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Localization of antibody-combining sites within the variable portions of heavy and light chains
Abstract:
The Fab'-fragment of a mouse IgA-myeloma (protein-315) was split by pepsin to yield a smaller fragment that retained the anti-2,4-dinitrophenyl activity of the intact protein. This fragment, which we call Fv, has a molecular weight of about 30,000 (half that of Fab'), and is composed of two polypeptide chains (molecular weight 14,000) held together by noncovalent bonds. The N-terminal sequence of Fv suggests that it is composed of the N-terminal half of Fab', and consists of the variable portions of the heavy and light chains. Since Fv has about one binding site with the same association constant as Fab', this experiment provides direct evidence that the antibody site in this protein is contained entirely in the variable portion, and is independent of the constant portion, of the molecule.
Insights
Researchers isolated an Fv fragment from mouse IgA myeloma protein, demonstrating that the antibody binding site resides solely within the variable regions of the heavy and light chains.
Area of Science:
- Immunology
- Molecular Biology
- Protein Chemistry
Background:
- Mouse IgA myeloma protein (protein-315) possesses anti-2,4-dinitrophenyl activity.
- The Fab'-fragment of this protein contains the antibody's antigen-binding site.
Purpose of the Study:
- To determine the minimal fragment of the antibody retaining antigen-binding activity.
- To investigate the role of variable and constant regions in antibody function.
Main Methods:
- Pepsin digestion of the Fab'-fragment to generate smaller fragments.
- Molecular weight determination of the resulting fragment (Fv).
- N-terminal sequencing of the Fv fragment.
Main Results:
- A smaller fragment, termed Fv, was isolated with retained anti-2,4-dinitrophenyl activity.
- The Fv fragment has a molecular weight of approximately 30,000 and consists of two non-covalently linked polypeptide chains.
- N-terminal sequencing indicated the Fv fragment comprises the variable portions of the heavy and light chains.
- The Fv fragment exhibited one binding site with an association constant similar to that of the Fab'-fragment.
Conclusions:
- The antibody binding site is exclusively located within the variable regions of the heavy and light chains.
- The constant regions of the antibody molecule are not essential for antigen binding activity.