right) forms. The biological strategy validation of these products is shown below.

IL-1 beta detection with Non-Recombinant Mouse Monoclonal Anti-Human IL-1 beta/IL-1F2 in lysates of THP-1 cells treated or untreated with PMA/LPS, WB IL-1 beta detection with Recombinant Mouse Monoclonal Anti-Human IL-1 beta/IL-1F2 in lysates of THP-1 cells treated or untreated with PMA/LPS, WB

Western blot shows lysates of THP 1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nM PMA for 24 hours and 10 µg/mL LPS for 3 hours. PVDF membrane was probed with: (Left) 1 µg/mL of Non-Recombinant Mouse Anti-Human IL 1 beta /IL 1F2 Monoclonal Antibody (Catalog # MAB601) and (Right) 1 µg/mL of Recombinant Mouse Anti-Human IL 1 beta /IL 1F2 Monoclonal Antibody (Catalog # MAB601R) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for IL 1 beta /IL 1F2 at approximately 36 kDa (as indicated). This experiment was conducted under reducing conditions.



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Novus’ Quality Guarantee: All our products are backed by the 100% Novus Guarantee, stating that the product you purchase is fully guaranteed to work in the application and species listed on the Novus website and datasheets or you will receive a free replacement or a full refund.

Novus provides over 6,000 recombinant antibody products covering more than 70 unique targets, providing quality antibodies to support researchers across the globe. With Novus products, you can reduce variability in your testing – saving you time, money, and increasing reproducibility.

 

 

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References:

Aggeler, B. "Recombinant Antibodies – Four species, endless possibilities." R&D Systems. www.rndsystems.com/webinar/recombinant-antibodies-four-species-endless-possibilities

Johns Hopkins Bloomberg School of Public Health. "Monoclonal Antibodies: Scientific uses and advantages of rAbs and aptamers." AltWeb. altweb.jhsph.edu/mabs/rabsaptamer.html

The Protein Man. "A Discussion of Protein Research | Recombinant Antibodies: An Overview." G-Biosciences. info.gbiosciences.com/blog/recombinant-antibodies-an-overview