Antibody Basics

What are Antibodies and What are Antigens?

Antibodies are large glycoproteins also known as immunoglobulins (Ig) which are produced, cell-surface expressed and secreted by immune cells, specifically B lymphocytes. Antibodies recognize foreign invading microorganisms by specifically binding to a pathogen’s proteins or antigens, facilitating their neutralization and destruction. Antigens are classically defined as any foreign substance that elicits an immune response. The antibody specificity for any given antigen is underscored by its unique structure, which allows antigen binding with high precision. Because antibodies are highly specific, they serve as very useful tools in scientific research to elucidate the location, abundance, and function of proteins in dynamic biological systems.

Where Antibodies Come From?

The principal role of the immune system is to confer protection from foreign/infectious pathogens and tissue injury. It consists of two main branches: innate (unspecific) and adaptive (acquired) immunity. Antibody production and secretion depends on cells belonging to the adaptive immunity branch.

Innate Immune System

  • Relies on physical barriers (e.g., mucus layer, intact epithelium, and beating cilia) and chemical barriers (e.g., antimicrobial peptides, low pH, and hydrolytic enzymes) to prevent infection and injury.
  • Provides a fast generic or nonspecific response to pathogen- or damage- derived antigens, commonly referred to as pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPS). These responses are mediated by interactions with Toll-like receptors (TLRs).
  • Involves soluble (e.g., cytokines/chemokines, and complement factors) and cellular (e.g., phagocytic, killer and antigen presenting cells) responses.

Adaptive Immune System

  • Provides pathogen-specific immune response through the activation of lymphocyte receptors (T cell receptors and B cell antigen receptors).
  • Involves cellular responses mediated by effector T lymphocytes and antibodies produced by B lymphocytes.
  • Results in immune memory to specific antigens which may be recalled upon re-exposure for a faster and more effective response.

Antibody Production by the Adaptive Immune System

The adaptive immune system has evolved to generate fine-tuned responses to pathogens and other foreign substances. The main effectors of adaptive cellular and humoral immune responses are the T and B lymphocytes, respectively. B lymphocytes develop from hematopoietic stem cells in the bone marrow which give rise to immature IgM expressing B cells. Following migration to the spleen, immature B cells further differentiate into mature or naïve B cells which express both IgM and IgD membrane bound immunoglobulin types. Mature B cells circulate peripherally through the lymphatic system where they interact with foreign antigens. Once a naïve B cell encounters an antigen, its activation may give rise to a plasma B cell or a memory B cell. Memory B cells have surface bound antibodies, whereas the plasma B cells secrete antibodies that are specific for the activating antigen. Memory B cells are rapidly activated upon re-exposure to the same antigen providing a faster and more effective response.

What is the Antibody or Immunoglobulin Type?

Antibody Structure

All antibodies share the same basic structure which consists of four polypeptide chains, two light chains (L chains) and two heavy chains (H chains), held together by disulfide bonds. These four polypeptide chains form a symmetrical molecule commonly depicted as having a "Y" shape and comprised of two identical halves, each bearing identical antigen binding sites.  Based on amino acid sequence variability, two main regions (variable and constant) are identifiable within each antibody polypeptide chain. These regions are commonly depicted as variable light (VL), constant light (CL), variable heavy (VH), and constant heavy (CH). Antibodies differ the most in their amino acid sequence at their variable regions which underscore their antigen specificity. Antigen binding sites are formed from the amino terminals or variable regions of the heavy (VH) and light (VL) chains.

Antibodies consists of four polypeptide chains, two light chains (L chains) and two heavy chains (H chains), held together by disulfide bonds. The fragment antigen-binding region or Fab is formed by the full light chain (VL and CL) and by the heavy chain’s full variable (VH) region and a portion of its constant (CH) region. The fragment crystallizable region or Fc consists only of constant heavy chains (CH).

Antibody Class

Antibody molecules have one of two light chain types, lambda (λ) or kappa (κ). The antibody’s light chain type is not associated with differences in antibody function beyond imparting antigen specificity. In contrast, an antibody’s heavy chain composition determines various functional properties such as its interaction with other proteins (Fc receptor binding), complement activation, avidity, and half-life. The main heavy chain classes in mammals are m, d, g, a and e, which determine the antibody classes or isotypes IgM, IgD, IgG, IgA and IgE, respectively. IgM is the main antibody isotype present during a primary immune response. The IgG antibody isotype predominates during secondary immune responses and is the most common circulating antibody in the immune system. Antibody isotypes differ in structure and immunological functions.

