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Probing Antibody-Antigen Interactions

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  • Authors: Guocheng Yang1, Stefanie N. Velgos2, Shanta P. Boddapati3, Michael R. Sterks4
  • Editors: James E. Crowe Jr.5, Diana Boraschi6, Rino Rappuoli7
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Chemical Engineering, Arizona State University, Tempe, AZ 85287-6006; 2: Mayo Clinic Arizona, Phoenix, AZ 85054; 3: Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR 97239; 4: Department of Chemical Engineering, Arizona State University, Tempe, AZ 85287-6006; 5: Vanderbilt University School of Medicine, Nashville, TN; 6: National Research Council, Pisa, Italy; 7: Novartis Vaccines, Siena, Italy
  • Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
  • Received 28 January 2013 Accepted 11 April 2013 Published 07 February 2014
  • Michael R. Sierks, sierks@asu.edu
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  • Abstract:

    Antibodies are biological molecules generated by the host immune system in response to the invasion of foreign bodies or antigens. Therefore, antibodies must possess high specificity toward target antigens in order for the antigen to be recognized and subsequently destroyed. Because of this specificity, antibodies or antibody fragments that maintain binding specificity are heavily used in diagnostic assays and are becoming increasingly important in many therapeutic applications. Classical immunoassays such as radioimmunoassay and enzyme-linked immunosorbent assay are effective analytical techniques that have been widely used to screen and determine antibody specificity. Because of increased demands for antibodies with well-defined specificities, other techniques have been developed that facilitate generation and characterization of antibody-binding specificities under different conditions, such as when the protein is expressed on a cell surface or the target antigen is hard to isolate. Here, we describe three alternate techniques that provide unique abilities to characterize antibody-antigen binding events: (i) surface plasmon resonance, (ii) fluorescence activated cell sorting, and (iii) atomic force microscopy. These different techniques take advantage of various changes in physical and/or chemical properties of the analytes that occur upon binding, such as refractive index, surface charge, and changes in structure. These techniques provide unique powerful advantages over traditional immunoassays including real-time and label-free detection, low sample volume and concentration requirements, and molecular-level detection sensitivity. This article provides an overview of how these alternate approaches to studying antibody-antigen interactions can be used to facilitate rapid development of new antibody-based reagents for diagnostic and therapeutic applications.

  • Citation: Yang G, Velgos S, Boddapati S, Sterks M. 2014. Probing Antibody-Antigen Interactions. Microbiol Spectrum 2(1):AID-0010-2013. doi:10.1128/microbiolspec.AID-0010-2013.

Key Concept Ranking

Enzyme-Linked Immunosorbent Assay
0.45979866
Atomic Force Microscopy
0.4393351
Scanning Probe Microscopy
0.40720776
0.45979866

References

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2014-02-07
2017-09-25

Abstract:

Antibodies are biological molecules generated by the host immune system in response to the invasion of foreign bodies or antigens. Therefore, antibodies must possess high specificity toward target antigens in order for the antigen to be recognized and subsequently destroyed. Because of this specificity, antibodies or antibody fragments that maintain binding specificity are heavily used in diagnostic assays and are becoming increasingly important in many therapeutic applications. Classical immunoassays such as radioimmunoassay and enzyme-linked immunosorbent assay are effective analytical techniques that have been widely used to screen and determine antibody specificity. Because of increased demands for antibodies with well-defined specificities, other techniques have been developed that facilitate generation and characterization of antibody-binding specificities under different conditions, such as when the protein is expressed on a cell surface or the target antigen is hard to isolate. Here, we describe three alternate techniques that provide unique abilities to characterize antibody-antigen binding events: (i) surface plasmon resonance, (ii) fluorescence activated cell sorting, and (iii) atomic force microscopy. These different techniques take advantage of various changes in physical and/or chemical properties of the analytes that occur upon binding, such as refractive index, surface charge, and changes in structure. These techniques provide unique powerful advantages over traditional immunoassays including real-time and label-free detection, low sample volume and concentration requirements, and molecular-level detection sensitivity. This article provides an overview of how these alternate approaches to studying antibody-antigen interactions can be used to facilitate rapid development of new antibody-based reagents for diagnostic and therapeutic applications.

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FIGURE 1

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. doi:10.1128/microbiolspec.AID-0010-2013.f1

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 2

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. doi:10.1128/microbiolspec.AID-0010-2013.f2

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 3

Schematic illustration of a FACS system. (A) A flow cytometer showing the light path, sensors, optics, and filters used (figure adapted from [ 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. doi:10.1128/microbiolspec.AID-0010-2013.f3

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 4

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. doi:10.1128/microbiolspec.AID-0010-2013.f4

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 5

Association curves demonstrate that H1v2 binds AB17-28 (A) and C1 binds AB29-40 (B).doi:10.1128/microbiolspec.AID-0010-2013.f5

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 6

Association ( ) and dissociation ( ) rate constants and dissociation constants ( = / ) of H1v2 (A) and C1 (B) to monomeric AB1-40. doi:10.1128/microbiolspec.AID-0010-2013.f6

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 7

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. doi:10.1128/microbiolspec.AID-0010-2013.f7

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 8

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. doi:10.1128/microbiolspec.AID-0010-2013.f8

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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FIGURE 9

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.” doi:10.1128/microbiolspec.AID-0010-2013.f9

Source: microbiolspec February 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.AID-0010-2013
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