Preparation of Fab and Fab2 Fragments From IgG

As well as their function in binding to antigen, immunoglobulins have other roles within the immune system that necessitate interactions with other proteins such as those of the complement and phagocytic systems. In techniques such as immunofluorescence or in in vivo applications, these interactions can be problematic but can be solved by the use of selective proteolysis to remove parts of the molecule. The polypeptide chains making up the IgG molecule

Fig. 2. Diagram of a molecule of IgG. The molecule is symmetrical, containing two identical heavy (H) chains and two identical light (L) chains. The VL and VH domains form antigen binding sites at the end of either arm. The Fc region is composed of the CH2 and CH3 domains from each of the larger heavy chains and is separated from the two Fab regions by the flexible hinge region which contains the disulphide bonds linking the two heavy chains. Different immunoglobulin classes (G, M, A, E, and D) have different heavy chains. There are two types of light chain (k and X) N, amino terminus; CT, carboxy terminus.

Fig. 2. Diagram of a molecule of IgG. The molecule is symmetrical, containing two identical heavy (H) chains and two identical light (L) chains. The VL and VH domains form antigen binding sites at the end of either arm. The Fc region is composed of the CH2 and CH3 domains from each of the larger heavy chains and is separated from the two Fab regions by the flexible hinge region which contains the disulphide bonds linking the two heavy chains. Different immunoglobulin classes (G, M, A, E, and D) have different heavy chains. There are two types of light chain (k and X) N, amino terminus; CT, carboxy terminus.

fold into a set of distinct domains (Fig. 2) with the two identical antigen binding domains being constituted by the VH and VC domains towards the N-terminus of the arms.

The other interactions are mainly associated with the CH2 and CH3 domains and these are separated from the rest of the molecule by a flexible region known as the hinge region, which is less compact allowing access of proteolytic enzymes. Papain will cleave the N-terminal side of the disulphide bond cluster creating two identical antigen-binding fragments (Fab) and one Fc fragment. Pepsin will cleave the C-terminal side of the disulphide bonds, and also within the Fc part to leave one major product, divalent with respect to antigen, known as (Fab')2.

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