CrownVivo™ antibodies are functional grade isotype controls, manufactured specifically for in vivo animal studies including antigen neutralisation and pathway/blockade depletion assays during development and validation of antibody biotherapeutics. These isotype controls are designed to avoid cross-reactivity with common pre-clinical species and are supplied in the exact format needed for in vivo, ex vivo, or in vitro studies.
CrownVivo™ antibodies are made to be highly concentrated, with low endotoxin levels and no preservatives.
CrownVivo™ Isotype Control Antibodies
- Clonality: Monoclonal
- Endotoxin Level: <1 EU/mg as determined by the LAL method
- Purity (SDS) Page: >95% by SDS Page and SEC-HPLC (>95% by SDS Page, >90% by SEC-HPLC for Product Code C0005)
- Concentration: >4 mg/ml
- Buffer: Aseptically packaged in PBS (40mM KH2PO4, 150mM NaCl, pH 7.4) with no carrier protein or preservatives added.
Production and Quality Control
Isotype controls are expressed in mammalian cells and raised against antigens such as hen egg lysozyme (HEL) that do not exist in humans or other common pre-clinical species. CrownVivo™ isotype controls maintain LOT to LOT conformity and reliability. All isotype control antibodies are:
- Affinity purified
- 0.22µm sterile filtered
- Purity is guaranteed to be 95% or greater as measured by SDS-PAGE and SEC-HPLC*. *(>95% by SDS Page, 90-95% by SEC-HPLC for Product Code C0005)
- Analyzed by mass spectrometry for the molecular weight of light and heavy chains
- Packaged in PBS with no added preservatives or carrier proteins at high concentrations
In Vivo Testing
Crown Biosciences has extensive experience conducting in vivo animal studies for clients with isotype control doses ranging from 1-10mg/kg, in animal strains such as NOD/SCID, SCID/Beige, NPG and C57BL/6. The majority of testing is performed with 3.4 or 4 mg/kg, i.p. or i.v. for up to 4 weeks.
Examples of animal studies comparing isotype control to vehicle and/or test antibody are shown below.
Choosing an Isotype Control
After selecting your primary antibody and determining what applications you’ll run, the next step is to identify whether you need a standard research grade or in vivo grade antibody. Here is how you can choose which isotype control is needed in your experiment.
Research Grade Isotype Controls
Standard research grade isotype controls are suitable for basic research applications like western blot, immunohistochemistry, immunoprecipitation, flow cytometry, and ELISA. They’re usually supplied in small quantities and contain a preservative to extend their shelf life. Since they are not used in vivo, endotoxin and purity usually aren’t measured and it’s common that these may be polyclonal and the buffer may contain preservatives.
In Vivo Grade Isotype Controls
For in vivo research, isotype controls are most often high purity monoclonal antibodies, with very low endotoxin levels, and supplied in PBS with no carriers or additives. You may also need bulk quantities for performing in vivo studies.
What are Isotype Control Antibodies?
Isotype control antibodies are negative controls used to accurately measure antibody drug effects and efficacy for in vitro and in vivo monoclonal antibody (mAb) studies. Isotype controls match the test/primary antibody characteristics, but are raised against antigens not found in common preclinical species; this means that isotype controls lack specificity for the target antigen.
In both in vivo and in vitro assays, a signal can result from various binding events. In order to accurately interpret assay results, it is critical to differentiate between binding events such as:
- mAb binding in an antigen-dependent specific manner
- mAb binding in a non-antigen dependent manner, due to interaction with Fc receptors (FcR) or other proteins
Isotype control antibodies provide an ideal negative control for mAb efficacy studies, enabling researchers to accurately distinguish between non-specific background and specific antibody signal.
Why Use Isotype Control Antibodies?
In general, antibodies are very similar to each other structurally. The majority of an antibody molecule is the “constant region”, where antibodies of the same isotype share amino acid sequences. This constant region helps to mediate the antibody mechanism of action (MOA) through binding to FcRs. Only a small, hypervariable region of the mAb actually determines antigen-specific binding and intended therapeutic effect.
When testing antibodies preclinically, you need to control for any variable that might impact the interpretation of study results. Mediation of MOA and engagement of immune cells via the constant and Fc regions can have an impact on mAb efficacy. Therefore, these factors need to be accounted for within study design.
Using a matched isotype negative control (with the same host species, isotype, conjugation, light chain, and concentration as the test/primary mAb) can account for these added variables and enable more precise data analysis. Simple negative controls like PBS cannot induce the same effects, potentially creating misleading data when compared to the test article.
Further Information
*CrownVivo™ isotype control antibodies are manufactured by Crown Biosciences, Inc., a JSR Life Sciences Company.
Originally posted on: https://resources.mblintl.com/learning-center/research-area/in-vivo-grade-isotype-controls/
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