Antibody Class Heavy Chain Class Molecular Weight (kDa) % Total Serum Antibody Functional Properties

IgM antibodies are the first class of Ig made by B cells, main Ig secreted during a response to new antigens, and is secreted as a pentamer.

μ (mu) 900 6
  • First class of Ig made by B cells
  • Main Ig secreted during immune response to new antigen
  • Secreted as a pentamer

IgG antibodies are the main Ig in blood, is secreted as a monomer, and is secreted in large amounts during a secondary immune response to the same antigen.

γ (gamma) 150 80
  • Main Ig class in blood
  • Secreted as a monomer
  • Secreted in large amounts upon secondary exposure

IgA is the main Ig present in body fluids such as saliva and mucous where is present as a dimer while in the blood IgA is present as a monomer.

α (alpha) 385 13
  • Main Ig present in body fluids (e.g., saliva, mucous, and milk)
  • Present as a dimer in body secretions and as a monomer in blood

IgE is secreted as a monomer, binds to the Fc receptor on basophils and mast cells, and is the main Ig produced in response to allergens.

ε (epsilon) 200 0.002
  • Secreted as a monomer
  • Binds to Fc receptors on basophils and mast cells

IgD is secreted in small quantities and mainly serves as a membrane bound antigen receptor.

δ (delta) 180 1
  • Secreted in small quantity
  • Serve mainly as membrane bound antigen receptors


Antibody Binding: Antibody Antigen Interactions

Antibody's Mechanisms of Action

An antibody's function in the immune system is to specifically bind and eliminate foreign particles. Antibodies may remove foreign antigens through several mechanisms.

Neutralization- Antibody binding serves to neutralize foreign particles preventing interaction with cells and consequent cellular damage and invasion.
Opsonization- Antibodies may act as tags which recruit immune cellular mediators to eliminate the foreign substances through phagocytosis.
Complement- Antibodies may coat foreign particles and activate the complement system leading to direct lysis of the foreign substance or further inducing its opsonization.  

What is the Difference between Antibody Affinity and Avidity?

Antibodies bind reversibly to unique regions or epitopes within specific antigens through weak non-covalent interactions which include hydrogen, ionic, hydrophobic, and Van der Waals bonds. The strength or affinity of antibody binding is determined by the net force of weak interactions between a single antibody binding site and its epitope.

Antibody binding affinity is determined by the net force of weak interactions between a single antibody binding site and its epitope while antibody avidity depends on the net strength of all interactions with an antigen.

Antigens may be multivalent, which refers to the presence of several identical epitopes per antigen. Multivalent antigens may interact with multiple antibody binding sites. For any given antibody molecule its avidity is defined by the net strength of all interactions with an antigen. Antibodies like IgG, IgE, and IgD bind their epitopes with higher affinity than IgM antibodies. However, each IgM molecule may interact with up to ten epitopes per antigen and therefore have greater avidity. Because of high affinity, the IgG antibody isotype is the most common type of antibody used in molecular and cell biology methods.

How are Polyclonal and Monoclonal Antibodies Different?

During an immune response to a foreign substance, activation of B cells leads to their expansion and formation of plasma B cells which secrete antigen specific antibodies. However, antigens are structurally complex and consists of multiple antigenic determinants or epitopes. In any given antigen, a B cell will recognize one of a multitude of epitopes, and different B cell clones will secrete antibodies specific to different epitopes within the same antigen. Antibodies secreted by B cells from the same clone are monoclonal antibodies as they bind specifically to the same epitope. However, the overall humoral response contains antibodies secreted by multiple B cell clone types and is naturally polyclonal. B cell secreted antibodies are predominantly found in the serum fraction of the blood, and serum containing antigen-specific antibodies is often referred to as antiserum.

Monoclonal and polyclonal antibodies are powerful tools that facilitate the analysis of complex biological processes. Several advantages and disadvantages are associated with each type of antibody when used as a molecular biology tool.

How to choose between Polyclonal vs Monoclonal Antibody

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