Choosing And Using Isotype Controls For In Vivo Experiments

What is an isotype control antibody?

Selecting and using the correct isotype control antibody is an essential part of all in vivo experiments. These antibodies are used as a negative control – they match the exact host species, isotype, and subclass of the primary targeting antibody, but have no specificity to proteins present in the target organism or species. Isotype control antibodies are designed to be non-binding in in vivo animal models but retain all the non-specific characteristics of the primary antibodies used in an experiment.

Why is it important to incorporate an isotype control treated group in an in vivo experiment?

Incorporating an isotype control treated group is required to generate reliable data because it allows the researcher to accurately differentiate between results observed from primary antibody binding in an antigen-specific manner and results observed from non-antigen specific binding or other nonspecific effects of antibody injection.

Primary monoclonal antibodies administered to animals can interact non-antigen specifically with Fc receptors on cells, including B cells, dendritic cells, NK cells, macrophages, and more. Sometimes Fc receptor binding is involved in the primary antibodies’ mechanism of action, such as antibody directed cytotoxicity (ADCC). Nevertheless, non-antigen specific Fc receptor engagement can result in observable biological effects or phenotypes not related to antigen-specific binding. An isotype control antibody matching the isotype and subclass of the primary antibody will also bind to Fc receptors, allowing the researcher to control for those effects.

In addition to matching the isotype and subclass, an ideal isotype control must also match the host species of the primary antibody. Many primary monoclonal antibodies used for in vivo research in mouse models are created using hybridoma technology in rats or hamsters. When a xenogeneic rat or hamster IgG is injected into a mouse, an immune response may develop against the injected antibody. Multiple injections increase this possibility. As with Fc receptor binding, this can also result in observable phenotypes not related to antigen-specific binding. Including a group of mice treated with an isotype control antibody matching the host species of the primary antibody allows the researcher to control for these non-antigen specific effects.

Frequently asked questions about isotype controls

1. Can an untreated group or a PBS treated group be used in place of an isotype control treated group?

   Using an untreated group or PBS treated group as a simplified negative control is not advised. An isotype control antibody must be used to accurately discriminate between results observed due to primary antibody binding in an antigen-specific manner and results observed due to non-antigen specific binding or other nonspecific interactions.

2. If mice are treated with multiple primary antibodies of different host species, isotypes, or subclasses, do the mice in the control group need to be treated with multiple isotype control antibodies or is a single isotype control antibody sufficient?

   Since the host species, isotype, and subclass of an antibody dictates its potential non-specific effects, the isotype control group must be treated identically to the primary antibody-treated group. For example, if the primary group is injected with a rat IgG2a and a rat IgG2b antibody, then the isotype control group must also be injected with a rat IgG2a and a rat IgG2b antibody.

Why choose Bio X Cell isotype control antibodies?

Bio X Cell provides an extensive range of isotype control antibodies formulated for both in vivo and in vitro applications. Our isotype controls feature:

• Exceptional Purity Our optimized proprietary antibody manufacturing method ensures an ultra-pure antibody solution without added proteins or chemicals. Each lot is QC tested for purity using SDS-PAGE.
• Ultra-low Endotoxin Levels The level of endotoxin is QC tested for each lot. Our InVivoMAb products are ≤ 2EU/mg and InVivoPlus products are ≤ 1EU/mg. If endotoxin levels below 1EU/mg are required, please simply contact our technical support for details.
• Pathogen Free Each lot of InVivoPlus product is screened for an exhaustive panel of murine pathogens. The results are detailed on product-specific datasheets, to help you adhere to IACUC and Animal Facility requirements.
• Low Protein Aggregation Our proprietary antibody manufacturing method ensures an antibody solution with very low levels of protein aggregation. Additionally, each lot of InVivoPlus product is QC tested for aggregate level and guaranteed to be below 5% of the total protein.

How to choose appropriate isotype control antibody?

The isotype control antibody must match the host species, isotype, and subclass of the primary antibody. For the convenience of our customers, we have included this essential product information as well as the recommended isotype control for all of our primary antibodies on each product page. The table below is a comprehensive list of Bio X Cell’s isotype control antibodies suitable for both in vivo and in vitro research.