What Are Antibody Isotypes and Why Do They Matter for Immunoassays?

Antibody isotypes are classes of antibodies defined by the constant region of their heavy chain. Humans have five main isotypes, IgM, IgD, IgG, IgA, and IgE, each with a distinct structure, role in the immune response, and location in the body. In immunoassays like western blotting and IHC, isotype matters because your secondary antibody must recognize both the host species and the isotype of your primary, and isotype controls help confirm that your signal is specific.

What Are Antibody Isotypes?

When we think of antibodies, we typically consider the variable regions of these proteins, which is what results in different antibodies’ ability to bind different targets. The constant region, however, exhibits much less variation – yet some diversity still exists. Unlike variable regions of antibodies, which are highly heterogeneous, constant regions of antibodies exhibit certain overarching patterns that result in a categorization known as antibody isotypes.

Unlike the stochasticity of variable regions on antibodies, constant regions develop into specific isotypes during B-cell maturation. After B-cell maturation is complete and the antibodies are produced, additional isotypes may be formed through a process known as class switching.

Particularly, there are five main isotypes in humans: IgM, IgD, IgG, IgA, and IgE. Each of these isotypes has its own specific purpose during an immune response and exhibits structural variation in the heavy chain of the antibody. The resulting isotypes differ from each other with respect to quaternary structure, which informs their native biological purposes. Across different regions of the body, different isotypes may be more or less abundant. Beyond humans, IgY antibodies – which are found in species like birds and reptiles – have become popular isotypes for antibody production due to heterogeneity with mammalian isotypes, which can help reduce potential background noise caused by homology when performing assays in mammalian tissues.

What do different isotypes do?

Of the five canonical isotypes, each serves a unique immunological purpose. IgM and IgD are the two isotypes produced by naive B-cells. IgD antibodies act as surface receptors that aid in B-cell maturation, whereas IgM antibodies are the first line of defense after an immune response. IgM antibodies are the most aggressive responder to pathogens, forming pentameric structures that can trap pathogens to thwart infection. After class switching, IgA, IgG, and IgE antibodies are secreted to act as adaptive immune defenders against pathogens. IgG has the longest half-life and circulates the blood to recognize known pathogens, whereas IgA is secreted at the body’s ports of entry, adopting a dimeric structure. IgE antibodies are the rarest class, serving a similar function to IgG antibodies, and this isotype is particularly remarked in allergic responses. Even more granularly, IgA and IgG antibodies are broken down into sub-classes – such as IgA1 and IgA2 which differ slightly structurally to be tuned to particular types of targets. IgG can be broken down into many different understood subclasses, including IgG1, IgG2, IgG3, IgG4. Isotypes are species-specific, and nuances exist within the subclassification between species. For example, mouse IgG2 can is recognized as distinct structures of IgG2a and IgG2b.

Where in the body are different isotypes found?

Most antibodies circulate through the blood, but there is a particular nuance to where each isotype is most abundant. While IgM and IgD antibodies are most closely associated with naive B-cells and may be found on the cell surface, IgM antibodies are also released into the blood to trap pathogens. IgG antibodies circulate throughout the blood to act as surveillance against known pathogens. IgE antibodies are particularly bound to mast cells and basophils. IgA antibodies, on the other hand, specifically reside at the entryways of the body, such as mucosal membranes, saliva, breast milk, and tears, acting almost like a “bouncer” to prevent pathogens from even entering the bloodstream.

How Do Antibody Isotypes Affect Your Immunoassays?

When planning for an experiment that uses antibodies, such as a western blot or immunohistochemical staining, the isotype of your primary antibody matters for how you are able to detect your target of interest. Particularly, it is important to match your secondary antibody to your primary antibody. Typically, we think of host species when considering this matching process, but it is essential to ensure that the isotype matches too. For example, a mouse IgE primary antibody will not be reactive with anti-mouse IgG1 and vice versa. 

Using Isotype Controls to Confirm Specific Signal

Within a more technical context, isotype controls can be added to your immunoassay workflow. Isotype controls are antibodies raised against targets that would be impossible to detect within your samples, yet contain the same heavy chain isotype as your primary of interest. Adding an isotype control to your workflow will allow you to parse out how much of your primary’s signal is specific, as it allows you to measure the signal contributed by the constant antibody regions alone, irrespective of the variable region that should bind your target.

Finding the Right Antibody Isotype at HUABIO

At HUABIO, we offer antibodies primarily of the IgG isotype, with various sub-classes represented in our catalog (such as IgG1 and IgG2b) as well as different secondaries suitable for many different existing isotypes (e.g., IgM, IgY, IgE). Need help matching your primary to the proper secondary? Interested in learning more about isotype controls? Email our human-monitored support inbox [email protected], and we’ll help you find the right antibody for your needs!