Understanding intermolecular reactions and how they affect antibody-antigen interactions is necessary during immune based labeling, such as immunohistochemistry, immunofluorescence and multiplex labeling. The antibody-antigen binding complex is dictated by several different intermolecular reactions and is the key concept behind therapeutic analysis when using this method. These interactions can affect the specificity, strength, and respective quality which is obtained during microscopy. Visualizing and imaging dozens of different proteins and antigens on a single tissue slice utilized during the multiplex labeling platform requires multiple antibody labels and subsequent rounds of stripping. This removal of antibody labels from the specimen requires an understanding of these intermolecular reactions. These reactions include Ionic Bonds, Covalent Bonds, Hydrogen (H-Bonds) and van der Waals interactions. Ionic and Covalent bonds are not themselves intermolecular, but these intramolecular forces must be understood first to characterize intermolecular interactions.
Electron distribution is at the heart of any intermolecular reaction and is related to the whole spectrum of these reactions. Uniform distributions are associated with nonpolar molecules, and uneven distributions, associated with polar molecules. These characteristics of polar and nonpolar can be seen as opposite ends of the same spectrum. Ionic bonds are at the extreme end of polar molecules and have defined charges that exhibit the strongest bonds. Molecules with these strong, polar, ionic bonds are usually in salt form and are readily soluble in water. It is rare for antibody-antigen complexes to involve ionic bonds directly. Rather, ionic characteristics define the environment which the antibody-antigen complex finds itself in. Depending on the salt concentration of the environment, partial charges of other biomolecules can be neutralized or weakened.
Covalent bonds can span the whole range of the polarity spectrum. Covalent bonds are strong bonds involving the overlap of electron orbitals between atoms to stabilize their valences. Covalent bonds can be used to construct both polar and nonpolar molecules, which presents a wide variety of possible configurations. The polarity of these bonds is determined by the electronegativity of atoms involved. For instance, a carbon atom bound to another carbon atom contains a nonpolar covalent bond because they have equal electronegativities and therefore an equal distribution of electrons. However, a carbon atom bound to an oxygen atom contains a polar covalent bond because oxygen has a higher electronegativity and therefore has a denser electron distribution around it. Molecules with polar covalent bonds have partially charged regions. These regions will orient themselves in space with other molecules that have similar characteristics according to these partial charges. Antibody-antigen complexes are not covalently bound, but partial charges due to the polar covalent bonds of the antibody and the antigen create this electrostatic interaction.
The polarity of some covalent bonds also defines Hydrogen bonds. Hydrogen bonds are commonly described as a hydrogen atom bound to either nitrogen, oxygen, or fluorine. The electronegative differences between hydrogen and these other elements generate a noticeable, and strong, partial charge of the molecules they are a part of. When multiple molecules that contain these covalent bonds come together, the large partial charges snap these molecules together in space electrostatically but not covalently. This is the Hydrogen bond. This is the basis behind capillary action in plants and is the main driver of antibody-antigen complexes and interactions. They are stronger than nonpolar covalent bonds, but not as strong as ionic bonds. They are impermanent and must be considered during immune based labeling.
Van der Waals Interactions
On the weak end of molecular bonds and interactions are van der Waals interactions. These interactions are associated with nonpolar, or weakly polar, covalent bonds. Nonpolar molecules have covalent bonds with relatively even electron distributions. However, sporadic changes in the electron distributions caused by environmental conditions can generate very small partial charges especially in larger hydrocarbons. When long fatty acid molecules come in contact with each other, these sporadic charges cause the molecules to conglomerate in a weak electrostatic manner. These are van der Waals interactions and work closely with Hydrogen Bonds to stabilize the antibody-antigen complex.
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