Overview of Antibodies
Antibodies, also known as immunoglobulins (Ig), are highly specific proteins secreted by B lymphocytes that recognize and neutralize foreign pathogens. They are essential molecular tools in research, diagnostics, and therapeutic development, playing a critical role in the life sciences.
Fig. 1. Antibody Structure Diagram (Source: DOI: 10.1007/978-3-319-42316-6_10)
Detailed Antibody Structure: Heavy and Light Chains
The basic structure of an antibody resembles a “Y” shape, formed by two heavy chains (HC) and two light chains (LC) connected by disulfide bonds.
Heavy Chain
Determines the antibody isotype, such as IgG, IgA, IgM, IgD, or IgE.
Each heavy chain consists of one variable region (VH) and three or four constant regions (CH1, CH2, CH3/CH4).
The Fc region, primarily composed of the heavy chain constant regions, mediates immune effector functions, such as complement activation and Fc receptor binding.
Heavy Chain Isotypes (Human Examples)
| Antibody Type |
Heavy Chain Type |
| IgG |
γ chain (gamma) |
| IgA |
α chain (alpha) |
| IgM |
μ chain (mu) |
| IgD |
δ chain (delta) |
| IgE |
ε chain (epsilon) |
Light Chain
Divided into two types: κ chain (kappa) and λ chain (lambda).
Each antibody molecule contains either κ or λ chains, never a mix of both.
Each light chain includes one variable region (VL) and one constant region (CL), primarily contributing to antigen recognition.
The proportion of light chain types varies across species; κ chains are more common in humans, while λ chains are more prevalent in mice.
Fab and Fc: Functional Components of Antibodies
Antibodies can be cleaved by proteases (e.g., papain, pepsin) into two main functional fragments:
- Fab (Fragment antigen-binding): Composed of the variable regions of the heavy and light chains, responsible for antigen binding.
- Fc (Fragment crystallizable): Consists of the heavy chain constant regions, interacting with immune system components (e.g., Fc receptors, complement proteins) to trigger effector responses, such as ADCC and CDC.
Antibody Classes (Five Major Types)
Antibodies are classified into five types based on the structure of their heavy chain constant regions, each with distinct functions and distributions:
| Type |
Molecular Structure |
Functional Characteristics |
Distribution |
| IgG |
Monomer |
Most abundant in serum, crosses the placenta, neutralizes pathogens, and mediates opsonization |
Blood, tissue fluids |
| IgA |
Monomer/Dimer |
Protects against viruses and bacteria, key in mucosal immunity |
Saliva, breast milk, tears |
| IgM |
Pentamer |
First responder in primary immune responses, strong complement activation |
Blood |
| IgD |
Monomer |
Expressed on B cell surfaces, function less clear |
B cell surfaces |
| IgE |
Monomer |
Involved in allergic reactions and anti-parasitic immunity |
Mucosal tissues, binds mast cells |
Fig. 2. Diagram of Five Antibody Classes (Source: ScienceDirect Topics, Immunoglobulin D Antibody - an overview)
Antigens: Basics and Classification
Antigens are molecules recognized by antibodies or T cells, with the characteristics outlined below:
| Type |
Definition |
| Immunogen |
Triggers an immune response and is recognized by the immune system |
| Antigen |
Recognized by the immune system but may not trigger a response |
| Hapten |
Not immunogenic alone, requires conjugation to a larger molecule to elicit a response |
| Epitope |
The specific molecular determinant recognized by antibodies or T cells |
Fig. 3. Antigen and Epitope Diagram (Source: Wild, D. (Ed.). (1996). Immunoassays: A practical approach. Oxford University Press)
Guidelines for Selecting Experimental Antibodies
To ensure reliable experimental results, consider the following when selecting antibodies:
- Match to Experimental Type: Choose antibodies validated for specific applications, such as Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), ELISA, or Flow Cytometry (FACS).
- Match to Protein Characteristics: Consider species, modification state (e.g., phosphorylation, acetylation), and protein localization (e.g., membrane or nuclear proteins).
- Reliable Validation Strategies: Prioritize antibodies validated through knockout (KO), complementary assays, or IP-WB confirmation for high confidence.
Antibody Sources and Their Application Differences
Polyclonal Antibodies (pAb)
Source: Derived from animal serum (e.g., rabbit, goat) after antigen immunization.
Characteristics: Recognize multiple epitopes, ideal for low-expression or variable samples.
Monoclonal Antibodies (mAb)
Source: Produced by hybridoma cell lines.
Characteristics: Recognize a single epitope, offering high batch-to-batch consistency, suitable for quantitative experiments and mechanistic studies.
Antibody Purification Methods and Optimization
Experimental antibodies typically require purification from supernatant or serum. Common purification methods include:
| Method |
Principle |
Application |
| Protein A |
Binds IgG Fc region |
Human IgG1/IgG2/IgG4 |
| Protein G |
Binds a broader range of Fc regions |
Mouse IgG, multiple species |
| Affinity Chromatography |
Targets antigen-specific binding |
Enhances purity and specificity |
| Ion Exchange Chromatography |
Separates based on charge differences |
Specialized antibody separation needs |
Note: When selecting Protein A or G, consider the antibody species and isotype to ensure optimal binding efficiency and purity.
Enhancing Antibody Specificity: The Importance of Fine Purification
High-specificity antibodies are critical for neutralization, blocking, and multicolor flow cytometry experiments. During preparation, use:
- Rigorous affinity purification protocols;
- Molecular sieving to remove impurities;
- Low-endotoxin processing;
- Strictly validated antigen sources.
These steps ensure antibodies perform with high signal-to-noise ratios and low background in critical experiments.
Conclusion
Understanding antibody structure, function, antigen recognition, and purification is key to designing robust experiments and achieving reproducible results. Researchers are encouraged to plan antibody selection and validation systematically from the outset to build a strong foundation for their studies.
Explore abinScience’s high-quality antibody portfolio, featuring over 15,000 antibodies, including monoclonal, labeled, phosphorylated, nanobodies, and functional in vivo antibodies. These tools, designed for virology, neuroscience, and cancer research, offer exceptional specificity and sensitivity, supporting diagnostics, vaccine development, and therapeutic discovery.
Visit abinScience Now
References
