Incorporating one of Quanta BioDesign's dPEG® linkers, Notre Dame researchers created novel synthetic allergens for investigating mast cell degranulation. Researching type-1 hypersensitivity (allergic reactions) under laboratory conditions in a way that reflects hypersensitivity in the wild is challenging because in natural conditions epitopes (the part of an antigen recognized by an immune system) are heterogeneous, and the IgE antibodies formed to heterogeneous epitopes are themselves variable. To address this issue, Michael W. Handlogten, Tanyel Kiziltepe, and Başar Bilgiçer, all of the University of Notre Dame, designed and synthesized homotetravalent and heterotetravalent synthetic allergens containing Dansyl, DNP, and DNP-Pro haptens (Figure 1) in different arrangements.
Synthetic Allergens Used a dPEG® Linker
Applying standard Fmoc-amino acid chemistry on solid support in the synthesis, the synthetic allergens used lysine to form a branched structure and Fmoc-N-amido-dPEG®8-acid (PN10273, from Quanta BioDesign, Ltd.) to provide spacing between the lysines and the haptens. (See Figure 2, below.)
Quanta BioDesign’s PN10273 was chosen because it “does not form non-specific interactions with proteins, is flexible enough to minimize steric constraints for hapten binding and enhances the solubility of the hydrophobic haptens” (page 93). Moreover, the dPEG®8 linker length when attached to the lysine residues in the branched structure provided what had previously been determined to be the optimal separation distance between haptens (page 93). From each of the haptens in Figure 1, the researchers synthesized three different homotetravalent (HmTA) and two heterotetravalent (HtTA) synthetic allergens, presenting two of the haptens, each with a valence of two. HtTA-1 presented DNP and Dansyl, while HtTA-2 presented DNP-Pro and Dansyl.
Homotetravalent vs. Heterotetravalent Synthetic Allergens
Using the monoclonal antibodies IgEDNP and IgEDansyl, the research group confirmed that both antibodies bind simultaneously to the synthetic allergens. They then compared the extent of mast cell degranulation that occurred with the synthetic allergens as compared to known positive controls. (Mast cell degranulation is the first major immune system response in a hypersensitivity reaction.) They found that homotetravalent-DNP-Pro was unable to stimulate any mast cell degranulation, while homotetravalent-DNP and homotetravalent-dansyl both provoked responses that were only marginally better than the positive controls. In contrast, the two heterotetravalent synthetic allergens provoked much stronger responses than the homotetravalent allergens and the positive controls. HtTA-1 (DNP, Dansyl) induced degranulation over a wide concentration range when both antibodies were present, but failed to induce degranulation under conditions where only one antibody was present. HtTA-2 (DNP-Pro, Dansyl) had a similar ability to induce degranulation. The mast cell degranulation response demonstrated a normal (Gaussian) distribution across the concentration range tested for each synthetic allergen. Moreover, for both heterotetravalent allergens, the maximum degranulation response occurred when the total IgE on the mast cell surface was 25% IgEDansyl, 25% IgEDNP, and 50% orthogonal IgE.
This paper provides important insights into hypersensitivity reactions and demonstrates experimentally the importance of allergen valency, affinity, and cooperativity in mast cell degranulation resulting from allergen-IgE binding. Moreover, the authors have provided a valuable, flexible platform on which they and others can build other functionalities (labels, drug conjugates, and so forth) to extend this area of allergy research.
Michael W. Handlogten, Tanyel Kiziltepe, and Başar Bilgiçer. Design of a heterotetravalent synthetic allergen that reflects epitope heterogeneity and IgE antibody variability to study mast cell degranulation. Biochem. J. (2013) 449 (91–99) doi:10.1042/BJ20121088. Also available through PubMed. To see a current publications list from Professor Başar Bilgiçer's lab, click here.
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Robert H. Woodman, Ph.D.
Robert Woodman earned his B.S. in Microbiology from the University of Southern Mississippi and his Ph.D. in biochemistry from The Ohio State University. He is a Sr. Production Development Scientist and the Quality Control Manager for Quanta BioDesign, Ltd. Robert has used his abilities in organic chemistry to develop new dPEG® products, and is now using his biochemistry training to develop new applications for these products. You can connect with Robert through LinkedIn.