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Category: Clinical Microbiology
Probing Antibody-Antigen Interactions, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817411/9781555817350_Chap22-1.gif /docserver/preview/fulltext/10.1128/9781555817411/9781555817350_Chap22-2.gifAbstract:
Antibodies bind antigens via noncovalent bonds, such as hydrogen bonds, ionic, hydrophobic, and Van der Waals forces, and their interactions depend strongly on the distance between two interacting molecules. While each individual bond is weak, the collective noncovalent bonds between the antibody and antigen can be strong when all the interacting molecules work together synergistically. Because there are a very large number of these interactions and the antigen and antibody are large, flexible, dynamic molecules, binding between an antibody and antigen is a very complex process. The binding interactions may also be time dependent, because formation of an antibody-antigen complex may involve a sequential series of interactions which induce conformational changes that generate some bonds while breaking others. Because of the dynamic and transient state of antibody-antigen interactions, measurements of the antibody-antigen interactions can be quite complicated and inconsistent, and the results may vary depending on the sample treatment conditions and technique utilized ( 1 ).
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Schematic illustration of a BIAcore SPR instrument. (A) The BIAcore system contains three main elements crucial to its operation: the integrated microfluidic cartridge (IFC) (light gray L-shaped block), sensor chip, and optical detection unit. (B) A simplistic overview of how SPR works. (C) The transformation of the shift in incident angles to the response signal in an SPR sensogram.
An SPR sensogram depicting the different stages of a binding event. After the target has been immobilized, a baseline RU is established by using only running buffer in the flow system. Upon injection of the analyte, the RU on the sensogram gradually increases, indicating that the system is in the association phase where the analyte binds to the target. It is important to note that some bound molecules have already begun to dissociate during analyte injection. The RU reaches saturation at steady state, where the associating and dissociating molecules are in equilibrium. Once the analyte injection is completed and is replaced by running buffer, the system is in the pure dissociation phase, which is marked by a decrease in RU. The sensor chip regeneration is performed, and baseline RU should be restored.
Schematic illustration of a FACS system. (A) A flow cytometer showing the light path, sensors, optics, and filters used (figure adapted from Applied Microbiology and Biotechnology [ 11 ] with permission of with permission of Beckman-Coulter). (B) A simplified illustration of the principles of flow cytometry with the use of FACS. FL, fluorescent light.
Schematic illustration of an AFM. (A) A typical AFM setup comprises an AFM cantilever, a laser source, a photodetector, and a piezoelectric scanner. (B) AFM topographical imaging is acquired when the tip raster scans the sample surface in the X-Y direction, while the tip deflection is recorded in the Z direction. (C) AFM force-distance curve. The force plot is generated when the tip (i) approaches the surface (red dash line), (ii) contacts the surface and moves with the surface up to a preset point. In the retracting phase (green solid line), the tip retracts from the surface and experiences (iii) one or (iv) more bond-breaking events before completely dissociating from the surface and (v) returning to its original point.
Association curves demonstrate that H1v2 binds AB17-28 (A) and C1 binds AB29-40 (B).
Association (k a) and dissociation (k d) rate constants and dissociation constants (K D = k a/k d) of H1v2 (A) and C1 (B) to monomeric AB1-40.
Illustration of magnetic bead-based (MACS) biopanning. Yeast cells expressing antibody fragments (scFv’s) are grown in medium containing galactose to induce expression and incubated with biotinylated antigen and streptavidin or neutravidin magnetic beads. Yeast cells are loaded into a magnetic column, and those expressing antigen-binding scFv’s are retained. After multiple washes, bound yeast cells can be plunged out and the process can be repeated several times to enrich for antigen binders.
Overview of the AFM-biopanning process. The target antigen is first adsorbed on the mica surface, and the selection of phage is performed by using multiple rounds of biopanning. Any unbound phage is subsequently washed and removed, and bound phages are eluted from the mica surface. The eluted phages are then amplified, purified, and used for subsequent rounds of panning.
Single-phage recovery using AFM. (Top) Phage DNA recovery on the AFM tip using PCR. (Bottom) AFM image of the sample before (A) and after (B) phage “pickup.”