Antibody Experts Cite NeuroMab Characterization as Industry Leading

The Importance of a Well-Characterized Antibody Product

In this installment of Antibodies in the Literature, we highlight a recent publication by co-authors from McGill University, The Development Studies Hybridoma Databank, University of Iowa, The Michael J Fox Foundation for Parkinson's Research, Emory University, Harvard Medical School, Stanford University School of Medicine, Frederick National Laboratory, Specifica, CiteAb, and Abcam, who describe the critical need for well-characterized antibodies in biomedical research and specifically call out our NeuroMabs as a "great model" for antibody characterization (1).

Antibodies are critical tools routinely used in a variety of immunoassays and protocols in biomedical research, clinical pathology, diagnostics, microscopy, imaging, drug development, CAR-T cell therapy, gene therapy, animal health and clinical research. The ability to detect, enrich, localize, and interrogate the function of a target protein is key to many scientific studies. Determining the concentration of a specific protein in a biological matrix (serum, plasma, urine) is a fundamental need in diagnostics, drug discovery and development, immunology and toxicology and has a profound impact on human health.

Most laboratories around the world purchase their tool antibodies from companies that either make antibodies or resell antibodies they obtained by a variety of business agreements. Companies that sell antibodies are not bound to any industry regulations or by any regulatory agency. Antibody companies have routinely sold antibodies that are not necessarily well characterized (2). There is a long history of consumers using antibodies that simply do not function as the seller has claimed, and for decades, the consumer operated under the "buyer beware" circumstance without even knowing it (3). The use of antibody products that were not well characterized resulted in colossal waste of time and money in laboratories across the world.

Validation vs Characterization

The term "validation" with respect to an immunoassay or any method that uses antibodies as tools, refers to confirmation of utility and performance of the method itself. The antibody is simply a part of that method. The term "characterized," with respect to an antibody purchased by the consumer, refers to data supplied by the antibody vendor to the public, typically in marketing materials. An antibody may have more than one use, and the antibody vendor may provide data that demonstrates that the antibody may function in on or more applications. For example, an antibody may be useful as a capture reagent in ELISA, in western blotting and in flow cytometry.

Antibodies purchased from antibody vendors can be used in FDA regulated studies. For example, laboratories that operate under GLP (Good Laboratory Practice) do commonly purchase antibodies on the commercial marketplace. They use antibodies to develop a number of different types of assays that may include PK (pharmacokinetics), ADA (anti-drug organizations) and biomarker assays. The GLP labs, typically CRO's (contract research organizations) or drug development companies, will develop an immunassay mehtod and then validate that method under the umbrella of GLP.

The Characterization of NeuroMabs

The monoclonal antibodies that are called "NeuroMabs" originated in an NIH sponsored initiative at the University of California, Davis. The initiative sought to address the lack of characterized antibodies on the market at that time (2005) and to put into place the framework for the development of well-characterized antibodies that can be used with confidence with respect to anticipated performance. This initiative also sought to reduce the enormous amount of wasted effort associated with the use of commercial antibodies that simply did not work in the hands of the end user. NeuroMabs are a part of a larger industry effort to provide end users with quality antibody products. Figure 1 details how the NeuroMab effort fits in the industry timeline of initiatives aimed at the production of a well-characterized antibody product.

The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use agains mammalian brain (i.e., NeuroMabs) (4). The screening process of monoclonal antibody clone candidates are screened by ELISA against the immunogen and then screened against transfected heterologuous cells expressing the antigen of interest that have been fixed and permeabilized using a protocol that mimics that used to prepare brain samples. Additional testing includes immunohistochemistry and Western Blots in rodent brains but also including KO mice, and samples from human brains when possible (4).

Figure 1. Timeline of key antibody characterization projects (1).

The NeuroMab Catalog - A Commitment to Quality and Well-Characterized Antibody Products

Our NeuroMab catalog has almost 500 unique monoclonal antibodies. Especially popular NeuroMab antibody products are LRRK2 (clone N241A/34) and PSD-95 (clone K28/43). Clone N241A/34 has been cited in the literature at least 150 times and clone K28/43 has been cited in the literature at least 940 times! Our Anti-Dardarin/LRRK2, C-terminus mouse monoclonal primary antibody from NeuroMab is produced in-house from hybridoma clone N241A/34. It is KO (knock-out) validated, detects drosophilia, human, mouse, non-human primate, and rat. Our anti-Dardarin/LRRK2, C-terminus is purified by Protein A chromatography. It is great for use in IHC, ICC, IP, ELISA, WB. Our Anti-PSD 95 mouse monoclonal primary antibody from NeuroMab is produced in-house from hybridoma clone K28/43. It is KO validated, detects chicken, human, mouse, non-human primate, and rat PSD 95, and is purified by Protein A chromatography. It is great for use in IHC, ICC, IP, WB. 

