Understanding Immunoglobulin Isotypes

Understanding Immunoglobulin Isotypes

Introduction 

Antibodies mediate adaptive immune responses, protecting the body from internal and external threats. They are also powerful research tools for scientists, helping them identify key players in biological mechanisms, disease biomarkers, and therapeutic targets. The power of the antibody lies in its variability, giving it the flexibility to bind an incredibly broad swath of antigens. In addition to binding site diversity, antibodies also differ in other areas, including the hinge and constant domain. This creates a multitude of isotypes and subclasses with differing functions.

The Anatomy of an Antibody

Antibodies are Y-shaped proteins comprising four polypeptide chains. Light chains are roughly 25 kDa in size and exist as λ and κ types. Heavy chains are roughly 50 kDa in size and determine the isotype of an antibody. μ, δ, γ, α, and ε heavy chain types have been discovered, corresponding to IgM, D, G, A, and E isotypes, respectively.¹ 

Both light and heavy chains have a single variable domain (V) that binds antigen. Light chains also have a single constant domain (C), while heavy chains have three. Disulfide bonds link light and heavy chains together, as well as the two heavy chains. Heavy chains also contain a flexible domain between their first and second constant domains, forming a hinge region. This hinge separates antibodies into Fab (antigen-binding) and Fc (crystallizable) regions.¹

Antibody Isoforms

Most commonly-used mammalian model species, including human, primate, rat, and mouse, have five antibody isotypes. Rabbit, lacking IgD, is the exception. Antibodies of different isotypes operate in different locations and modulate different mechanisms.¹

IgM

Exists as a pentamer. Is the first antibody produced during immune activation.

IgD

Monomer. Regulates B cell responses. Not as well understood as the other isotypes.²

IgA

Exists as a monomer and a dimer. Blocks bacteria from crossing epithelial boundaries.

IgG

Monomer. The main antibody in the blood. Humans possess four subclasses.³

IgE

Monomer. Triggers mast cell-mediated local reactions against pathogens and allergens.³

IgG Subclass Selection

As the main antibody subtype in human circulation, IgG is the primary choice for developing therapeutic antibodies. Of its four subclasses (IgG1, IgG2, IgG3, and IgG4), IgG1 is most often selected due to its strong ability to engage immunostimulatory Fcγ receptors and to trigger robust immune effector responses.⁴ By contrast, IgG2 and IgG4 are preferred when minimal immune cell activation is needed—for example, when an antibody should block but not deplete its target. Subclass selection is based on desired immune function. For example, IgG4 is widely used for anti-PD-1 therapies since it elicits little effector function, reducing the risk of cell depletion or toxicity.⁴

Selecting Antibodies for In Vivo Studies

Choosing the appropriate antibody isotype is crucial for in vivo model studies, as it directly influences immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and Fc receptor engagement. Structural differences in the hinge region, disulfide bonding, and constant domains affect antibody flexibility, stability, and half-life—factors that significantly shape antibody behavior in biological systems. Isotype selection should align with the desired immune effector response, as the Fc region governs interactions with immune cells and complement, influencing whether the antibody elicits functions such as cell depletion. Because antibody isotypes vary across species, selecting the appropriate isotype analog is essential for translational relevance. These considerations are critical for accurately modeling antibody function and drawing meaningful conclusions from in vivo studies.

Antibody-Mediated Immune Responses

In addition to marking targets for adaptive immune cells, antibodies also exert humoral responses. Antibodies can neutralize pathogens by binding to them, preventing them from entering host cells. Antibody binding can also trigger macrophage-mediated opsonization, where macrophages engulf antibody-bound objects and destroy them. Finally, antibody binding can trigger activation of the complement cascade, a pathway that stimulates immune activation and destroys microbes and damaged cells.¹

Antibodies can restore or augment the body’s natural anti-pathogen mechanisms. To that end, understanding how antibodies operate within the immune system is critical for maximizing their research and therapeutic potential. Scientists must consider several factors, including target biology, cellular distribution, target environment, desired effector function, and ease of development/ manufacture when studying antibody-based therapeutic approaches.

IgG Subclasses Across Species

IgG is further subdivided into subclasses based on structural and functional discrepancies in the heavy chain constant region. While IgG is present in all mammals, subclass diversity differs between species.

ADCC: Immune cell-mediated lysis, primarily by natural killer (NK) cells through engagement of FcγRIII 

CDC: Target cell lysis mediated by activation of the complement cascade via binding of C1q to the antibody Fc region 

ADCP: Engulfment and clearance of antibody-coated cells by phagocytes (P) such as monocytes and macrophages via engagement of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) 

Strong Activity: +++ 

Moderate Activity: ++ 

Low Activity: + 

Minimal or No Activity: - 

Unknown Activity: ?

References

1. Janeway CA Jr, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001.
2. Gutzeit C, et al. The enigmatic function of IgD: some answers at last. Eur J Immunol. 2018;48(7):1101-1113. 
3. Vidarsson G, et al. IgG subclasses and allotypes: from structure to effector functions. Front Immunol. 2014;5:520. 
4. Yu J, et al. How to select IgG subclasses in developing anti-tumor therapeutic antibodies. J Hematol Oncol. 2020;13:45.