The Fab (Fragment, antigenbinding) fragment of an antibody is a key component responsible for the specific recognition and binding of antigens. Here's a more detailed look at the Fab fragment:

Structure of the Fab Fragment

Composition: The Fab fragment is composed of the variable regions of both the heavy and light chains of the antibody. It includes:
Light Chain: The variable light chain domain (VL).
Heavy Chain: The variable heavy chain domain (VH) and part of the constant region of the heavy chain (CH1).
Formation: The Fab fragment is generated by enzymatic cleavage of the fulllength antibody (immunoglobulin) using enzymes such as papain. This cleavage results in the production of two Fab fragments and one Fc fragment from each antibody molecule.

Function of the Fab Fragment

Antigen Binding: The Fab fragment contains the antigenbinding site, which is formed by the combination of the variable regions of the heavy and light chains. This region specifically binds to a particular epitope on the antigen.
Specificity: The variability in the amino acid sequences of the Fab regions allows antibodies to recognize a vast diversity of antigens with high specificity. Each antibody has a unique Fab region that binds to a unique epitope.

Key Characteristics

AntigenBinding Sites: Each Fab fragment has one antigenbinding site. Since an antibody molecule has two Fab fragments, it has two antigenbinding sites.
Flexibility: The Fab region allows the antibody to interact with antigens that may have different shapes and sizes due to its flexible structure, which is crucial for effective immune recognition.

Applications

Diagnostic and Therapeutic Use: Fab fragments are used in various diagnostic and therapeutic applications. For example:
Diagnostic Tests: Fab fragments are used in assays for detecting the presence of specific antigens.
Therapeutic Agents: Fab fragments are used as therapeutic agents in cases where a full antibody might be too large or may cause unwanted effects. For instance, Fab fragments can be used in cancer therapies to target and neutralize specific cancerrelated antigens.
Fab Fragments Production
Fab fragments can be produced using enzymatic digestion,phage display and recombinant DNA technology.
Enzymatic Digestion
Starting Material
Use a purified antibody.
Digestion with Papain
Treat the antibody with the enzyme papain, which cleaves above the hinge region.
This process yields two identical Fab fragments and an Fc fragment.
Purification
Separate the Fab fragments from the Fc region using techniques like protein A/G affinity chromatography, size exclusion chromatography, or ion exchange chromatography.
Phage Display Technology
Animal Immunization
cDNA Synthesis
Total RNA extraction.
RTPCR
PCR amplification with Actinspecific primers to identify the quality of cDNA
Fab Library Construction
Plasmid construction transformation
Packaging: >1×10^13 phage particles /ml.
Recombinant DNA Technology
Gene Cloning
Clone the DNA sequences encoding the variable and constant regions of the light and heavy chains.
Expression Systems
Insert the cloned genes into an expression vector.
Transfect into suitable host cells (e.g., E. coli, yeast, mammalian cells).
Production and Purification
Harvest and purify the Fab fragments from the culture supernatant using affinity chromatography.

Fab fragments and F(ab')2 fragments
Fab Fragments
Structure: Consist of one antigenbinding arm, made from one constant and one variable domain from each of the heavy and light chains.
Production: Generated by papain digestion of an antibody, splitting it above the hinge region.
Function: Bind to a single epitope, useful for blocking and detection without effector functions.
F(ab')₂ Fragments
Structure: Comprise two linked Fab regions, retaining both antigenbinding arms.
Production: Created by pepsin digestion, which cuts below the hinge region, keeping the two Fab portions connected.
Function: Bind two identical epitopes, providing bivalent binding without the Fc region, often used in therapeutic and diagnostic applications for enhanced avidity.

Summary
The Fab fragment of an antibody plays a crucial role in the immune response by specifically binding to antigens. Its ability to recognize and bind to specific epitopes is fundamental to the antibody's function in targeting pathogens or other harmful substances.
Reference:
https://www.alpha-lifetech.com/fab-antibody-discovery-service/