Related Products and Services

Generation of Positive Controls - Anti-Drug Assay Development (ADA) We understand the crucial role positive control antibodies play in the drug testing and approval process. It is hard to overstate the importance of safe, effective drugs, and we provide top quality controls for ADA assays. Our scientists have years of experience in the design and production of these essential reagents.

These antibodies are designed to streamline assay development and sample testing, providing researchers with the precision they need to enhance drug safety and efficacy studies in biologic drug development. The antibodies are compatible with any immunoassay platform including ELISA, Gyros, Meso Scale Discovery (MSD), AphaLISA and ELLA.

Recombinant Chimeric Chicken Antibodies!

A chimeric antibody is a recombinant hybrid antibody that combines domains from distinct species, such as mice and chickens, to create an antibody with improved functionality.

Advantages of our Chimeric Chicken Antibodies

1. Reproducibility: Because they are recombinant, chimeric chicken antibodies offer a consistent and reproducible source of antibody.
2. Animal-friendly: Our chicken chimerics are produced in cultured mammalian cells using serum-free and protein-free growth medium. No animals are used in their production.
3. Known binding properties: The mouse variable regions - which contain the antigen binding sites are from well-characterized and highly validated NeuroMab antibodies.
4. Reduced background: Using mouse antibodies on mouse samples can be problematic if the mouse secondary antibodies react with endogenous mouse IgG. Chicken chimerics use an anti-chicken secondary, greatly reducing background signal.
5. Easier Multiplexing: The use of a unique species such as chicken allows researchers to perform a triplex immunofluorescence experiment alongside the standard mouse and rabbit primaries without worrying about the cross reactivity of another mammalian antibody.

Buffers:

Coating Buffers: Coating Buffers optimize antibody or antigen adsorption onto ELISA plates, stabilize tertiary structures, extend shelf-life, and minimize capture reagent usage.

Blocking Buffers: Block Buffers target antibody-sandwich or antigen-down ELISAs, preserve hydration of dried coat proteins, mitigate non-specific binding, enhance protein stability, and decrease assay variability for prolonged shelf-life.

Sample and Assay Diluents: ICT's Sample Diluents are suitable for antibody-sandwich or antigen-down ELISAs. They minimize non-specific binding interactions during the assay process, reduce sample variation and matrix interference, and decrease background noise.

• Protein-Free Sample Diluent: a protein-free matrix used for the dilution of biological samples (e.g., serum, cell culture media) into the useful range of antibody-sandwich and antigen-down ELISA format assays. This unique buffer contains a hetergeneous mixture of proprietary molecules that help reduce background noise associated with non-specific bridging of signal-generating conjugates to the plate well surface.
  
• Neptune Assay Diluent: formulated to address non-specific binding issues with human, porcine, or bovine serum or plasma samples. This non-mammalian assay additive works to enhance the specific anti-analyte/antigen antibody signal.
  

• Assay Diluent Optimization Pack: our optimization pack contains 4 x 100 mL ELISA development buffers (General Assay Diluent, IgM-Reducing Assay Diluent, Neptune Assay Diluent, and Antigen-Down Assay Diluent) and offers an economical method of addressing matrix effects and optimizing signal-to-noise ratio in ELISA development projects.

• Sample Diluent Optimization Pack: our optimization pack provides 4 x 100 mL bottles of Sample Diluent formulations (General Serum Diluent, Plasma Sample Diluent, Neptune Sample Diluent, and Protein-Free Sample Diluent) for an economical and fast method to screen for the best Sample Diluent for an assay.

Substrates and STOP Solutions: Substrates improve ELISA sensitivity, minimize background noise, ensure reproducible linearity, and are eco-friendly, while STOP solutions standardize readings, halt color development, and reduce variation for enhanced reproducibility.

References

1. Kahn RA, Virk H, Laflamme C, Houston DW, Polinski NK, Meijers R, Levey AI, Saper CB, Errington TM, Turn RE, Bandrowski A, Trimmer JS, Rego M, Freedman LP, Ferrara F, Bradbury ARM, Cable H, Longworth S. Antibody characterization is critical to enhance reproducibility in biomedical research. Elife. 2024 Aug 14;13:e100211. doi: 10.7554/eLife.100211. PMID: 39140332; PMCID: PMC11324233.
2. Saper CB. An open letter to our readers on the use of antibodies. J Comp Neurol. 2005 Dec 26;493(4):477-8. doi: 10.1002/cne.20839. PMID: 16304632.
3. Couchman JR. Commercial antibodies: the good, bad, and really ugly. J Histochem Cytochem. 2009 Jan;57(1):7-8. doi: 10.1369/jhc.2008.952820. Epub 2008 Oct 14. PMID: 18854593; PMCID: PMC2605718.
4. Gong B, Murray KD, Trimmer JS. Developing high-quality mouse monoclonal antibodies for neuroscience research - approaches, perspectives, and opportunities. N Biotechnol. 2016 Sep 25;33(5 Pt A):551-64. doi: 10.1016/j.nbt.2015.11.007. Epub 2015 Nov 28. PMID: 26644354; PMCID: PMC4884554.